Mass Frontier 5.0 User Guide Version A

Mass Frontier 5.0 User Guide Version A
Xcalibur™
Mass Frontier™ 5.0
User’s Guide
XCALI-97181 Rev A
July 2006
© 2006 Thermo Electron Corporation. All rights reserved.
OrbiTrap and LTQ FT are trademarks and Xcalibur is a registered trademark of
Thermo Electron Corporation. Mass Frontier, Fragmentation Library, and Spectral Tree are
trademarks of HighChem, Ltd. Microsoft, Excel, and Windows are registered trademarks of
Microsoft Corporation. Pentium is a registered trademark of Intel Corporation.
This document is provided to customers who have purchased Thermo Electron Corporation
equipment to use in the operation of such Thermo Electron Corporation equipment. This document
is copyright protected and any reproduction of this document or any part of this document is strictly
prohibited, except as Thermo Electron Corporation may authorize in writing.
Technical information contained in this publication is for reference purposes only and is subject to
change without notice. Every effort has been made to supply complete and accurate information;
however, Thermo Electron Corporation assumes no responsibility and will not be liable for any
errors, omissions, damage, or loss that might result from any use of this manual or the information
contained therein (even if this information is properly followed and problems still arise).
This publication is not part of the Agreement of Sale between Thermo Electron Corporation and the
purchaser of an LC/MS system. In the event of any conflict between the provisions of this document
and those contained in Thermo Electron Corporation’s Terms and Conditions, the provisions of the
Terms and Conditions shall govern.
System Configurations and Specifications supersede all previous information and are subject to
change without notice.
Printing history: Revision A printed July 2006
Written by Robert Mistrik.
Contents
Preface ............................................................................................. ix
About This Guide ......................................................................ix
Related Documentation .............................................................ix
Special Notices...........................................................................ix
Contacting Us.............................................................................x
Assistance .................................................................................x
Changes to the Manual and Online Help.................................x
Thermo Electron Corporation
Chapter 1
Introducing Mass Frontier.................................................................1
General Information ...................................................................2
System Requirements ..................................................................3
Installation ..................................................................................4
Installing Xcalibur....................................................................4
Installing SQL Server and Mass Frontier..................................4
Activating Mass Frontier ..........................................................5
Starting Mass Frontier..............................................................8
Program Limitations ...................................................................9
New Features in Mass Frontier 5.0............................................11
Modules Overview ....................................................................12
Chapter 2
Structure Editor .................................................................................27
Structure Editor Window..........................................................28
Restoring Defaults..................................................................29
Opening and Saving Structures ..............................................29
Structure Data Formats..........................................................30
Structure Layout .......................................................................31
Text ..........................................................................................32
Template Structures ..................................................................33
Selecting Atoms and Bonds .......................................................34
Atom Properties ........................................................................36
Bond Properties ........................................................................38
Copying Structures ...................................................................39
Pasting Structures......................................................................40
Moving, Resizing, Rotating, and Mirroring Structures..............41
Cleaning Structures...................................................................43
Checking Structures ..................................................................44
MS Calculations........................................................................45
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Contents
Unspecified Bond Location.......................................................46
Unspecified Charge Site ............................................................47
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Chapter 3
Spectral Trees................................................................................... 49
Tree Arrangement .....................................................................51
Tree Node Items .......................................................................52
Tree Layout...............................................................................53
Tree Generation........................................................................54
Manual Creation and Editing of Trees ......................................57
Copying and Pasting Trees........................................................58
Tree Chromatograms ................................................................59
Chapter 4
Database Manager........................................................................... 61
Open a Database Manager Window..........................................63
Records in the Database Manager ..........................................65
Additional Information Associated with a Record ..................66
Mass Differences .......................................................................68
Comparing Spectra ...................................................................69
Cutting, Copying, and Pasting Records ....................................70
Structures in Database Manager ................................................72
Working with Spreadsheets .......................................................75
Search Utilities ..........................................................................77
Spectrum Search ....................................................................78
Tree Search ............................................................................80
Substructure Search................................................................82
Substructure Search Rules ......................................................85
Name Search..........................................................................86
Molecular Formula Search .....................................................86
Molecular Mass Search...........................................................86
ID Number Search.................................................................87
CAS Number Search..............................................................87
Retention Time Search...........................................................87
Search Constraints .................................................................87
Mass Spectral Data Exchange Between Modules .......................89
Fragment Assignment to Spectral Peaks ....................................90
Chapter 5
Library Utilities ................................................................................. 91
Microsoft SQL Server 2000 Desktop Engine ............................92
NIST/EPA/NIH Mass Spectral Database .................................93
Library Installation....................................................................94
Creating User Libraries .............................................................96
Uninstall A Library ...................................................................98
Adding Records to a User Library .............................................99
Thermo Electron Corporation
Contents
Deleting Library Entries..........................................................100
Saving Changes in Libraries ....................................................101
SQL Server and Library Tools.................................................102
Spectral Libraries..................................................................102
Fragmentation Libraries .......................................................103
SQL Server...........................................................................103
Backup and Restore Libraries ...............................................104
Thermo Electron Corporation
Chapter 6
Fragments and Mechanisms.........................................................107
Features...................................................................................108
General Fragmentation and Rearrangement Rules................108
Fragmentation Library Mechanisms .....................................108
Charge Localization Concept ...............................................108
Unimolecular Linear Reaction Mechanisms .........................108
Even- Electron Rule .............................................................109
Bond Cleavages Only ...........................................................109
Ionization Methods..............................................................109
Formally Possible Solutions..................................................109
Fragmentation, Rearrangement and Resonance Reactions.......110
Starting Generation.................................................................112
Fragments & Mechanisms Window ........................................115
Reaction Restrictions...............................................................117
Knowledge Base Page ...........................................................117
Ionization & Cleavage Page .................................................119
H-Rearrangement Page ........................................................121
Resonance Page....................................................................123
Additional Page....................................................................124
Sizes Page.............................................................................125
Generated Fragments Linked with Spectrum ..........................126
Eliminating Generated Fragments Not Present in
a Spectrum ...........................................................................127
Simulation of MSn Experiments..............................................128
Unexplained Peaks ..................................................................129
Too Many Proposed Fragments for a Peak..............................130
Bar Code Spectra ....................................................................133
Chapter 7
Fragmentation Library ....................................................................137
Fragmentation Library Toolbar...............................................139
Drawing Fragmentation Reactions ..........................................140
Active Record..........................................................................142
Additional Information ...........................................................144
Saving Records........................................................................145
Mechanism Extraction ............................................................146
Reaction Symbols....................................................................148
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Contents
Using Library Reactions in Fragmentation Prediction .............150
Search Utilities .......................................................................152
HighChem Fragmentation Library..........................................154
Chapter 8
Fragments Comparator .................................................................. 155
Chapter 9
Mass Spectra Classification........................................................ 159
Spectra Classification ..............................................................160
Principal Component Analysis (PCA) .....................................162
Neural Networks (Self-Organizing Maps) ...............................164
Fuzzy Clustering .....................................................................165
Spectra Transformation...........................................................166
Chapter 10 Spectra Classifier........................................................................... 169
Spectra Classifier Window ......................................................170
Classifying Mass Spectra .........................................................173
Maintaining Groups of Spectra ...............................................175
Chapter 11 Spectra Projector ........................................................................... 177
Generating Spectra Projector Window ....................................178
Spectra Projector Window ......................................................179
3-D Projection Mode..............................................................181
Opening and Saving of Classification Results..........................182
Accessing Spectra from Spectra Projector ................................183
Adding An External Spectrum.................................................184
Chapter 12 Neural Networks ............................................................................ 185
Generating Neural Networks Window....................................187
Neural Networks Window ......................................................189
Working with Neural Networks..............................................191
Chapter 13 Chromatogram Processor ............................................................. 193
Chromatogram Processor Window .........................................195
Data File Formats ...................................................................196
Opening Chromatograms .......................................................197
TIC Page ................................................................................198
Info Page.................................................................................199
Spectra Averaging....................................................................200
Background Subtraction..........................................................201
Processing Extracted Spectra ...................................................202
Selected Ion Chromatogram....................................................203
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Contents
Thresholding, Baseline Correction and Smoothing .................205
Thresholding........................................................................205
Baseline Correction and Noise Elimination..........................206
Smoothing ...........................................................................207
Automated Component Detection and
Spectra Deconvolution.........................................................208
RCD Algorithm ...................................................................211
JCD Algorithm ....................................................................213
TECD Algorithm.................................................................216
Direct Infusion Algorithm....................................................217
Processing Xcalibur MSn Data ...............................................219
Chapter 14 Components Editor..........................................................................221
Searching Components ...........................................................223
Chapter 15 Mass Settings ..................................................................................225
Resolution...............................................................................226
Precision .................................................................................228
Chapter 16 Microsoft Office in Mass Frontier................................................229
Data Exchange between Excel and Mass Frontier....................231
Exporting Data to Excel ..........................................................232
Importing Spectra from Excel .................................................233
Using Excel as Spectrum Editor ..............................................234
Chapter 17 Formula Generator ..........................................................................235
Formula Generation from a Peak ............................................236
Formula Generator Options....................................................238
Chapter 18 Report Creator..................................................................................241
Report Creator Window .........................................................242
Creating Reports .....................................................................243
Index..................................................................................................245
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Preface
About This Guide
Welcome to the Mass Frontier™ software, which is part of the
Thermo Electron Xcalibur® mass spectrometry data system.
Mass Frontier 5.0 provides tools for the management, evaluation, and
interpretation of mass spectra.
This guide describes how to use Mass Frontier for mass spectral
interpretation.
Related
Documentation
Special Notices
In addition to this guide, you can use the Help available from within the
Mass Frontier software.
This guide contains special notices in the text, which can include the
following:
IMPORTANT Highlights information necessary to avoid damage to
software, loss of data, invalid test results, or information critical for
optimal performance of the system.
Note Highlights information of general interest.
Tip Helpful information that can make a task easier.
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Preface
Contacting Us
Assistance
There are several ways to contact Thermo Electron Corporation.
For new product updates, technical support, and ordering information,
contact us in one of the following ways:
Visit Us on the Web
www.thermo.com/finnigan
Contact Technical Support
Phone:
Fax:
E-mail:
1-800-685-9535
1-561-688-8736
[email protected]
Find software updates and utilities to download at
http://mssupport.thermo.com.
Contact Customer Service
In the US and Canada for ordering information:
Phone:
1-800-532-4752
Fax:
1-561-688-8731
Web site: www.thermo.com/finnigan
Changes to the Manual
and Online Help
To suggest changes to this guide or to the online Help, use either of the
following methods:
• Fill out a reader survey online at www.thermo.com/lcms-techpubs
• Send an e-mail message to the Technical Publications Editor at
[email protected]
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Chapter 1
Introducing Mass Frontier
The Mass Frontier 5.0 software can help you manage, evaluate, and
interpret mass spectra. The software provides a number of tools for
processing and organizing mass spectral and chromatographic data.
Because of the large volume of information a mass spectrometer produces,
management capabilities are essential for mastering your analytical
workloads. In contrast to other software systems, Mass Frontier offers
features for the interpretation of mass spectra when an unknown is not
contained in your libraries.
This chapter contains the following topics:
• General Information
• System Requirements
• Installation
• Program Limitations
• New Features in Mass Frontier 5.0
• Modules Overview
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Introducing Mass Frontier
General Information
General Information
Mass Frontier is based on ten modules, which are accessible as windows. All
the modules are seamlessly integrated within an intuitive multidocument
interface (MDI). All the windows are located in your program desktop.
However, four of the fourteen modules (Fragments & Mechanisms,
Neural Networks, Spectra Projector, and Components Editor) cannot be
directly opened from the program desktop by clicking a button or menu
item; they must be generated from user-supplied input.
Although each of the modules is independent, the program automatically
establishes a link between several modules. For example, the program
can make a link between the Database Manager window and the
Fragments & Mechanisms module. In this case, the peaks in a mass
spectrum, displayed in the Database Manager module, are linked with
mass-to-charge ratios in the Fragments & Mechanisms module. Records in
Database Manager can also be linked with objects in the Spectra Classifier
and Spectra Projector modules. Double-click these objects to get the spectra
(Spectra Classifier) or spectrum (Spectra Projector) that are linked with a
particular record in Database Manager. In addition, spectra, structures, and
library entries can be exchanged between modules by using the Copy and
Paste commands.
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System Requirements
Introducing Mass Frontier
System Requirements
At a minimum, Mass Frontier requires the following:
• Microsoft® Windows® 2000 SP 3 or XP SP 1
• Computer with Pentium® 255 MHz processor or higher
• 128 MB of RAM (512 MB recommended)
• SVGA monitor
• 2 GB available hard disk space
• Microsoft Office XP
• Microsoft Internet Explorer 4.01 SP 1 or higher
• Xcalibur 2.0 installed with local user access
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Introducing Mass Frontier
Installation
Installation
Installing Xcalibur
Before you install Mass Frontier software, make sure the Xcalibur data
system is installed on your system with local user access. Mass Frontier
cannot be activated without it.
To install the Xcalibur software
1. Insert the Xcalibur Core Data System Software CD into your
CD-ROM drive.
2. Choose Start > Run from your Windows desktop.
3. Type D:\setup.exe, where D is the letter of the CD-ROM drive.
Click OK to begin the installation.
4. Follow the instructions on the screen until you reach Finish.
Installing SQL Server and
Mass Frontier
Before running the Mass Frontier installation, install the Microsoft SQL
Server desktop engine and run the automated configuration procedure from
the Mass Frontier Setup Launcher.
Installation of the Microsoft SQL Server desktop engine is a complex
procedure, which depends on the system setup. If the installation fails, make
a note of the error message and contact the HighChem database group at
[email protected]
To run Mass Frontier, you must have Full Control permission
on the Mass Frontier application directory (by default,
X:\Program Files\HighChem\Mass Frontier 5.0). Windows XP does not
have this permission set for Limited Account users. If you are a Limited
Account user, ask your system administrator to grant Full Control
permission on the Mass Frontier directory for your account.
To install the MSDE and Mass Frontier software
1. Insert the Mass Frontier CD in the CD-ROM drive. The Setup
Launcher starts automatically.
If the Setup Launcher does not start automatically, choose Start > Run
from your Windows desktop. Type D:\setup.exe, where D is the letter
of the CD-ROM drive. Click OK to begin the installation.
2. Click the Install MSDE button to start the installation of the Microsoft
SQL Server desktop engine. The installation takes several minutes.
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Introducing Mass Frontier
Installation
3. Click the Configure MSDE button to start the automated
configuration of the Microsoft SQL Server desktop engine.
4. If the installation and configuration of the Microsoft SQL Server
desktop engine are successful, the installation of Mass Frontier starts
automatically. Follow the instructions that appear on the screen.
If the Microsoft SQL Server desktop engine cannot be properly
installed or configured, the installation ends. If you cannot
resolve the problem, contact the HighChem database group at
[email protected] After the problems have been resolved, click
the Install Mass Frontier button to continue with the Mass Frontier
installation.
Unless you specify a different location, Mass Frontier is installed at
X:\Program Files\HighChem\Mass Frontier 5.0\MassFrontier.exe
where, X is the CD-ROM drive on your computer.
Activating Mass Frontier
Mass Frontier requires an activation key for each system where you install
the software. The activation keys are not transferable from one system to
another. If you have purchased multiple copies of Mass Frontier, perform
the following procedure for each copy of Mass Frontier that you install.
When you first start Mass Frontier, the License page appears (Figure 1) and
prompts you for an activation key.
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Introducing Mass Frontier
Installation
Figure 1.
License page, showing the serial number and the types of activation
keys available
1. On the License page, highlight the text that appears in the Serial
Number box, and then press CTRL+C to copy the serial number to the
Windows clipboard.
2. Obtain an activation key by e-mail or fax using the following
procedures.
3. On the License page, paste the new activation key in the Activation
Key box, and then click Activate.
Note The serial number begins with a number, whereas the activation
key begins with a letter. Both use only capital letters. The number “0”
and the letters “O” and “I” are not used.
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Introducing Mass Frontier
Installation
To obtain an activation key by e-mail
Send an e-mail message containing the following information to
[email protected]:
1. Type license request in the subject line of the e-mail, and paste the text
from the License page Serial Number box into the body of the e-mail.
2. Locate the bar code on the back of the Mass Frontier CD case. Type the
serial number that appears below the bar code into the body of the
e-mail message.
3. In the body of the e-mail message, include your name, company name,
and phone number, and provide the software version, for example:
Mass Frontier 5.0
Full Version Single License
To obtain an activation key by fax
1. On the Windows desktop, double-click the Xcalibur shortcut icon to
open the Xcalibur Home Page.
2. Choose Tools > Configuration to open the Xcalibur Configuration
dialog box.
3. On the Customer Information page, enter all the information that a
Technical Support representative might need to contact you.
4. Place the pointer below the last line of text in the Address box, and then
press CTRL+V to paste the serial number from the Windows clipboard
into the Address box.
5. Below the serial number, specify the product name and license version
that you want, for example:
Mass Frontier 5.0
Full Version Single License
6. Click Print User Info to automatically create the form you need to fax
or mail to Technical Support. Click OK.
7. Send the printed request form to Thermo Electron using one of the
following fax numbers:
USA or Canada: 1-408-965-6120
International: international access + 1-408-965-6120
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Introducing Mass Frontier
Installation
Starting Mass Frontier
To start Mass Frontier, double-click the Mass Frontier shortcut icon
on the Windows desktop, or choose
Start > All Programs > HighChem Mass Frontier 5.0 > Mass Frontier
To add Mass Frontier to the Xcalibur Home Page Window Tool menu or
the Qual Browser window Tool menu, choose Tools > Add Tools from the
appropriate Xcalibur window. The Add Programs to Tool Menu dialog box
appears.
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Program Limitations
Introducing Mass Frontier
Program Limitations
Mass Frontier offers a number of features. However, there are some
limitations.
• Mass Frontier deals primarily with small organic structures rather than
peptides and other biologically-related molecules. The Structure Editor
and other modules dealing with structures have a limit of 199
non-hydrogen atoms per structure. If you try to exceed this number, a
message box appears, reminding you of this limit.
• Mass Frontier uses pure substances only. Mixtures are not accepted. The
program considers a mixture to be two or more structures, depicted in
the same window, that are not connected by a bond (represented as a
line). If you try to generate fragments and mechanisms from a mixture,
the message box alerts you that this action is not permitted. The Check
Structures option also detects mixtures as an error. However, library
utilities support mixtures, to assure backward compatibility with
commercial libraries. Mixtures can also be added to a user library.
Fragmentation Library does not support mixtures.
• The automated generation of fragments and mechanisms has been
greatly extended by numerous new aspects, but some restrictions still
remain. Mass Frontier can select reagent gases for Chemical Ionization.
However, the relative ionization potentials of reagent gases cannot be
modified. Negative ionization (deprotonation) is only supported using
the fragmentation library. There are no general rules for negative
ionization. Because soft ionization techniques are mainly low-energetic
experiments, which often yield complex skeletal and random
rearrangements, the predictability of these fragmentation and
rearrangements processes is not as high as that attained by electron
impact ionization. Improve the degree of predictability by using
compound-specific fragmentation mechanisms in the Fragmentation
Library module.
• Use Mass Frontier with neutral and single charged molecules. As a
consequence, you can attach the charge symbol (+ or -) to only one
atom. If the charge multiplicity is described in general. use the
unspecified charge location option in Structure Editor ([M+2H]2+,
[M+3H]3+). Biradicals are not supported by any module.
• Mass Frontier supports high resolution mass spectra with a m/z range of
1-3000 mass units. Mass spectra containing peaks with a mass-to-charge
ratio higher then 3000 are not displayed. Classification modules allow
only 800 peaks per spectrum. If a spectrum contains more than
800 peaks, the classification procedure selects the 800 most prominent
peaks.
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Program Limitations
• Classification methods supports only low resolution spectra. If you
try to classify high-resolution spectra, the program automatically
converts them into low-resolution spectra.
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1 Introducing Mass Frontier
New Features in Mass Frontier 5.0
New Features in
Mass Frontier 5.0
• Automated report creation in the Report Creator module
• Fragmentation Library in SQL Server format
• Retention time search in Database Manager module
• Component detection on chromatograms with multiple experimental
settings (polarity switching, IT/FT, direct infusion MSn)
• Three new component detection algorithms
• Advance chromatogram filtering (four algorithms)
• Chromatogram baseline correction (five algorithms)
• Chromatogram smoothing (three algorithms)
• Chromatogram three-dimensional view
• Automated spectra annotation capabilities with predicted fragments
• Formula Generator module
• Full support of Thermo Electron MS data, including data from the
OrbiTrap™ and LTQ FT™ mass spectrometers
• Unspecified bond location in Structure Editor
• Spectral and fragmentation libraries automatic backup and restore
procedures
• Fragmentation Library™ with more than 100000 mechanisms
• Spectral Tree™ MSn library of human and veterinary pharmaceuticals,
endogenous metabolites, drugs of abuse and doping agents (ESI +/-)
• OCX version (optional)
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Introducing Mass Frontier
Modules Overview
Modules Overview
Mass Frontier 5.0 consists of the following modules:
• Structure Editor
• Database Manager
• Chromatogram Processor
• Spectra Classifier
• Fragments Comparator
• Fragmentation Library
• Isotope Pattern
• Periodic Table
• Formula Generator
• Report Creator
• Spectra Projector
• Neural Networks
• Fragments & Mechanisms
The Fragments & Mechanisms, Spectra Projector, Neural Networks,
Fuzzy Clustering, Components Editor, and Hit Selector modules cannot
be opened directly from the program desktop. These modules must be
generated from data you supply.
Active module window
Report Creator
Formula Generator
Periodic Table
Isotope Pattern
Fragmentation Library
Fragments Comparator
Spectra Classifier
Chromatogram Processor
Database Manager
Structure Editor
Figure 2.
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Mass Frontier modules toolbar
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Introducing Mass Frontier
Modules Overview
Structure Editor (Chapter 2) is a structure drawing tool that automatically
calculates the mass of a selected fragment and the corresponding loss.
Many chemical structures created in this module are used throughout the
program.
Figure 3.
Thermo Electron Corporation
Structure Editor window
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Introducing Mass Frontier
Modules Overview
Database Manager (Chapter 4) provides a number of ways for organizing
and processing mass spectra, chemical structures and libraries. A spreadsheet
format is provided for data handling. Advanced database query and search
features give you instant access to the information needed for rapid
compound identification. User libraries containing spectra, MSn trees,
chromatograms, chemical structures and extensive compound characteristics
can be created with a mouse click. This module supports data exchange with
Microsoft Excel®.
Figure 4.
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Database Manager window
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Introducing Mass Frontier
Modules Overview
Use the Chromatogram Processor (Chapter 13) to extract and process mass
spectral scans of hyphenated chromatographic techniques such as GC/MS
or LC/MS. Component detection and spectra deconvolution algorithms
enable the automated extraction of individual spectra or MSn trees from
complex chromatographic data files. This module also provides visual tools
for annotating particular components or chromatographic peaks.
Figure 5.
Chromatogram Processor window
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Modules Overview
Use the Spectra Classifier (Chapter 10) to retrieve and organize spectra
intended for classification. Because spectra can be classified according to
various criteria (structural, physical and other properties), it is useful to
organize the spectra into different groups. Such groups of spectra can be
visually represented in different ways (using colors, symbols, and numbers)
to highlight similarities or dissimilarities among the spectral groups you
choose.
Figure 6.
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Spectra Classifier window
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Introducing Mass Frontier
Modules Overview
Fragments Comparator (Chapter 8) displays a series of fragments in a table
format. Columns are made up of individual compounds and rows show
either mass-to-charge ratios or the structures of fragments. Use this module
to compare the product ions of analogous molecules.
Figure 7.
Thermo Electron Corporation
Fragments Comparator window
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Modules Overview
Isotope Pattern displays the isotopic profile whenever a structure or
fragment is selected in the program. Isotope patterns can also be calculated
from a molecular formula you supply.
Save
Copy
Mass Settings
Spectrum Layout
Print
Zoom Out
Enter Molecular Formula
Figure 8.
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Isotope Pattern window
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Introducing Mass Frontier
Modules Overview
Use the Periodic Table to display the terrestrial isotopic abundance of
elements and their multi-atomic isotopic profiles.
Figure 9.
Periodic Table window
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Modules Overview
Use the Formula Generator (Chapter 17) to a calculate list of theoretical
molecular formulas that best fit an m/z value.
Figure 10. Formula Generator window
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Introducing Mass Frontier
Modules Overview
Use the Report Creator (Chapter 18) to create customizable reports from
modules displayed on the screen. Reports can be printed or exported as
PDF files.
Figure 11. Report Creator window
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Modules Overview
Spectra Projector (Chapter 11) displays the results of Principal Component
Analysis and Fuzzy Clustering classification methods. Mass spectral data can
be classified using two-dimensional (2-D) or two-dimensional (3-D)
projections in which each point represents a spectrum. If the class
membership of an unknown spectrum needs to be determined, open or
paste it into an existing projection.
Figure 12. Spectra Projector window
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Introducing Mass Frontier
Modules Overview
Neural Networks (Chapter 12) is an additional classification strategy in
Mass Frontier. Mass spectra are classified using a powerful method called
Self-Organizing Maps (SOM), which are a special class of neural network.
If two or more spectra activate the same neuron, the corresponding
compounds will exhibit similar physical or chemical properties, or biological
activities.
Figure 13. Neural Networks Window
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Introducing Mass Frontier
Modules Overview
The Fragments & Mechanisms (Chapter 6) module is an expert system
for automated generation of fragments and detailed fragmentation and
rearrangement mechanisms from a chemical structure you supply. This
module consists of a system that includes a set of known general reaction
mechanisms and comprehensive collection of library mechanisms which
enable automated prediction at an expert level. This module can be
generated either from the Structure Editor or the Database Manager
module.
Figure 14. Fragments & Mechanisms window
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Introducing Mass Frontier
Modules Overview
Use the Fragmentation Library (Chapter 7) module for the creation and
management of fragmentation mechanism databases. This module contains
an expert system that automatically extracts a decomposition mechanism for
each fragmentation reaction in the database and determines the compound
class range that the mechanism can be applied to. Mass Frontier uses this
expert system to apply database mechanisms to a user provided structure
and automatically predicts the fragmentation reactions for a given
compound.
Figure 15. Fragmentation Library window
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Chapter 2
Structure Editor
Mass spectra reflect the structural features of molecules which are
essential for the interpretation and the investigation of structure-spectra
relationships. Mass Frontier incorporates the Structure Editor structure
drawing tool, which enables the interactive handling of all kinds of
structural information. Use the Structure Editor for editing, importing,
exporting and checking chemical structures. The Structure Editor is the
gateway to four other modules in this program: Database Manager,
Fragments & Mechanisms, Fragmentation Library, and Isotope Pattern.
This chapter contains the following topics:
• Structure Editor Window
• Structure Layout
• Text
• Template Structures
• Selecting Atoms and Bonds
• Atom Properties
• Bond Properties
• Copying Structures
• Pasting Structures
• Moving, Resizing, Rotating, and Mirroring Structures
• Cleaning Structures
• Checking Structures
• MS Calculations
• Unspecified Bond Location
• Unspecified Charge Site
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Structure Editor
Structure Editor Window
Structure Editor
Window
The Structure Editor Window is shown in Figure 16.
Generate Fragments & Mechanisms
Substructure Stretch
Print Structure
Structure Layout
Redo
Save Structure
Select All Check Structure
Undo
Open Structure
Delete
Clean
Default Mode
Paste
Mirror
Copy
Rotate
Cut
Resize
Single Bond
Double Bond
Triple Bond
Chain
Benzene Ring
Six Membered Ring
Five Membered Ring
n-Membered Ring
Templates
Atom Properties
Bond Properties
Positive Charge
Negative Charge
Radical
Text
Ellipse
Select Unspecified Charge Site
Figure 16. Structure Editor window showing names of icon commands
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2 Structure Editor
Structure Editor Window
To open the Structure Editor window
• Click the Structure Editor
button on the main tool bar.
• Or choose Tools > Structure Editor.
• Open a structure by choosing File > Open > Structure. The Structure
Editor starts automatically.
Note Only one Structure Editor window can be open at any one time in
the program. If you click the Structure Editor button, or choose
Tools > Structure Editor and the Structure Editor is already open, this
window becomes active.
To begin drawing a chemical structure in Structure Editor, click a button on
the vertical bar. When you click one of these buttons, the shape of the
cursor changes to visually represent the engaged drawing mode. The vertical
buttons, in contrast to the horizontal buttons, are not represented in the
menu. If the function of a button is not apparent from its appearance, move
the cursor over the button and a hint appears.
Restoring Defaults
In the Structure Editor's default state, all buttons are switched off and no
atom or bond is selected. The plain cursor indicates that the Structure
Editor is in default state.
To restore the default state of the editor
1. Click the Default mode
Structure Editor window.
button in the upper left corner in the
2. Right-click and switch off the activated button and deselect all atoms.
Opening and Saving
Structures
To open or save a structure
1. Click the Open Structure
Structure Editor window.
or Save Structure
button in the
2. Choose File > Open > Structure or File > Save > Structure.
If you are opening a file which contains more then one structure (.sdf file),
only the first structure in the file is loaded into Structure Editor.
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Structure Editor
Structure Editor Window
Mass Frontier is a 32-bit application, enabling you to use long names to save
structures. You can also save structures by their actual names (for example,
1-Amino-2-hydroxyindane.mol).
Structure Data Formats
Structure Editor supports two kinds of structure formats: MDL MOL files,
(SDF files), with the .mol (.sdf ) extension, and HighChem MCS format
(Maximal Compressed Structure), with the .mcs extension. These formats
are also supported in the Database Manager module. Templates are stored
in MCS format, using the .tml extension.
Mass Frontier features the ability to restrict a search by a set of structural
constraints called the Good-Bad list. For example, you can instruct the
program to conduct a library search comparing an unknown spectrum only
with the spectra of ketones. This feature provides an endless range of
possibilities to target your search results with. The Good-Bad structures are
stored in the directory …\Constraints, and the structures are saved in MSC
format with the .mcs extension. The program automatically retrieves all
MCS structures from the ..\Constraints directory and puts them in a
Good-Bad box in the Constraints dialog window.
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Structure Layout
Structure Editor
Structure Layout
With Mass Frontier. you can change almost anything for structures, as well
as for other objects. Every layout setting change also affects printing and
copying to the Clipboard, except background color, which affects only
screen display in Mass Frontier. Use the various layout items to tailor the
graphics to your individual report or publication needs.
By default, the symbols for hydrogen atoms attached to carbon atoms are
not displayed (example a). To display them, select Show Carbon Symbols
(example b) in the Structure Layout dialog window. See Figure 17.
To open Structure Layout dialog window
1. Click the Structure Layout
button in the Structure Editor window.
2. Choose Options > Structure > Layout.
Hydrogen atoms are only displayed for carbons if the Show Carbon
Symbols box is selected. Otherwise, corresponding hydrogens are displayed
for heteroatoms only.
Note If you draw nonisotopic explicit hydrogen atoms (see example c in
Figure 17) these are removed in the Fragments & Mechanisms window
because they can make the mechanism network unclear, especially for
complex hydrogen rearrangement steps.
Figure 17. Structures showing different displays of hydrogens
The structure layout settings apply to all structures in Mass Frontier
simultaneously. This means that if you change a structure layout item,
all structures in the Structure Editor, Database Manager, Fragments &
Mechanisms, and Fragments Comparator modules are affected.
Note If you are printing in black and white and have set bright colors
for bonds or atoms, the lines and fonts might appear indistinct. To avoid
this, specify darker colors for all structural items, including spectra,
chromatograms, and mechanisms.
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Structure Editor
Text
Text
Structure Editor offers the possibility of labeling a structure or displaying a
text note on the screen or on the printout.
To enter a text note
1. Click the Text
button in the Structure Editor window.
2. Click anywhere in the drawing area to place the text.
3. Type the desired text.
4. Confirm the text by clicking outside the text area or click any button in
Structure Editor.
You can create up to 127 separate text notes. If you want to change the font,
color, size, or background of the text notes, use the Structure Layout
window on the Text tab.
Note Text notes are not associated with structures. As a result, the
Open, Save, Copy, and Paste actions apply only to structures. When
these actions are applied, the text notes are ignored even when a
structure is selected together with a text. Additional structure handling
routines such as resizing, rotating, or mirroring can be performed only
on structures.
Figure 18. Structure labeled with its chemical name
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Structure Editor
Template Structures
Template Structures
When you click the Templates
button in the Structure Editor window,
the Template dialog window appears. Mass Frontier comes with more then
200 predefined templates.
To insert a template structure into Structure Editor
1. Select a group of templates in the directory list box by using the arrow
keys on the keyboard or click the appropriate name of the group.
2. Click any atom or bond, depending on whether you want to attach the
template to an atom or a bond of a structure in Structure Editor.
3. The Template dialog window disappears and you can place or attach the
chosen template.
4. Switch off the template button or restore the default state of the Editor.
You can create your own group of templates or add a structure to an existing
group. The templates are organized by directory. The template root
directory is …\Templates. Every group of templates is stored in a separate
subdirectory of the template root directory. Subdirectories are named after
compound groups (for example, Steroids). The files within each
subdirectory are named after actual structures using the .tml extension (for
example, Cholesterol.tml). When you save a structure for template purposes
select the Template format with the .tml extension in the Save Structure
dialog window.
To build your own templates
1. Draw a template structure.
2. Click the Save Structure
button in the Structure Editor window or
choose File > Save > Structure.
3. Choose Template format in the Files of type: box in the Save Structure
dialog window.
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Structure Editor
Selecting Atoms and Bonds
Selecting Atoms and
Bonds
Any modification that you make to a structure applies only to the selected
atoms or bonds In addition, when a substructure search is initiated, the
program automatically uses the selected substructure in Structure. Before
you select one or more atoms, restore the default state of the editor.
To select a group of atoms that are next to each other
While pressing the mouse button, drag a rectangle around the atoms you
want to select.
The Windows convention for selecting multiple items applies. To select
atoms at different locations, use the keyboard Shift key. You can select a
group of atoms that are not adjacent in one of two ways:
• Click the atoms you want to select while holding down the Shift key.
• Or hold down the mouse button and drag a rectangle around the atoms
you want to select while holding down the Shift key.
To select all of the atoms and bonds in the structure:
• Click the Select All
button in the Structure Editor window or
choose Structure > Select All.
• Or double-click anywhere in the draw area within Structure Editor,
except on atoms and bonds.
Structure Editor offers two selection modes: Rectangle Selection and
Lasso Selection.
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2 Structure Editor
Selecting Atoms and Bonds
To choose the selection mode
• Right-click the Default Mode
button and choose the appropriate
selection mode from the popup menu.
• Or right-click anywhere in the draw area within Structure Editor, and
choose Rectangle or Lasso Selection from the pop-up menu that
appears in the draw area.
Lasso Selection
Rectangle Selection
Figure 19. Lasso and rectangle selection in Structure Editor
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Structure Editor
Atom Properties
Atom Properties
Use the Atom Properties dialog window to change the charge state or
isotope of an atom, or to change the element entirely.
To open the Atom Properties window
• Click the Atom Properties
button in the Structure Editor window
and then click the atom you want to change.
• Or restore defaults, and then double-click the atom you want to change.
In the Atom Properties dialog window, make changes by clicking the
appropriate element button, charge and radical box, or nucleon number
box.
Note All changes carried out in the Atom Properties dialog window
affect only a single atom.
To change an element that has a single character symbol, such as C, H, N,
O, B, F, K, P, S, I, V, W, Y, U and R; select all the atoms that you want to
change and click the appropriate key on the keyboard. All the selected atoms
transform into the element you have chosen.
You can set chlorine (Cl) or bromine (Br) atoms by selecting all the atoms
that you want to change and click either the C (for chlorine) key or B (for
bromine) key on the keyboard, while holding down the Shift key.
Figure 20. Atom Properties window showing 14C atom
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Structure Editor
Atom Properties
You can use a substituent instead of a specific element. A substituent is any
atom, functional group, or substructure substituted for another, or entering
a structure in place of some other part which is removed. The symbol ”R”
represents a substituent. A substituent can be with or without index.
Substructure search and fragment-generation algorithms consider
substituents with identical indexes as equal and substituents with different
indexes as not equal.
Figure 21. Atom Properties window showing substituent R1
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Structure Editor
Bond Properties
Bond Properties
The Bond Properties include bond multiplicity, bond style, and bond color.
To change the multiplicity of a bond, click the
,
, or
button in
the Structure Editor window and then click the bond you want to change.
To change the color or optical orientation of a bond, use the Bond
Properties dialog window.
To open the Bond Properties window
• Click the Bond Properties
button in Structure Editor and then
click the bond you want to change. The Bond Properties dialog window
appears.
• Or restore defaults, and then double-click the bond you want to change.
The Bond Properties dialog window appears.
Figure 22. Bond Properties window
Mass Frontier automatically recognizes aromatic bonds in an appropriate
six-membered ring or in polyaromatic structures. However, if unusual
semiaromatic or aromatic resonance structures are required, the aromatic
bond can be forced to select bonds by selecting Force Aromaticity.
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Copying Structures
Structure Editor
Copying Structures
Mass Frontier supports extensive use of the Windows Clipboard for the
exchange of structural information between modules. In addition, copy and
paste functions can be used inside Structure editor. To draw larger
structures efficiently, use the copy and paste functions.
To copy a structure or part of a structure to the Clipboard
1. Select the structure or part of the structure you want to copy.
2. Click the Copy
Edit > Copy.
button in the Structure Editor, or choose
Note Only the selected atoms and their associated bonds are copied.
In addition to structure exchange between modules, Mass Frontier allows
structure export to other programs that deal with structural information.
When you copy a structure, Mass Frontier automatically copies two
different formats to the Clipboard: structural information in MOL format
and graphics in Windows metafile format. If you paste a structure into
the structure editing software, the MOL format is used. If you paste the
structure into any text editor, spreadsheet or program that works with
graphics, the graphical information is used. All these actions occur
automatically.
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Structure Editor
Pasting Structures
Pasting Structures
If you copy a structure or fragment anywhere in the program or in a third
party structure drawing tool, you can paste it to Structure Editor. If
necessary, the structure can be changed or corrected and then returned to
where it originated which is useful for structure elucidation. For example,
you can copy a structure from the Database Manager window, paste it to
Structure Editor, make appropriate changes, and then move it back to
Database Manager. If the spectrum and the structural proposal are not
consistent, repeat the process.
To paste a structure to Structure Editor
Click the Paste
Edit > Paste.
button in the Structure Editor. Or, choose
If you have copied a structure or fragment in a program other than
Mass Frontier, you can only paste this structure if the external structure
drawing software supports MOL format and this format is activated. The
majority of structure drawing tools support MOL format and have this
format activated by default. If you paste a structure from an external source,
it might appear larger in Mass Frontier than in the original software. If this
occurs, make the structure smaller by using the Resize tool.
Figure 23. Structure Exchange by using the Copy and Paste commands
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2 Structure Editor
Moving, Resizing, Rotating, and Mirroring Structures
Moving, Resizing,
Rotating, and
Mirroring Structures
To move a structure in the Structure Editor
1. Select the atoms or bonds to move.
2. Point the cursor at any selected atom or bond.
3. Hold down the mouse button and drag the selected structure to the new
location.
4. Release the mouse button to drop the selected structure at the new
location.
To resize a structure in the Structure Editor
1. Select the structure or part of the structure to resize.
2. Click the Resize
button, or choose Structure > Resize.
3. Drag one of the small rectangles on the structure edge until the new size
is achieved.
4. Release the mouse button.
Note If you drag one of the diagonal rectangles, the aspect ratio is
kept constant during structure resizing.
Use the Rotate Structure option to twist a structure in any direction. The
center of rotation, indicated by a small circle with a cross in the middle ,
can be moved to any location.
To rotate a structure in the Structure Editor
1. Select the structure or part of the structure you want to rotate.
2. Click the Rotate
button, or choose Structure > Rotate.
3. Move the center of rotation to the desired position by dragging the
circle with a cross.
4. Drag any of the small rectangles on the structure edge to achieve the
new angular position.
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Structure Editor
Moving, Resizing, Rotating, and Mirroring Structures
To make a mirror image of your structure in the Structure Editor
1. Select the structure or part of the structure you want to mirror.
2. Click the Mirror
button, or choose Structure > Mirror.
3. Click one of the small rectangles on the structure edge.
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Structure Editor
Cleaning Structures
Cleaning Structures
Use the Clean function to achieve a professional look for your structures.
With Mass Frontier, you can clean up an individual part of a structure. For
example, you can restrict cleaning to certain functional groups, while the
main skeleton remains intact. However, the algorithm of cleaning 2-D
structures is a particularly difficult mathematical problem and has yet to be
completely solved. As a result, this function might, in some complicated
cases, lead to unsatisfactory structures. If this occurs, use the Undo function.
To clean a structure
1. Select the structure or part of the structure to clean up.
2. Click the Clean
button, or choose Structure > Clean.
Figure 24. Structures showing the results of using the Clean command
Note If you want to clean only part of a structure, the selected atoms
must be connected or a message box appears to remind you that only
connected atoms can be cleaned.
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Structure Editor
Checking Structures
Checking Structures
Structure Editor comes complete with a function for checking chemical
structures. Structure Checker searches for formal errors and unusual
structural features. If a structure is formally incorrect, or Structure Checker
considers there is some doubt about its validity, a Structure Check Results
window appears with a list of errors and warnings. When this window is
closed the program automatically selects the atoms and bonds, which are
considered incorrect. As mentioned in “Program Limitations” on page 9,
structures that are not connected are considered to be mixtures, which are
reported as errors.
Figure 25. Structure showing the result of the Check Structure command
To check a structure
1. Click the Check Structure
button in the Structure Editor
2. Choose Structure > Check Structure.
Note This option does not perform quantum mechanical or
thermodynamical calculations concerning possible structure stability.
After finishing a structure drawing, always check it for errors before
proceeding with any other procedure. Once fragments and mechanisms
generation is initiated, a structure is automatically checked for errors. If any
error is discovered, the program prevents you from continuing with the
generation.
Before running Generation of Fragments and Mechanisms and after
finishing structure drawing in Fragmentation Library window structure
check is automatically performed.
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MS Calculations
Structure Editor
MS Calculations
When you select a part of a structure, the Structure Editor automatically
displays the molecular formula and molecular mass of the selected
atoms (F:) in the status bar of the Structure Editor, together with
corresponding loss (L:). See Figure 26. Use this information for simple
consistency checking of mass spectrum and chemical structure.
Figure 26. Structure Editor showing the result of selecting part of a structure
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Structure Editor
Unspecified Bond Location
Unspecified Bond
Location
Mass spectrometry is often unable to provide information about the exact
position of a functional group on a chain or ring portion of a structure. In a
number of application areas, even incompletely characterized molecules can
be sufficient to study a particular phenomenon. A typical example is
metabolite characterization in an early drug discovery process, where
knowledge of the precise location of biotransformation action is of less
importance.
To display and calculate monoisotopic mass or the isotopic pattern of a
fragment or molecule with an unspecified bond location, the Structure
Editor provides Ellipse as a graphical tool. Ellipse visually defines the region
on a structure where a functional group could potentially be attached. In
order for the software to correctly interpret an unspecified bond location,
the ellipse must enclose one or more atoms of the core structure and a single
atom of a functional group that is not attached to the core structure. The
ellipse is green for better visual orientation. See Figure 27.
Ellipse tool
Figure 27. Structure Editor with unspecified bond location
Note The Ellipse tool is available only in the Structure Editor module,
You cannot use a structure with an unspecified bond location in other
modules. In addition, the generation of fragments and mechanisms from
such structures is not supported.
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2 Structure Editor
Unspecified Charge Site
Unspecified Charge
Site
Use Mass Frontier to process structures where the charge site is not
specified. This kind of molecule representation is important when working
with ionic structures, because the favored ionization site or the explicit
charge site during fragmentation reactions is often unclear. Several ion types
can be chosen in the program.
To create an ionic structure with an unspecified charge site, select one of the
ion types from the box at the bottom of Structure Editor. Every structure
with an unspecified charge site has a specific symbol on the upper left part
of the structure.
Figure 28. Structure Editor window showing Unspecified Charge Site option
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Chapter 3
Spectral Trees
Mass Frontier uses tree representation for MSn spectra. The tree structure
best reflects the hierarchical spectra dependencies in tandem experiments.
Mass Frontier provides a graphical user interface for the management and
processing of spectral trees. Spectral trees can be reconstructed from data
files, automatically extracted from data dependent chromatographic
components, or manually created by the user. Trees are supported by
all the spectral modules except Spectra Classifier, which uses total composite
spectra generated from trees. You can store and search trees in libraries,
annotate every node spectrum, or create chromatographic libraries with
spectral tree components. Exchange trees between modules by using the
copy and paste commands just as for single spectra. In addition, spectral
trees can be exported or imported from or to Excel in text format.
Mass Frontier uses a newly developed algorithm, which is integrated in the
database search procedures, for the comparison of spectral trees.
Note Most management and processing actions distinguish between a
single spectrum and a tree. Therefore, when dealing with data that
contains trees, determine whether a particular action needs to be applied
to the entire tree or the selected single spectrum. The displayed
information is also associated either with the tree or the spectrum, or
both depending on the information type.
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Spectral Trees
Figure 29. Spectral Tree window showing MS3 spectra representation
This chapter contains the following sections:
• Tree Arrangement
• Tree Node Items
• Tree Layout
• Tree Generation
• Manual Creation and Editing of Trees
• Copying and Pasting Trees
• Tree Chromatograms
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Spectral Trees
Tree Arrangement
Tree Arrangement
Mass Frontier uses tree representation for MSn spectra. A spectral tree
consists of levels, nodes, node connectors, and node items. The levels
symbolize MSn stages starting at n=1. The node connector is a graphical
symbol of the precursor m/z value, or the precursor m/z range if the isolation
width is included. The node is a holder of node items. The node item stands
for the product or calculated spectra of identical precursor m/z value or
m/z range or for a chromatogram. Node product spectra, or parallel spectra,
represent spectra acquired at various collision energies and isolation widths,
or using wide band activation or they can be zoom spectra, source CID
spectra, or any other spectra that enhance reproducibility in compound
identification. If a node contains more than two parallel spectra, the average
and composite spectra are automatically calculated. In addition to spectra,
each node can contain a chromatogram.
Figure 30. MSn Data Tree Structure
Note Chemical structures are associated with nodes and not with entire
trees so that you can assign fragments for product spectra. The top-level
node MS1 holds the structure of the neutral compound.
Complete trees can be stored in a library and updated at any time. Any
complementary information associated with a single stage spectrum or
a chromatogram can be associated with a node spectrum or node
chromatogram.
Note To display or edit complementary information for a particular
node spectrum, first select it.
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Spectral Trees
Tree Node Items
Tree Node Items
Some mass spectrometric techniques generate spectra whose appearance is
dependent upon the experimental conditions and sample preparation. To
allow the management and search of diverse product spectra with an
identical precursor ion for a single chemical entity, Mass Frontier uses
spectral trees that can contain nodes with several node items. The node item
stands for any product or calculated spectrum of an identical precursor
m/z value or m/z range (node spectra) or for a chromatogram.
Node product spectra represent spectra acquired at various collision
energies and isolation widths; by using wide band activation; or they can
be zoom spectra, source CID spectra, or any other spectra that enhance
reproducibility in compound identification. If a node contains more than
two parallel spectra, the average and composite spectra are automatically
calculated. In addition to spectra, each node can contain a chromatogram.
There are several reasons for using node spectra. They can significantly
contribute to correct compound identification in a tree library search, they
allow the study of fragmentation processes by changing the experimental
conditions, and they permit the efficient organization of product spectra.
The spectral node strategy strengthens the robustness of all the
mathematical processing methods and, compared to simple spectra
averaging, does not distort the highly nonlinear peak ratio progress.
Node spectra are easily accessible from a library and can be created or edited
by using the graphical interface. Every tree item has its own annotation
caption that can be edited by double-clicking it.
Select node items by clicking the mouse on the edge of the spectral or
chromatographic node item, or by browsing in the box displayed below the
tree. All processing actions are accessible from the pop-up menu that
appears when you double-click the node.
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Tree Layout
Spectral Trees
Tree Layout
A spectral tree consists of levels, nodes, node connectors, and node items.
Node items are divided into five groups that are differentiated by the
displayed color: Single, Average, Composite, and Source CID spectrum,
and Chromatogram. Change color settings in the Options > Spectrum
Layout dialog window in the MS Tree page. Because display and editing
actions affect the selected node item, the item selection is also distinguished
by color.
Figure 31. MS Tree page showing customizable spectral tree layouts
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Spectral Trees
Tree Generation
Tree Generation
A spectral tree can be created in the following four ways:
• Reconstruction from raw files where each file contains a single
MSn experiment (direct infusion).
• Reconstruction from a single raw file that contains various
MSn experiments in one run (direct infusion).
• Spectral tree deconvolution from a data-dependent MSn chromatogram.
See Chapter 13, “Chromatogram Processor.”
• Manual creation using tree editing utilities.
Reconstruction from raw files where each file contains a single
MSn experiment (direct infusion)
The spectral tree reconstruction feature reads Xcalibur .raw files stored in a
directory and automatically creates a tree according to the precursor m/z and
isolation width values. All files (spectra) in a directory need to be from an
identical chemical entity (compound or chromatographic component) with
one directory per tree per compound. This feature works on Xcalibur .raw
files acquired using direct injection and a single MSn stage. Each file in a
directory can be acquired separately at a specific collision energy; a wide
band activation spectrum, zoom or source CID spectrum; or the sample
preparation (pH, concentration, buffer, and so on) can be diverse.
Example files are installed automatically into the
\Mass Frontier 50\Chromatograms\Cloramphenicol directory.
To import trees using the tree reconstruction feature
1. Click the Import button in the Tree pane in Database Manager
2. Choose Import Tree.
3. Select the directories to import.
4. Click Add and then click OK.
Use this procedure to create an MSn library.
Reconstruction from a single raw file that contains various
MSn experiments in one run (direct infusion)
You can create a spectral tree of a reference compound by reading various
MSn spectra acquired in one run using direct infusion and stored in a single
raw file. To do this, you must use the Component Detection & Spectra
Deconvolution feature in the Chromatogram Processor module.
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Spectral Trees
Tree Generation
To create a spectral tree from a single run using the tree reconstruction
feature
1. Click the Chromatogram Processor button in the main Mass Frontier
menu bar.
2. Select the direct infusion file to process and click the Open button. The
file opens in the Chromatogram Processor window.
3. Click the Component Detection & Spectra Deconvolution button
and select the Direct Infusion button from the pop-up menu.
4. If required, change the preset parameters and click Calculate.
5. Click the component triangle on the left side of the TIC pane and the
reconstructed spectral tree appears below.
Use this procedure to create an MSn library.
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Spectral Trees
Tree Generation
Spectral tree deconvolution from a Data Dependent MSn
chromatogram
Spectral trees can also be generated from chromatographic components.
Chromatographic data must be processed using a different feature. To
create a tree from a chromatogram, use the Component Detection &
Spectra Deconvolution feature in the Chromatogram Processor module.
For more information, see Chapter 13, “Chromatogram Processor.”
Figure 32. Select Directories window for tree reconstruction from raw files
Manual creation using tree editing utilities
The tree reconstruction automatically assigns node spectra to specific nodes
utilities in addition to creating levels, nodes and node connections.
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3 Spectral Trees
Manual Creation and Editing of Trees
Manual Creation and
Editing of Trees
Spectral trees can be manually created and edited in Database Manager in
the Spectral Tree pane. All editing actions are accessible from the pane
buttons and from the pop-up menu that appears when you right-click.
Note All actions described below are applied only to the selected node or
spectrum.
You might want to construct a tree from scratch. You can start creating an
empty tree framework by adding new nodes and then successively pasting
and importing spectra into the nodes. To add a new node, right-click the
parent node and choose Add > Add Node. An empty node can be filled
with a spectrum from the Clipboard or imported from a file. Right-click the
node and choose Paste > Paste Parallel Spectrum. This item is displayed if
the Clipboard contains a spectrum. To add another empty node product
spectrum (parallel spectrum), right-click the node and choose Add > Add
Parallel Spectrum. A node can contain only one empty parallel spectrum.
If you add more than one parallel spectrum to the node, the software
automatically generates the average and composite parallel spectra, which
are differentiated by color. If you import a .raw file into the node, the
program calculates the average spectrum from all scans. Do not import a
chromatogram .raw file into a spectrum node. To create a node with a
chromatogram, you must paste a chromatogram from the Chromatogram
Processor window.
A tree would be incomplete without precursor m/z values and the isolation
width of the product nodes. There are two ways to define the precursor
m/z value. You can either manually type the value in the box in the
Structure pane or paste or draw the ionic product fragment in the Structure
pane and click the Restore Precursor m/z button in the bottom right corner
of the Structure pane. The default value of the isolation width is determined
automatically, but can be manually changed in the box in brackets.
Edit the node caption of every parallel spectrum or chromatogram by
double-clicking the caption and typing the new formatted text into the
annotation dialog window.
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Spectral Trees
Copying and Pasting Trees
Copying and Pasting
Trees
Use Mass Frontier for the exchange of trees between windows, records,
chromatographic components or programs (for example, Excel) by using the
copy and paste commands. When using the paste command, you must
distinguish between a single spectrum and a tree. Be sure to use the correct
paste option when exchanging trees.
Trees can be pasted into Excel in numerical format and then edited and
copied back into Mass Frontier. The tree numerical format supports every
types of spectral trees and parallel spectra, except chromatograms. You can
change the precursor m/z value, modify spectra, and edit the node caption
by using Excel.
Figure 33. Exchanging spectral trees between modules
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3 Spectral Trees
Tree Chromatograms
Tree Chromatograms
Mass Frontier supports spectral trees that contain nodes with data-reduced
chromatograms. Chromatograms can be assigned to every node. You can
store product ion chromatograms for a particular precursor m/z value. In
contrast to spectra, only one chromatogram per node is allowed. Tree
chromatograms with components and selected scans are fully searchable. A
node chromatogram can be reviewed or reloaded from the original data file
in Chromatogram Database Viewer, which is launched from the button of
the same name in Database Manager. Review node chromatogram
components or selected scans in the Chromatogram Database Viewer and
the Components Editor window.
Figure 34. Spectral Tree window showing LC/MS/MS chromatogram assigned
to the top level (full scan) node
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Chapter 4
Database Manager
Use the Database Manager to manage spectral and structural information
in the SQL desktop engine database. This module provides library
maintenance utilities that enable you to create and organize spectral and
chromatographic libraries with chemical structures. In addition, because the
program supports ion structures and tree spectra representation, you can
also create true MSn libraries. Advanced library query and search features
provide access to the information needed for compound identification and
to help interpret unknown spectra. A flexible set of search restrictions is
available to target your search results, useful when using large libraries.
Structural and spectral data are organized in spreadsheet-like windows,
together with a variety of supplementary information. The number of
Database Manager windows that can be opened simultaneously is limited
only by system resources. This means unconstrained flexibility in handling
spectral and structural data. It is easy to move spectra-structure pairs from
one Database Manager window to another, enabling you to organize your
libraries, search results and any other data.
The Database Manager provides a customizable tool for creating reports,
which can be either printed directly or copied and pasted to a word
processor for more advanced reports.
For each record in the Database Manager you can view the mass spectrum; a
list of peaks; the compound identification information and the neutral losses
spectrum, if the molecular mass is available; or compare two spectra. View
this information by selecting the appropriate tab.
This chapter contains the following sections:
• Open a Database Manager Window
• Mass Differences
• Comparing Spectra
• Cutting, Copying, and Pasting Records
• Structures in Database Manager
• Working with Spreadsheets
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Database Manager
• Search Utilities
• Mass Spectral Data Exchange Between Modules
• Fragment Assignment to Spectral Peaks
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4 Database Manager
Open a Database Manager Window
Open a Database
Manager Window
To open a Database Manager window
Do one of the following:
• Click the Database Manager
window.
button on the tool bar in the main
• Choose Tools > Database Manager. An empty Database Manager
window opens.
• Start any search from the Search menu and, if a spectrum or structure is
found, a Database Manager window opens containing search results.
If an empty Database Manager window has already been opened, you
cannot open another. Each search dialog window has a box at the bottom,
with the caption Merge Results into Active Database Manager. As the caption
indicates, when you activate this box, the search results are merged at the
end of the Spreadsheet in the active Database Manager window. If
unchecked, a new Database Manager window opens and the search result
records are added to it.
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Database Manager
Open a Database Manager Window
Remove record from Spreadsheet (not from library)
Spectrum and Info Grid Layouts
Library Utilities
Show Chromatogram Viewer
Open
Edit Components
Save
Show Spectral Tree
Print Report
Show Structure
Report
Assign Fragment Structure to Peak
Create new
record in
Text Box
Spreadsheet
Spectrum Processing
Search
Cut
Add Selected Records to Spectra Classifier
Copy
Paste
Generate Fragments & Mechanisms
m/z value of the precursor
Lower boundary of the
isolation width
Upper boundary of the
isolation width
Restore Precursor m/z
Delete Node or Parallel Spectrum
Paste Parallel Spectrum of Chromatogram
Copy Tree or Parallel Spectrum
Cut Tree or Parallel Spectrum
Print Spectral Tree
Import Tree or File
Next
Previous
Copy Selected Rows
in Spreadsheet
Paste Structure
Copy Structure
Clear Structure
Edit Structure
Copy Selected Tab (Spectrum)
Figure 35. Database Manager window
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Open a Database Manager Window
Records in the Database
Manager
Spectral tree associated
with active record
A single record in the Database Manager contains one spectrum with a
structure (if available) or one spectral tree with associated structures (if
available). and complementary information associated with the record.
Each record is visually represented as a single line in the spreadsheet. The
hand icon
always points to the active record. The active record is also
highlighted on the Spreadsheet. The structure, spectrum, spectral tree and
any other available information are displayed for the active record only. The
data associated with the active record are displayed in the upper half of the
window.
Selected spectrum in
active record
Structure associated with
selected spectrum
Active record
Figure 36. Database Manager window showing the selected tree items in active record
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Database Manager
Open a Database Manager Window
Additional Information
Associated with a Record
In addition to the mass spectrum or spectral tree and the chemical structure,
each record includes number of compound identifications, property and
origin information. This additional information, which is associated with
the record, is found in the Info tab in the Database Manager window. The
Info tab contains editable fields organized by group and subgroups. Fields
with information that is automatically calculated from a chemical structure
(formula, molecular mass, and so on) or acquisition settings adopted from a
file cannot be edited. Text in uneditable fields is unavailable. The
information in the Info tab is divided into three groups:
• Main (compound identification, compound characteristic, biochemical
information, sample preparation, comments, instrument, contributor)
• Chromatographic Info
• Spectral Info (mass resolution, MSn info, scan, file)
It is important to understand the connection between data in the Info page
and the spectral tree hierarchy. The Main group is associated with the entire
tree, the Chromatographic Info is associated with a tree node (one
chromatogram per tree node is allowed) and Spectrum Info is associated
with the selected parallel spectrum. Every parallel spectrum contains its own
data in the Spectrum Info group. This is because the parallel spectra can be
acquired under different experimental conditions (collision energy, isolation
width, and so on) that must be stored independently.
Figure 37. The relationship between tree hierarchy and Info page
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Open a Database Manager Window
If a record is saved in a library, all data in the Info tab is automatically stored
in the SQL database. To permanently store any change, update, or deletion
in the Info tab, you must save the corresponding record in the library.
The Main group contains the Name subgroup with three fields: IUPAC,
Synonyms and Commercial Product. The library name search covers all
three fields. The Spreadsheet column Name is associated with the IUPAC
field in the Info tab.
Figure 38. Database Manager window showing the Info page
The predefined list of additional information associated with a record might
contain a large number of unused items, Mass Frontier allows items to be
selected which appear in the Info tab for each library separately.
To exclude or include items from the predefined list of additional
information
Click the Layout
button in the Database Manager window and
choose Info Grid Layout. The Info Grid Layout dialog window appears.
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Mass Differences
Mass Differences
Use the Mass Differences page in the Database Manager to select any peak
and see the mass differences to the right and left (possible parent-daughter
ions). Use the track bar at the top of the page to move the m/z scale. If
molecular mass information is available, the zero value of the m/z scale
automatically starts at the molecular mass; that is, a standard neutral loss
spectrum is displayed. In this case, a blue selection bar in the track bar
marks the shift of the m/z scale with respect to molecular mass. Enlarge the
graphic to see the number for less prominent peaks when a large number of
peaks are close to each other.
Figure 39. Database Manager window showing the Mass Differences page
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Comparing Spectra
Database Manager
Comparing Spectra
Use the Compare Spectra tab in the Database Manager to compare two
spectra. The bottom spectrum is from the active record in the Spreadsheet.
The middle spectrum displays the difference between the top and bottom
spectrum. The top spectrum can be added from an active record by clicking
the Add button or pasted from the Clipboard by clicking the Paste button.
The peaks in the top and bottom spectrum have different colors; thus, the
color of a difference peak in the middle spectrum is taken from the
spectrum that has a more abundant peak at a particular m/z value.
Figure 40. Database Manager window showing the Compare Spectra page
Note After a spectrum search has been completed, the query spectrum is
automatically pasted to the top spectrum in the Compare Spectra tab
where you can view of the peak differences of spectra in the match list
and query spectrum.
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Database Manager
Cutting, Copying, and Pasting Records
Cutting, Copying, and
Pasting Records
Use the cut, copy and paste functions to copy or move records from one
Database Manager window to another. Records can also be copied or
moved to different locations within a spreadsheet in the same Database
Manager window. This permits clear organization and maintenance of your
experimental data or search results.
Mass spectra can be exchanged between the Database Manager and Excel by
using the import and export features.
The cut and copy functions apply only to selected records. With the
spreadsheet, you can select multiple contiguous records to copy or move
many records at once.
Copy Tree or Chromatogram
Paste Tree or Chromatogram
Copy Record
Paste Record
Copy Selected Tab (Chromatogram,
Spectrum Text, Mass List)
Paste Structure
Copy Structure
Copy Selected Rows in
Spreadsheet
Figure 41. Database Manager window showing the diverse Copy and Paste buttons
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Cutting, Copying, and Pasting Records
To cut or copy records
1. Select one or more records in the Spreadsheet.
2. If the Database Manager is an active window, click the Cut or Copy
button in the Database Manager or choose Edit > Cut or Edit > Copy
in the main menu.
If you cut or copy a record the selected mass spectrum, in graphic format, is
simultaneously copied to the Windows Clipboard, in addition to the record.
If you have selected more then one record, only the graphic of the spectrum
in the first record is copied to the Clipboard. This graphic spectrum
representation can then be pasted to any Windows application. Graphics are
copied to the Clipboard in 32-bit format (enhanced Windows metafile),
which is sometimes not supported by older 16-bit applications.
To paste records
1. Select one record in the Spreadsheet where you want to paste records.
2. Click Paste in any Database Manager window or choose Edit > Paste in
the main menu.
A single spectrum copied in the Chromatographic Processor can be pasted
to the Database Manager to extract spectral scans and move them to the
Database Manager for further processing.
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Database Manager
Structures in Database Manager
Structures in
Database Manager
Chemical structures are used at every stage in the Database Manager module
and can be added to corresponding records. You can create libraries with
structures including isotopes, ions, radicals and optically active compounds.
Structures in the Database Manager can be used in connection with the
Fragments & Mechanisms module to check the consistency of a mass
spectrum and chemical structure. Use the Database Manager to perform
structure elucidation through modification of input structure and
regeneration of product fragments.
When working with spectral trees structures are associated with nodes and
not with an entire tree, these structures can be added to the Database
Manager record in one of three ways:
• Opening the Structure Editor directly from structure pane and drawing
a new structure.
• Pasting the structures.
• Loading the structures from an external file.
To add structure to a record that contains a tree, select the appropriate node
by clicking any spectrum in that node. You can assign fragments for product
spectra for elucidation and database maintenance of CID spectra. The
top-level node MS1 holds structural information about neutral compound.
Structure displayed in the Database Manager is associated with selected
node. Once again, structure you see or edit in the structure pane in the
Database Manager is associated with selected node.
You can add structure not only to a spectrum or tree node but also to any
peak by clicking the Assign Structure to Any Peak button. A New
Structure Editor window opens and you can draw a fragment. After you
finish drawing, the program tries to automatically connect your structure
with a spectral peak according the fragment's m/z value. If you need to
connect a drawn fragment to a different peak, drag the connecting circle to
move it to any other peak. The drawn fragment can be resized. Note that
structures assigned to spectral peaks are not searchable.
You can copy a structure you want to paste into the Database Manager from
anywhere in the program. To paste a structure into the Database Manager,
use the Paste Structure button in the top-right corner. The Copy and
Paste buttons on the button bar are intended for records, not single
structures.
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Structures in Database Manager
To paste a structure to a record
1. Activate the record you want to paste the structure into.
2. If processing a tree, select node you want to paste the structure into.
3. Paste the structure by clicking the Paste Structure button.
If a structure has been added to a record or an existing structure has been
replaced, the word Updated appears at the bottom of the structure pane. If
anything is changed in the record, including the structure, a small circle is
displayed in the ID Num. column in the spreadsheet. After a structure is
added or changed, the molecular formula and molecular mass are
automatically calculated and updated.
If a structure has been added to a tree node or an existing structure has been
replaced, the Restore Precursor m/z
button at the bottom of the
structure pane becomes active if the structure molecular mass differs from
the existing precursor m/z value. Use this button to set the precursor
m/z value of the active product spectrum according to the molecular mass of
the drawn structure.
Figure 42. Detachable Structure window showing the precursor ion structure,
the precursor m/z value (316.1) and the isolation width m/z range
(314.5-318.8)
Import structures to the Database Manager by loading them from an
external .mol file or .sdf file. In contrast to the pasting of a structure, when
you load from an external file, you can add structures to more than one
record at a time. You can, for example, assign a large number of structures
to library spectra when importing an external library to Mass Frontier.
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Structures in Database Manager
To import structures from a file
1. Select the records to which you want to add structures.
2. Load the structures by choosing File > Open > Structure.
When adding more than one structure to your records, the structures are
added in the same order as they are present in the file, from the first to the
last selected record. If the number of structures in an external file is greater
than the number of selected records, structures are added only to the
selected records. If the number of selected records is greater than the
number of structures in the file, all the structures are added, and some
records remain without structures.
Chemical structures can be added not only to a spectrum or tree node but
also to any peak. To add a chemical structure to a peak, click the Assign
Structure to Any Peak button. A New Structure Editor opens and you can
draw a fragment. After you finish drawing, the program automatically
attempts to connect your structure with a spectral peak according to the
fragment's m/z value. If, for whatever reason, you need to connect the drawn
fragment to a different peak, drag the connecting circle with the mouse to
the required peak. The drawn fragment can be resized.
A structure can also be assigned to a peak by using the Data tab, where all
peaks in the spectrum are listed. Click on the line where the peak is listed,
then click the
button in the Fragment column and the Structure Editor
opens.
Note Structures assigned to spectral peaks are not searchable.
Figure 43. The spectral peaks with assigned fragments
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4 Database Manager
Working with Spreadsheets
Working with
Spreadsheets
The spreadsheet is organized by rows where each record is represented by a
single row. The columns contain supplementary record information. In the
spreadsheet, move columns by dragging the appropriate column header to a
new position. If you want to move a row, use the cut and paste functions as
dragging is not supported.
When you open a Database Manager window, the spreadsheet is empty.
You can add records to the spreadsheet by conducting a search, opening
spectra or references, or by pasting records or stand-alone spectra. For an
active record, the associated spectrum and structure (optional) appear in the
upper half of the window. The hand-shaped cursor in the first column
indicates which record is active. You can select more then one record, but
the row with the hand icon is always the active one.
A Database Manager window can contain 999 records, but you can open as
many Database Manager windows as your system allows.
Record data from the spreadsheet can be exported to Excel in text format.
Sometimes you might need to simultaneously preview all structures rather
than record information in spreadsheet. To display a grid of structures,
select the Structure tab next to the Spreadsheet tab. In this window, the
structures are organized in small cells. If structures need to be enlarged,
resize any structure cell by dragging the edges of the column or row headers.
Note In the Structure view option, you can select only a single record.
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Working with Spreadsheets
Figure 44. Database Manager windows showing Spreadsheet page and
Structure page
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Search Utilities
Database Manager
Search Utilities
For retrieving spectra and structures from libraries, Mass Frontier has several
query and search features. You can search every library simultaneously. The
following search options are available:
• Spectrum Search
Searches for the library spectra most closely matching an unknown
spectrum.
• Tree Search
Searches for the library spectral tree most closely matching an unknown
spectral tree or subtree.
• Substructure Search
Searches for an exact match for the query structure (structure search)
or searches for an exact match for the structure subset (substructure
search).
• Name Search
Incremental name search.
• Molecular Mass Search
Searches for compounds with a given molecular mass (unit resolution
only).
• Formula Search
Searches for compounds with a given molecular formula.
• ID Number Search
Searches for library entries with a given ID number.
• CAS Number Search
Searches for compounds with a given Chemical Abstract Service registry
number
• Retention Time Search
Searches for library entries with a range of given retention times.
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Database Manager
Search Utilities
Every search dialog window contains a box with the caption Merge results
into Active Database Manager Window. If the box is clicked, the search
results beyond the last record into the Spreadsheet are stored in an active
Database Manager window. If you uncheck this box, a new Database
Manager window opens and the search results are put into it. If this box is
disabled, there is no active Database Manager window and a new window
opens.
To start a search
1. Click Search in the main menu.
2. Choose the specific search you want to conduct.
3. When the dialog windows appears, select the active library or libraries to
be searched.
If the search is successful, the results are stored in the Spreadsheet in the
Database Manager window.
Spectrum Search
Mass Frontier uses a search algorithm developed by HighChem. This
algorithm is based on the optimized dot-product function together with an
additional term, based on ratios of peak intensities.
The query spectrum can originate from the Database Manager, Component
Editor, or from the Chromatogram Processor by selecting a spectral scan in
a chromatogram. In addition, the query spectrum can be pasted from the
Clipboard to the search dialog window. In this case, the spectrum can be
copied to only Mass Frontier.
Use the Search Spectrum option to choose between Identity or Similarity
searches. Use identity searching to locate a library spectrum that closely
matches an unknown. Use similarity searching to retrieve spectra library
entries of similar compounds when the unknown is either not in the library
or its spectrum is so badly distorted that a reliable match is not possible.
After a spectrum search has been performed, the search results (or match
list) are stored in the Spreadsheet. The usual result of the spectrum search is
a match list of 100 records. After a search has been completed, a match
factor column, with the caption Match is automatically added to the
Spreadsheet. The match factor is a number from 1 to 999 that specifies the
measure of similarity between the query spectrum and the library reference
spectrum. A Match factor of 999 means a perfect match. To draw your
attention to a match greater than 930, a lightning-shaped icon is displayed
in the Match column in the spreadsheet.
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Database Manager
Search Utilities
Figure 45. Spreadsheet page of the Database Manager window showing
spectra search results with Match factors
If an Identity search does not provide an acceptable match (for example, the
unknown has not been positively identified), use a Similarity Search. In this
case, the algorithm does not use the high mass peak index, but instead uses
wider abundance ranges. The match list resulting from a similarity search
can be valuable in deducing structure, especially in establishing a structural
proposal for an unknown spectrum.
To establish a structural proposal for an unknown, switch to the Structures
tab. From the match list of similar compounds, you might recognize some
common structural features which are displayed in the structures grid. You
can copy the structures to the Structure Editor and put the pieces of the
structural puzzle together and so create an initial structure. This structure
can be pasted back into a Database Manager window to the record holding
the unknown spectrum. After this, a comparison of the peaks in the
spectrum with generated fragments can provide valuable information about
the consistency of the proposed structure and unknown spectrum. If the
m/z values of the fragments do not match the spectrum, modify the
structure and repeat this procedure. However, if the structures in the hit list
are highly diverse and dissimilar, combine this approach with other
methods.
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Database Manager
Search Utilities
Tree Search
Mass Frontier allows the processing of MSn spectra in hierarchically
consistent trees that can be searched in spectral or chromatographic libraries
using various options that can be found in the Advance tab in the Spectra or
Tree Search dialog window. Each node in spectral trees can consist of four
types of spectra (average, composite, parallel and source collision induced
dissociation) and you can specify in which type spectra is searched.
Figure 46. Spectra type options for searching in tree nodes
This option is useful when dealing with source collision induced
dissociation (CID) spectra. If you have a library which consists exclusively of
source CID spectra and your unknown spectrum is also a source CID,
exclude other spectra types from a search. If no such library is available, you
can search source CID spectra in product CID spectra, but be careful
regarding the search results. Source CID spectra might contain
fragmentation products from all the ions present in the source including
adduct or cluster ions, while product CID spectra are preferably generated
from protonated or deprotonated ions.
There are two combinations relating to the tree search:
• Search single spectrum in library trees
• Search tree in library trees
If you search a single spectrum in library trees, the spectrum is compared to
every spectrum in the tree hierarchy and the match factor is individually
calculated. A single spectrum can be searched only in the top level (full scan,
source CID), everywhere except for the top level (first stage), or everywhere
in the tree.
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Search Utilities
Figure 47. Search options for searching single spectrum in trees
If you search a tree (unknown) against spectral trees in a library, the
spectrum is compared according to a special logic. The corresponding
spectra on an identical level (MSn stage) with a common precursor m/z are
compared using an algorithm based on the optimized dot-product. If a
spectrum only appears on one side, it is ignored and does not negatively
effect the search result. If there are several corresponding spectra (such as
single node with average, composite, parallel, or other spectra), the best
match is accepted (optimistic approach). The total match factor is calculated
from all non-zero match factors.
Figure 48. Metric principles of tree comparison in library search
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Search Utilities
A spectral tree can be searched using two options. The top tree level
(full scan, source CID) can be included or excluded from a search and the
MSn stage of the tree spectra can be identical (identity search) or not
identical (subtree search). See Figure 49.
Figure 49. Search options for spectra tree search
Substructure Search
A structure or substructure query can be taken from the Structure Editor,
Database Manager or a fragment copied in a Fragments & Mechanisms
window.
Note When initiating a substructure search from the Structure Editor,
the (sub)structure query must be selected.
Structure and substructure searches are important for retrieving library
entries. The structure search is the most straightforward method for finding
compounds in a library. Because the rules of systematic nomenclature do
not necessarily lead to a unique name for each compound, name search can
be, in many cases, ineffective. It is easier to draw or import a structure query
than to type a complicated name.
While a structure search provides an exact match of query and library
structure, a substructure search retrieves compounds that contain a common
structural subset, called substructure. The exact substructure must be
embedded in each molecule retrieved. The exact match, in structure and
substructure searches, has a notable exception—that stereo bonds are
ignored, because optical activity does not play a significant role in mass
spectrometry. All other structural features such as bond multiplicity, atom
state and skeletal arrangement must match exactly. Charge, radical and
unspecified charge site can be optionally ignored and isotopes can be
optionally disregarded.
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Figure 50. Substructure search options
Substructure search offers two additional search options: Substructure Best
Match and Substructure Match Ring Bonds. A substructure can sometimes
fit at several locations of a larger structure. The Substructure Best Match
option that represents the closest match found and appears in red in the
Structure tab. Note that this option can lengthen calculation times. Because
fragmentation mechanisms on rings significantly differ from acyclic
moieties, search substructures that exactly match the ring membership for
each bond. Set this option in the Search Type box. Note that a substructure
search can also lengthen calculation times.
Use a Substructure search for a variety of purposes such as to study mass
spectra of structurally related molecules or to positively identify an
unknown. If you need help in interpreting a mass spectrum, compounds
retrieved by substructure search can provide analogies to the fragmentation
processes in the spectrum under consideration.
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Search Utilities
Figure 51. Structures page of the Spectra Manager window showing the
results of a substructure search
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Substructure Search Rules
Database Manager
Search Utilities
When searching structures with an unspecified charge site or substituents,
the search rules in Figure 52 apply.
Figure 52. (Substructure Search Rules
A structure similarity search can be performed using substituents.
Note A substructure search ignores the number and position of
hydrogen atoms.
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Name Search
The name search option provides incremental search capabilities. As you
start typing a portion of a name, a list of compounds that are spelled
similarly is displayed with a slight delay. The chemical structure is displayed
for the highlighted name. Use the up and down keys to browse the
displayed names. The library name search covers all three name types:
IUPAC, Synonyms, and Commercial Product.
Figure 53. Dialog window showing interactive name search
Molecular Formula Search
Use the molecular formula search option to search for all compounds with a
specific molecular formula. You can use lowercase letters when typing the
formula unless this would lead to an ambiguous query; for example, si could
be interpreted as Si or SI). In this case, be careful to avoid misinterpretation.
Molecular Mass Search
In this search, the term molecular mass is used to mean monoisotopic
molecular mass. The monoisotopic mass of an ion is the mass of the isotopic
peak which is composed of the most abundant isotopes of its elements.
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ID Number Search
Database Manager
Search Utilities
Each library entry is individually numbered. In this mode, you can search
for a single ID number or for a range of ID numbers. The ID search dialog
window contains two boxes (From and To) for you to type the ID range. If
you want to retrieve a single ID number, leave the To box blank. The
maximal ID number found is displayed in the Max. ID box.
Note If you delete a record from a library, the ID numbers of records
with a higher ID than the deleted record are decremented. This means
that if you delete one or more records, there are no ID number gaps in
the library.
CAS Number Search
In contrast to earlier versions of Mass Frontier, the Chemical Abstract
Service (CAS) registry number search option is now available for all
libraries. Do not use dashes when typing CAS numbers.
Retention Time Search
Each spectrum of a tree or stand-alone spectrum of a record can include a
retention time value (Spectrum Info/Scan Info/RT item in Info tab). Using
a retention time search, you can retrieve spectra whose retention time values
fit into a range of retention values you have defined.
Search Constraints
All searches, except Name Search, can be restricted by a set of constraints.
The dialog windows of these searches contain a panel with buttons for
activating constraints and for editing search constraints. When you click the
Edit button, a dialog window appears to enable you to search selected
libraries for matches with a specific set of criteria. Four constraint types are
available: Molecular Mass range, Number of Atoms range, Allowed
Elements and Good-Bad list. Searches conducted with activated constraints
can be time consuming because each library entry is examined for matching
criteria. Use Search Constraints when you are working with large libraries
and you want to retrieve matches that are of interest for your specific
problem.
Use the Good list (required substructures) to focus your search results on
the particular compound classes you are most interested in. The Bad list
(forbidden substructures) eliminates all structures containing unwanted
functional groups from a match list. For example, the Good-Bad List can be
used in a search of acids with a specific molecular formula, or you can search
for spectra similar to an unknown, with the requirement that ketones not
appear in the match list. Figure 54 depicts a substructure search using the
Search Constraints option. The figure shows a Structure Constraints dialog
window set for C, N, and O as allowed elements. The Good-Bad list is set
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for esters as required (good) and for tertiary amines as a forbidden (bad)
functional group. The substructure query is the triphenyl group and the
search results, with the described restrictions, are also shown.
Figure 54. Search Constraints dialog window showing allowed elements and functional groups
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Mass Spectral Data Exchange Between Modules
Mass Spectral Data
Exchange Between
Modules
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Database Manager serves as a gateway for several modules. Mass spectra can
be imported directly from a file, the Chromatographic Processor, Excel, or
Xcalibur. Spectra can also be exchanged between the Database Manager
modules. In addition, Mass Frontier is able to automatically establish a link
between Database Manager records and other modules that need to access
spectral or structural data. With the ability to link equivalent spectral
information, you can access the original data that was supplied as input
information for one of the many interpretation methods available in
Mass Frontier. The direct feedback between source (records and mass
spectra) and results (mechanisms, bar code spectra, classes and projections)
helps you to keep track of all the modules that originate from a single
source. This feature makes it easier to use more modules simultaneously,
thus enabling additional problem-solving approaches.
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Database Manager
Fragment Assignment to Spectral Peaks
Fragment Assignment
to Spectral Peaks
Mass Frontier enables the automated prediction of fragments from a
structure you provide. If you start the generation of fragments from the
Database Manager and the active record contains a structure (an MS1
structure for Spectral Tree), the generated fragments are automatically
linked with peaks in the spectrum (spectra in the Spectral Tree) according to
their m/z values. After a generation, highlighted (explained) peaks are
displayed in a different color (the default is red) in the original mass
spectrum. Clicking a highlighted peak reveals all the mechanisms leading to
it. In addition, generated fragments (a corresponding Fragments &
Mechanisms window must be open) can be assigned automatically to peaks
in a spectrum by clicking the Spectra Processing button and choosing
Auto Fragment Annotation of Peaks.
Figure 55. Database Manager window
In addition to automated peak annotation, you can also manually assign a
fragment structure to any peak by clicking the Assign Fragment Structure
to Peak button.
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Chapter 5
Library Utilities
Mass Frontier provides a number of methods for creating and maintaining
mass spectral and chromatographic libraries with chemical structures. To
help you visually distinguish between libraries, each library has its own icon.
You can select an icon for your libraries. National Institute of Standards and
Technology (NIST) main and replicate and HighChem MSn libraries icons
are assigned automatically by the program and cannot be changed. Up to
255 libraries can be installed at once.
Both low- and high-resolution mass spectra are supported. Mass spectra can
be stored as individual single spectra or as MSn trees with multiple node
spectra. Data-reduced chromatograms with selected scans and components
can be stored and searched. Any information in a library record can be
updated, except the NIST database which is read-only.
This chapter contains the following sections:
• Microsoft SQL Server 2000 Desktop Engine
• NIST/EPA/NIH Mass Spectral Database
• Library Installation
• Creating User Libraries
• Uninstall A Library
• Adding Records to a User Library
• Deleting Library Entries
• Saving Changes in Libraries
• SQL Server and Library Tools
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Microsoft SQL Server 2000 Desktop Engine
Microsoft SQL
Server 2000 Desktop
Engine
Mass Frontier library utilities are based on the Microsoft SQL Server 2000
desktop engine. The SQL Server 2000 desktop engine is a data engine built
and based on core SQL server technology. It is a reliable storage engine and
query processor for desktop applications. The SQL Server 2000 desktop
engine is a royalty-free database engine that is fully compatible with the
SQL Server. The SQL Server 2000 desktop engine is included in the Mass
Frontier installation process.
The SQL Server 2000 desktop engine is Mass Frontier's background
application. Library utilities are seamlessly integrated in a graphical interface
and you do not need to directly interact with the database engine. The
Mass Frontier database concept offers a broad spectrum of functionality and
provides flexible features, but these are connected with an overhead problem
that affects all modern database systems. Therefore, complex searches in
large amounts of data might require longer search times than in previous
versions of Mass Frontier.
There is a restriction in the SQL Server 2000 desktop engine that allows the
creation of databases with a maximum file size of 2 GB. Therefore, libraries
created in Mass Frontier cannot exceed this file size. If you try to store data
above this limit, Mass Frontier informs you that this action cannot be
completed and to store the extra data in a new library. This problem occurs
more often with chromatographic libraries than with spectral libraries. For
example, the NIST 2002 database with 175000 spectra takes up 800 MB in
the SQL Server 2000 desktop engine format. However, if you need to create
large spectral or chromatographic databases, the SQL Server 2000 desktop
engine can be upgraded to later SQL Server editions (Enterprise, Standard,
or Personal) that have terrabyte capacity. Mass Frontier libraries are fully
compatible with other editions of the Microsoft SQL Server. Later SQL
Server editions must be setup individually by a database expert. If you
require additional information on how to upgrade the SQL Server 2000
desktop engine, contact Microsoft or the HighChem, Ltd. database group.
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5 Library Utilities
NIST/EPA/NIH Mass Spectral Database
NIST/EPA/NIH Mass
Spectral Database
Mass Frontier can work as a stand-alone application, or combined with
the NIST/EPA/NIH Mass Spectral Database 2002 (NIST 2002 Library).
If the NIST 2002 Library has not been simultaneously installed with
Mass Frontier, the library can be either installed, if the library is in
Mass Frontier format, or imported from NIST file format. During import
of a NIST library, the data is converted to the SQL Server database format
used by Mass Frontier. This process can take several hours. If you select the
NIST Main library, the NIST Replicate library is installed automatically.
The NIST Replicate library cannot be installed as a stand-alone library
without the Main library.
To import NIST library or any other library in NIST format, choose
Library > Spectral Library > Import NIST Library.
Figure 56. Import NIST or Mass Frontier 5.0 User library dialog window
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Library Installation
Library Installation
To install a user, NIST, or HighChem MSn library into Mass Frontier,
choose Library > Spectral Library > Install Library from the main menu.
When the Install Library dialog window appears, follow these installation
steps:
1. Select the drive where the library is located.
2. Click the Find Library on X: button (X is the driver you have selected
in step 1). The program scans the drive for libraries. If a library is found,
proceed with step 3.
3. Select the library you want to install.
4. Choose an icon for the library.
5. Click Install Library.
Figure 57. Install Library dialog window
If the library has been successfully installed, the icon, name, and path of the
library are displayed in the List of Installed Libraries grid in the Install
Library dialog window.
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Library Utilities
Library Installation
Note A library can be installed in Mass Frontier only if the data is in the
Mass Frontier SQL Server database format. For libraries in NIST format
(NIST Library, user libraries from Mass Frontier 3.0 and earlier
versions) use the Import NIST Library option instead. Choose
Library > Spectral Library > Import NIST Library.
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Library Utilities
Creating User Libraries
Creating User
Libraries
Experimental data, as well as reference spectra, can be stored in user libraries
to organize your mass spectral, chromatographic and structural data. With
Mass Frontier, you can create highly annotated user libraries with structures.
Both low- and high-resolution mass spectra are supported. Mass spectra can
be stored as individual single spectra or as MSn trees with multiple node
spectra. Data reduced chromatograms with selected scans and components
can be stored in user libraries and searched.
To create a user library, choose the Library > Spectral Library > Create
User Library item in the main menu. When the Create User Library dialog
window appears, follow these steps:
1. Enter or select the directory for the user library. We recommend
accepting the default directory.
2. Enter the library name. Only the characters a-z, A-Z, space and 0-9 can
be used.
3. Select the library icon.
4. Click the Create & Install User Library icon.
If the library has been successfully created the icon, name and path of the
library are displayed in the List of Installed Libraries grid. The program
creates an individual subdirectory for each new library, complete with the
name of the library. All the library data are stored in an .mdf file. The full
library path is displayed in the Directory column.
An unlimited number of user libraries can be created, but no more then 255
libraries can be installed in Mass Frontier at the same time. If you require
more installed libraries, you can temporarily uninstall a library you do not
need and create or install the library you want to work with.
Note A higher number of spectral libraries slows the program start,
consume system resources and potentially lengthen the search processes.
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Creating User Libraries
Figure 58. Create User Library dialog window
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Uninstall A Library
Uninstall A Library
Because database files can be copied or deleted only if the library is
uninstalled, you might need to uninstall a library. In addition, a large
number of libraries can slow the overall performance of Mass Frontier so the
uninstallation of unused libraries can be beneficial. If you uninstall a library,
the library is not deleted. The library reference is merely removed from the
program without the loss of spectral or structural information. An
uninstalled library can be reinstalled at any time. With Mass Frontier, you
can install or uninstall a library as many times as you want.
To uninstall a library
1. Choose Library > Uninstall Library. The Uninstall Library window
appears. See Figure 59.
2. Select the library to uninstall.
3. Click Uninstall.
Note Database files with the .mdf extension cannot be copied or deleted
when the library is installed. If you need to make a backup copy or you
want to permanently delete a library from your hard drive, you must
uninstall it first.
Figure 59. Uninstall Library dialog window
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5 Library Utilities
Adding Records to a User Library
Adding Records to a
User Library
Mass Frontier provides a way of adding new records (spectra, trees,
chromatograms, structures and compound identification information) to a
user library. This option creates new library entry with a new ID. To update
an existing record, use the Save Changes in Library option.
To add one or more records from the Database Manager to a user library
1. Select the records you want to add to the user library from the
spreadsheet in the Database Manager.
2. Choose Library > Add To Library.
3. When the Add To Library dialog window appears, select the library you
want to add to the records by choosing the appropriate button.
4. Click Add.
Note Only selected records in a Database Manager Spreadsheet can be
added to a user library.
A spectrum you intend to add to a library must not contain more than 3000
peaks and the m/z value might not be greater then 3000. A single structure
accompanying a spectrum in a user library must not contain more then 199
non-hydrogen atoms.
Check whether the structure associated with the spectrum you want to add
to a user library is already present in the library. Perform redundancy checks
by clicking the Check button in the Check Structure Redundancy window.
When this option is selected, the program compares each structure to be
added to the library with structures in the selected library. Use this option if
the structure is present, otherwise this option is ignored.
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Deleting Library Entries
Deleting Library
Entries
Deleting a library record causes the information to be irreversibly lost.
There is no undo function for deleted library records. You cannot delete
library entries from a NIST library.
To delete one or more records
1. Retrieve the records you want to delete using any search.
2. Select the records you want to delete in the Database Manager
Spreadsheet.
3. Choose Library > Delete from the Library menu.
4. When the Delete From Library window appears, review the spectra and
structures carefully.
5. If you want to delete all records at once that are displayed in Preview,
choose Delete without confirmation. If you want to delete record by
record, select Confirm Each Spectra/Structure Pair Before Deletion.
6. Click Delete.
Note If you delete a record from a library, the ID numbers of records
with a higher ID than the deleted record are decremented. This means
that if you delete one or more records, there are no ID number gaps in
the library.
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Saving Changes in Libraries
Saving Changes in
Libraries
Any record information (spectra, trees, chromatograms, structures and
compound identification information) can be changed and saved in a
library. If anything is changed in the record, a small circle is displayed in the
Database Manager in the ID Num. column in the spreadsheet. Changes in
records are not automatically updated in the database unless you save them.
To save changes in library, choose Library > Spectral Library > Save
Changes in Library.
If you try to save a newly created record, you are redirected to the Add New
Records to Library option.
Note If you try to close a Database Manager window, or Mass Frontier
with changed unsaved records, you are prompted to save them.
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SQL Server and Library Tools
SQL Server and
Library Tools
To open the SQL Server & Library Tools dialog window, choose
Library > Library Tools from the main menu tool bar. The SQL
Server & Library Tools dialog window contains three tabs:
• Spectral Libraries
• Fragmentation Libraries
• SQL Server
Spectral Libraries
The Info grid lists all the attached spectral and chromatographic libraries to
the SQL Server. Libraries incompatible with the current version of
Mass Frontier can be detached or removed from your system by using the
context menu which you access by right-clicking.
In the Update group, you can compress or re-index selected libraries to
shrink their sizes, or defragment indexes if the performance decreases. Note
that these actions might require additional time to perform, depending on
the amount of stored data.
Note Make backup copies of your libraries before reannexing and
compressing spectral libraries.
Figure 60. Spectral Libraries page of the SQL Server & Library Tools dialog
window
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SQL Server and Library Tools
Fragmentation Libraries
The Info grid lists all the spectral and chromatographic libraries attached to
the SQL Server with their paths. Libraries incompatible with the current
version of Mass Frontier can be detached or removed from your system by
using the context menu which you access by right-clicking.
In the Update group, you can compress or re-index selected libraries to
shrink their sizes, or defragment indexes if the performance decreases. Be
aware that these actions might require additional time to perform,
depending on the amount of stored data. If you build large fragmentation
libraries, perform these actions regularly.
Note Make backup copies of your libraries before re-indexing and
compressing fragmentation libraries.
Figure 61. Fragmentation Libraries page of the SQL Server & Library Tools
dialog window
SQL Server
Note Use this page only if you are familiar with SQL Server database
technology.
The Monitor box displays information about the SQL Server status and
contains buttons for starting, running and stopping the SQL Server service.
Use the Extended Stored Procedures box in the Reload box to redefine the
extended stored procedures used by the SQL Server. These procedures are
essential for spectra search functionality. Use this action if a library search
does not work properly.
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SQL Server and Library Tools
The Default Library box in the Reload box re-creates damaged internal
database files. Use this action if the message Cannot open library
DefMSMSLibrary! appears.
The Connected Libraries box in the Reload box reconnects all the spectral
libraries. Use this action if the SQL Server was stopped and restarted outside
of Mass Frontier; for example, in the task bar notification area.
Figure 62. SQL Server page of the SQL Server & Library Tools dialog window
Backup and Restore
Libraries
It is not advisable to back-up and restore spectral or fragmentation libraries
by a library files copy and paste operation. Use the backup and restore
procedures in Mass Frontier.
To backup a library
1. Open the SQL Server & Library Tools dialog window by choosing
Library > Library Tools.
2. Select the library type by clicking the Spectral Library or the
Fragmentation Library tab.
3. Click the Backup/Restore button and the dialog window appears.
4. Specify the library you want to back up and type the name, description
and destination of the backup files.
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SQL Server and Library Tools
Figure 63. Backup/Restore dialog window showing Backup page
You can restore library data from previous backups into a new library or into
an existing library.
Note If you restore library data into an existing library, its data is
irreversibly overwritten. Thermo recommends that you restore data into
a new library with a unique name.
To restore a library from backup files
1. Open the SQL Server & Library Tools dialog window by choosing
Library > Library Tools.
2. Select the library type by clicking the Spectral Library or the
Fragmentation Library tab.
3. Click the Backup/Restore button and the dialog window opens.
4. Specify the library name in the Library to Backup box where the data is
to be restored. Type a new library name here.
5. Select the library data to restore from the list of backups.
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SQL Server and Library Tools
Figure 64. Backup/Restore window showing Restore page
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Chapter 6
Fragments and Mechanisms
One of Mass Frontier´s features is the automated generation of possible
fragments at an expert level, including complete fragmentation and
rearrangement mechanisms, starting from a chemical structure you supply.
The Fragments & Mechanisms module provides information about basic
fragmentation and rearrangement processes that might occur in a mass
spectrometer.
Use generated fragments and corresponding mechanisms for:
• Checking consistency between a chemical structure and its mass
spectrum.
• Confirming library search identifications
• Recognizing the structural differences between spectra of closely related
compounds
• Interpreting the spectra of isotopically-labeled compounds
• Illustrating the structure-spectra relationship for educational purposes
This chapter contains the following sections:
• Features
• Fragmentation, Rearrangement and Resonance Reactions
• Starting Generation
• Fragments & Mechanisms Window
• Reaction Restrictions
• Generated Fragments Linked with Spectrum
• Eliminating Generated Fragments Not Present in a Spectrum
• Simulation of MSn Experiments
• Unexplained Peaks
• Too Many Proposed Fragments for a Peak
• Bar Code Spectra
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Features
Features
The Fragments & Mechanisms module is based on an expert system that
uses a mathematical approach for the simulation of unimolecular
ion-decomposition reactions. It is important for you to understand what the
system is and is not capable of, and what you can and cannot expect from
this module.
The expert system, which generates possible fragmentation and
rearrangement pathways, is based on the following assumptions.
General Fragmentation
and Rearrangement Rules
The system optionally predicts reaction pathways that are based on general
fragmentation and rearrangement rules. Compound-specific mechanisms
that cannot be applied generally are not included in this feature. Use this
feature in combination with a substructure search for identifying specific
compound classes.
Fragmentation Library
Mechanisms
The system optionally accesses an intelligent fragmentation mechanism
knowledge base for the prediction of unimolecular decomposition reactions.
HighChem Fragmentation Library currently contains around 19000
individual mechanisms. User mechanisms can be included in fragmentation
prediction as well.
Charge Localization
Concept
Every ion-decomposition reaction that is generated is based on the charge
localization concept. The location of the charge site in all precursor and
product ions is exactly determined by the program. Mass Frontier internally
generates resonance reactions which, by default, are not displayed. Note that
charge sites can be moved to distant locations by these reactions and in some
complicated structures it might appear that the charge localization concept
has been violated. If a reaction step is not clear, you can instruct the
program to display mechanisms along with resonance reactions. It is,
however, possible to use an unspecified charge location that is internally
transformed to all combinatorial structures with a localized charge.
Unimolecular Linear
Reaction Mechanisms
Mass Frontier generates only unimolecular reactions. The reaction pathways
are displayed as linear reaction mechanisms which incorporate one
intermediate left-hand site and one intermediate right-hand site for each
reaction step. Thus, only ionic products are included in reaction pathways
and neutral fragments are not displayed.
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Fragments and Mechanisms
Features
Even- Electron Rule
Reactions mechanisms are generated in accordance with the Even-Electron
rule. The Even-Electron rule states that the homolytic bond cleavages of an
even electron ion are energetically unfavorable. Therefore, Mass Frontier
never generates radical cations from an even-electron ion.
Bond Cleavages Only
Using the General Fragmentation and Rearrangement Rules option,
fragments can only be generated from bond cleavages. Bond creation is not
supported, with the exception of the creation of an H-X bond (where X=any
element) in hydrogen rearrangements. Thus, for this option the expert
system does not include ring contractions, cyclizations, or skeletal and
non-hydrogen rearrangements. The fragmentation library option supports
bond creation with all rearrangements and ring transformations.
Ionization Methods
Formally Possible
Solutions
Thermo Electron Corporation
Mass Frontier supports Electron Impact, protonation, deprotonation,
cluster ion formation, alkali metal adducts, and chemical ionization
methods.
The mechanisms generated by Mass Frontier contain formally possible
reaction steps. The determination of the stability of product ions from
thermodynamic data or rates of reaction is not performed. When evaluating
generated mechanisms, remember that short and uncomplicated reaction
pathways are more favorable than complex mechanisms involving
complicated, multistep hydrogen rearrangements.
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Fragmentation, Rearrangement and Resonance Reactions
Fragmentation,
Rearrangement and
Resonance Reactions
Mass Frontier utilizes general unimolecular reactions for prediction of
fragmentation, rearrangement and resonance mechanisms.
To preview unimolecular reactions used by the program
Click the Reaction Mechanisms Overview
button in any
Fragments & Mechanisms window. The Reaction Mechanisms Overview
window appears. See Figure 65.
Figure 65. Fragmentation, rearrangement, and resonance reactions
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Fragmentation, Rearrangement and Resonance Reactions
Table 1.
Thermo Electron Corporation
Reaction formalism used in Mass Frontier
α
α - cleavage
π
π electron ionization
σ
Sigma electron ionization
cr
Charge stabilization
-e-
Non-bonding electron ionization
es
Electron sharing
i
Inductive cleavage
+H+
Protonation
-H•
Hydrogen radical loss
-H:
Hydride abstraction
rHA
Radical site rearrangement
rHB
Charge site rearrangement (α, β)
rHC
Charge site rearrangement (γ)
rHR
Charge-remote rearrangement
rH1,2
Hydrogen shift to adjacent position
rr
Radical resonance
Lib
Fragmentation Library Mechanisms
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Starting Generation
Starting Generation
Fragmentation and rearrangement pathways can be generated from any
structure you supply, including ions and isotopically-labeled compounds.
The structure can originate from the Structure Editor, Database Manager,
or from a Fragments & Mechanisms window. Before the generation is
performed, the program checks the input structure for errors. If any errors
are found, a message box appears that informs you about these errors and
the generation ends.
When you start a generation from the Structure Editor, in contrast to the
copy function or substructure search, the structure does not have to be
selected. The program assumes that the complete structure is intended as
input for the generation.
If a generation is started from the Database Manager window, the program
automatically links the generated fragments in the Fragments &
Mechanisms window with the corresponding spectrum in the Database
Manager window. Peaks having the same m/z value as the generated
fragments are highlighted in the color set in the Spectra Layout dialog
window (by default, red). Selecting a highlighted peak reveals all the
pathways leading to it.
To start a generation of possible fragmentation and rearrangement
pathways
1. Click the
button in the Structure Editor, Database Manager, or
Fragments & Mechanisms window.
2. Or make the Structure Editor or Database Manager active and then
choose Tools > Fragments & Mechanisms.
When you start generation, a Reaction Restriction window appears to
specify the type of knowledge used for fragmentation.
During the generation of possible fragmentation and rearrangement
pathways, the Generation of Fragments & Mechanisms window appears.
See Figure 66.
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Starting Generation
Figure 66. Generation of Fragments & Mechanisms dialog window
The Already Generated m/z box that stores m/z values of ions that have
already been generated. Click the down arrow to display a list of ions that
have been generated. The total number of ions that have already been
generated appears next to the box.
While a generation is in progress, the Reactions Limit bar provides an
approximate indication of how many temporary internal reactions have
been generated from a particular structure. Large and structurally
complicated molecules can produce a large number of reactions. Because the
generated reactions consume a significant amount of memory, there is a
limit to the number of temporarily-generated reactions. If the reactions
limit is reached, the generation stops and a message box appears to remind
you of this. Even if the generation is stopped, the fragments and
mechanisms generated up to that point are displayed. The fragments and
mechanisms that the system generates first are the most important.
Therefore, when a generation is stopped, the most important fragments
have likely already been generated. However, if you are missing an
important fragment and you assume that it is because the generation was
interrupted, you can increase the number of internal reactions in the
Reaction Restriction window in the Size tab.
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Starting Generation
The message Mass Frontier is a multithreading application, so you can use this
program while reactions are being generated appears in the Info window. The
multithreading strategy allows more than one task to be performed
simultaneously. For example, while reactions are being generated, you can
search libraries or analyze your spectra. You can even run two or more
generations of fragments and mechanisms at the same time.
Click the Pause button to temporarily interrupt generation to redirect
processing power to other processes that might be running simultaneously
in Mass Frontier or to other Windows applications.
Click the Finish button to stop generation. Fragments, along with their
production mechanisms generated up to that point, appear.
Click the Cancel button to end a generation. It can take several seconds for
the window to disappear after the generation has ended.
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Fragments & Mechanisms Window
Fragments &
Mechanisms Window
Once reaction-generating process has finished, the Fragments &
Mechanisms window appears. See Figure 67.
A Fragments & Mechanisms window contains complete mechanisms of
ion-decomposition reactions, or the resulting fragments only. To display the
mechanism or fragment for a particular m/z value, choose the appropriate
m/z tab. If you do not like the tab control, replace it with a box in the
Options | Mechanism Layout dialog window.
A Fragments & Mechanisms window contains two Copy buttons. Use the
first one to copy mechanisms in the Enhanced Windows Metafile format
.emf. Use the second one o copy a selected fragment. If you copy a
fragment, you can paste it to the Structure Editor, Database Manager in
Mass Frontier, or to any Windows application.
Note Only a selected fragment can be copied to the Clipboard.
Several possible isobaric fragments can be generated for a particular
m/z value. If more then one fragment is generated for a m/z value, a
hand-shaped pointer
with the caption Select possible fragments with
m/z xy moves from right to left to draw your attention to this. Click the
numbered buttons next to the hand pointer to display the isobaric
fragments and their corresponding mechanisms.
The fragments are sorted according to the simplicity of their production
mechanism. Thus, fragment number 1 is produced by the simplest
(shortest) mechanism. The isobaric fragments are usually isomers of the
same fragments with a different charge, radical or, p-bond location.
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Fragments & Mechanisms Window
Open Mechanisms
Generate Fragments & Mechanisms from Selected Fragment
Save Mechanisms
Show m/z Values for Explained Peaks only
Print
Compare Fragments
Copy Mechanisms
Show Bar Code Spectrum
Copy Fragment
Exclude m/z (Bar Code Spectrum, Fragments Comparator, Clipboard)
Copy List of
Set m/z (Bar Code Spectrum, Fragments Comparator, Clipboard)
Fragment
Default m/z Values
Reaction
Reaction Mechanisms Overview
Layout
Ionization Method
Figure 67. Fragments & Mechanisms window
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Reaction Restrictions
Fragments and Mechanisms
Reaction Restrictions
Use Reaction Restrictions to change the default settings of the ionization
method and of basic fragmentation and rearrangement mechanisms. To set
the m/z precision of calculated fragments in the Mass Settings dialog
window, choose Options > Mass Settings.
To Restrict Restrictions
Choose Options > Reaction Restrictions.
The Reaction Restrictions window contains six tabs: Knowledge Base,
Ionization & Cleavage, H-Rearrangement, Resonance, Additional, and
Sizes.
If you have established restriction settings that you want to apply frequently
to a specific compound class, save the current reaction restrictions to a file
with the .rrs file extension by clicking the Save button in the Reaction
Restrictions dialog window. When you open a reaction restriction file by
clicking the Open button, the dialog window adopts the restrictions saved
in the file. You must click OK to make these restrictions active in Mass
Frontier.
Note All changes in the Reaction Restrictions window take effect after
the regeneration of fragments and mechanisms. The existing
Fragments & Mechanisms windows are not affected by changes in
Reaction Restrictions. In addition, the changes in the Reaction
Restrictions window affect all subsequent generations, unless you restore
default settings. Therefore, when changing the settings, remember to
restore the default reaction restrictions when the changes are no longer
needed.
Knowledge Base Page
You can specify the type of knowledge base the expert system uses for the
prediction of fragmentation pathways in the Knowledge Base box in the
Reaction Restrictions window. The options are General Fragmentation
Rules, Fragmentation Library, or both.
If you choose the Fragmentation Library, select which library to use from
the list of libraries. The preinstalled HighChem Fragmentation Library™
contains around 19000 mechanisms, and so calculation times are
significantly longer when it is used.
There are four options connected with the Fragmentation Library
knowledge base. Because some mechanisms of small fragments can fit to
virtually every user-provided structure and can generate many useless
fragments, there is an option to deactivate a record in the Fragmentation
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Library window. To use this option, select the Active Record Only option
in the Reaction Restriction window. If you do not, all mechanisms from the
selected libraries are considered by the expert system.
If a neutral molecule is provided, Mass Frontier automatically simulates the
ionization process according to the selected ionization mode in the
Ionization & Cleavage page. Click Library Ionization Only to disable this
automatic simulation and use a different ionization site by adding an
ionization mechanism to the library.
Because some fragmentation library mechanisms follow general
fragmentation rules, you can speed up the generation by enabling General
Fragmentation Rules in the Knowledge Base group and disabling general
fragmentation rules in Fragmentation Library Options group in the Ignore
General Frag. Rules in the Library Reaction box.
With Mass Frontier, you can work with structures with an unspecified
charge site in the fragmentation library or in the starting structure but it
dramatically slows the generation process. If an unspecified charge site is
used, the expert system must consider a large number of combinations for
every step. Therefore, avoid using unspecified charge sites in the
fragmentation library, if possible. To exclude mechanisms in the
fragmentation library that contain unspecified charge sites from the
generation process, click Charge Localization Concept Only.
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Reaction Restrictions
Figure 68. Reaction Restriction dialog window showing the Base page
Ionization & Cleavage
Page
In the Ionization & Cleavage tab in the Reaction Restrictions window,
choose among the ionization mode Electron Impact (EI) that produces
M+ ions, the protonation mode that produces [M+H]+ ions,
deprotonation - negative ionization ([M-H]-), cluster ion formation
([M+NH4]+, [M+H3O]+), alkali metal adducts ([M+Li]+, [M+Na]+,
[M+K]+), and Chemical Ionization (CI). The protonation and
deprotonation mode represents soft ionization techniques such as
Electrospray Ionization (ESI), Atmospheric Pressure Chemical Ionization
(APCI), Fast Atom Bombardment (FAB) and others. The chemical
ionization option offers three basic ionization reactions: protonation,
hydride abstraction and adduct formation. In addition, you can select one of
six most common reaction gases: methane (CH4), hydrogen (H2), isobutane
(i-C4H10), ammonia (NH3), water (H2O) and nitrogen monoxide (NO).
Note The deprotonation option does not support General
Fragmentation Rules in the Knowledge Base. Deprotonation can be
used only in connection with the Fragmentation Library.
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Reaction Restrictions
When comparing generated fragments and mechanisms with a mass
spectrum, always choose the correct ionization method. A warning message
appears if the reaction restrictions are set for protonation techniques or
chemical ionization and you are attempting to compare generated fragments
with a spectrum from the NIST library which contains EI spectra only.
However, if the spectrum is from a file, the mass spectrum type and
ionization techniques are not checked for consistency.
Figure 69. Reaction Restriction dialog strict showing the Ionization & Cleavage
page
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H-Rearrangement Page
Fragments and Mechanisms
Reaction Restrictions
The second tab in the Reaction Restriction window is H-Rearrangement.
This contains controls setting the four basic hydrogen rearrangements listed
in Table 2.
Table 2.
Hydrogen rearrangement types
rHA
Radical site rearrangement
rHB
Charge site rearrangement (α, β)
rHC
Charge site rearrangement (γ)
rHR
Charge-remote rearrangement
The hydrogen rearrangements that involve radical (odd electron ions) rHA
are set by default for hydrogen transfer from a steric optimal atom, usually
from an γ-atom (McLafferty rearrangement). Hydrogen shift to adjacent
position (rH1,2) is activated by default and cannot be deactivated.
There are two possible reasons for changing the default setup of
rearrangements. First, if you are missing an important peak and you suspect
an unusual rearrangement, you can compel hydrogen transfer from an α, β,
γ or δ-atom. Second, you might want to simplify a mechanism by
deactivating rearrangements that cause redundant reaction steps.
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Reaction Restrictions
Figure 70. Reaction Restrictions dialog window showing the Hydrogen
Rearrangement page
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Resonance Page
Fragments and Mechanisms
Reaction Restrictions
Mass Frontier generates fragmentation and rearrangement mechanisms
along with electron shift reactions (resonance reactions). Because these
reactions might, even for small structures, cause a large number of
by-products, by default, the resonance reactions are not depicted. To keep
the reaction network clear, the program performs a reduction of reaction
complexity, by not displaying resonance reactions. Thus, elementary
reaction steps that include resonance reactions are merged into a single step.
If you encounter unclear elementary reaction steps, instruct the system to
display all resonance reactions by clicking the Yes button in the Display
Resonance Reactions box in the Resonance page in the Reaction
Restrictions dialog window. See Figure 71.
Figure 71. Reactions Restrictions dialog window showing the Resonance page
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Additional Page
By default, cleavage on an aromatic ring is not activated. However, when
you dealing with small aromatic compounds, activate bond cleavage on
aromatic rings by clicking the Cleavage box in the Allowed on Aromatic
Systems box in the Additional page in the Reaction Restrictions dialog
window. See Figure 73. For example, when you generate fragments and
mechanisms of phenol, the important fragment corresponding to the peak
m/z 66 can be generated only if cleavage on aromatic systems has been
activated.
Figure 72. Structures of phenol, showing bond cleavage on an aromatic system
Cleavage on aromatic systems is deactivated by default. This is due to the
large numbers of fragments can be generated from large aromatic
compounds and the aromatic bond is a very strong bond.
Figure 73. Reaction Restriction dialog window, showing the Additional page
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Sizes Page
Fragments and Mechanisms
Reaction Restrictions
Use the Sizes page to limit the size and complexity of a reaction pathway
generation. See Figure 74. The Reaction Steps Max Number box gives the
number of cascaded fragment reactions. Increasing this number could
exponentially increase the number of fragments produced for a given
reaction path. Generally, keep this number small, and if additional
fragments need to be generated, select individual fragments to use as starting
points for additional reactions.
Large and structurally complicated molecules can produce a large number of
reactions. Because generated reactions take up a large amount of memory,
the number of temporarily generated reactions is limited. The system
generates the fragments and mechanisms so that the fragments that are
generated first are the most important. If the reactions limit is reached, the
generation stops, and a message box appears as a reminder. Even if the
generation is stopped, the fragments and mechanisms generated up to that
point are displayed. Therefore, when a generation is stopped, the most
important fragments have probably already been generated. However, if you
are missing an important fragment, and you believe this is due to the
generation being interrupted, increase the number of internal reactions in
the Reactions Limit box.
The m/z precision of calculated fragments can be set in the Mass Settings
dialog window. Choose Options > Mass Settings.
Figure 74. Reaction Restrictions dialog window showing the Sizes page
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Generated Fragments Linked with Spectrum
Generated Fragments
Linked with Spectrum
Mass Frontier has the ability to link generated fragments with a mass
spectrum. If you start a generation of fragments and mechanisms from the
Database Manager, the generated fragments are automatically linked with
peaks in a spectrum according to their m/z values to help explain peaks in
spectrum. After a generation, highlighted (or explained) peaks are displayed
in a different color (by default red) in the original mass spectrum. Selecting
a highlighted peak reveals all the mechanisms leading to it.
If an unexplained peak is likely to be an isotopic peak of an explained peak,
this is depicted in a third color (by default, green). Selecting such a peak
reveals all the mechanisms leading to the inferred fragment, which can
produce this isotopic profile.
Remember that the inability to predict energies and barriers in ionized
molecules prevents the prediction of all the peaks in a mass spectrum. Thus,
fragment predictability usually ranges between 50-90 percent. However, a
prominent unexplained peak can be valuable for the interpretation or
identification of an unknown. An unexplained peak can indicate a
compound-specific mechanism that occurs in molecules with similar
structural features or with a common substructure. There are a number of
mechanisms that have only been observed in a specific group of compounds
and cannot be applied generally when proposing fragmentation and
rearrangement pathways.
If you suspect a compound-specific mechanism of fragment formation,
verify your assumption by conducting a substructure search and then
comparing the explained and unexplained peaks in the spectra retrieved by
the substructure search.
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Eliminating Generated Fragments Not Present in a Spectrum
Eliminating
Generated Fragments
Not Present in a
Spectrum
The Fragments & Mechanisms module shows all m/z values that have been
generated with given Reaction Restrictions settings. If the generated
fragments are linked with a spectrum (that is, the fragments were generated
from a still existing Database Manager window which contains the given
spectrum), you can eliminate the m/z values that do not have corresponding
peaks in the spectrum. In some cases, Mass Frontier generates a large
number of theoretically possible fragments with a variety of m/z values. It is,
therefore, useful to display only those fragments that can be linked with a
spectrum (explained peaks).
To eliminate m/z values that cannot be linked with peaks in a given
spectrum
• Click the Show m/z Values for Explained Peaks Only
the Fragments & Mechanisms window.
button in
If you eliminate m/z values that do not correspond to a spectrum, these
values are not permanently deleted. While the button is in the down
position, Mass Frontier removes these values from the tab control or from
the box, depending on the selected settings of the m/z selector (Mechanism
Layout dialog window). Reset the button to its up position to restore the
original state and list all generated m/z values.
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n
Simulation of MS Experiments
Simulation of MSn
Experiments
With Mass Frontier, you can simulate fragmentation processes in MSn
experiments. See Figure 75. The fragments and mechanisms can be
generated not only from neutral compounds, but also from ions. You can
also select a product fragment (parent ion) in a Fragments & Mechanisms
window and begin the generation there. An unlimited number of
consecutive secondary ion decomposition reactions can be simulated.
Figure 75. Fragments & Mechanisms window showing the simulation of an MSn experiment
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Unexplained Peaks
Fragments and Mechanisms
Unexplained Peaks
The fact that a peak cannot be explained by Mass Frontier because the
corresponding fragment was probably formed by a compound specific
mechanism, can be helpful in the identification of characteristic structural
groupings that give rise to the peak. For example, the phthalates produce a
characteristic ion with m/z 149, which is formed by a highly specific
mechanism. The peak at m/z 149 can be easily recognized as a contaminant
from elasticized polymers. Mass Frontier is not able to explain this peak
because its corresponding fragment is formed by an unusual hydrogen
rearrangement and cyclization, which are not supported. To distinguish
between a randomly unexplained peak and a compound-specific peak, you
need to find some examples in the library. Using a substructure search, you
can retrieve compounds that contain a phthalate group as a common
substructure. After the generation of fragments and mechanisms of the
retrieved examples, the prominent peak corresponding to the phthalate
group remains unexplained in the majority of cases. For example, a
phthalate with a functional group at position 3, 4, 5, or 6 has its prominent
peak shifted to higher masses by the mass of this functional group. Such an
unexplained prominent peak, present in the spectra of structurally similar
compounds, can be a strong indicator of a compound-specific
fragmentation process. This information can help in the identification of a
substructure under investigation.
Figure 76. Spectra and structures of phthalate analogs
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Too Many Proposed Fragments for a Peak
Too Many Proposed
Fragments for a Peak
For a molecule that contains a larger number of locations for possible
reaction initiation (heteroatoms and π-bonds), Mass Frontier can generate
several theoretically possible ions with identical mass-to-charge ratios.
Usually, these ions are isomers of the same fragment, but sometimes they are
structurally different. If the program generates two or more different
fragments with identical mass value, you need to choose the correct one. As
before, a substructure search can provide the necessary information.
Figure 77 shows how to select the most likely fragment from several
possible candidates. The spectrum of 2,3,3a,4,5,6-Hexahydro-10methyl-1H-pyrazino[3,2,1-j,k]carbazole exhibits a base peak at m/z 198
(M-28). See Figure 77. For this peak, Mass Frontier selected twenty-two
possible ions that are mostly isomers of two fragments.
Figure 77. Spectrum of 2,3,3a,4,5,6-Hexahydro-10methyl-1H-pyrazino[3,2,1-j,k]carbazole
The first fragment is formed by a retro-Diels-Alder reaction involving an
α-bond on a carbazole group. The second fragment is the result of ethylene
loss from a piperazine group. See Figure 78.
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Too Many Proposed Fragments for a Peak
Figure 78. Two possible mechanisms of fragment formation
To determine the most likely fragmentation process for the formation of the
fragment m/z 198 (M-28), begin a substructure search of the investigated
structure, excluding the methyl group, to determine whether the spectra of
the retrieved compounds also contain a base peak or a prominent peak at
M-28. See Figure 79.
Figure 79. Spectra from a substructure search
When the peak M-28 is confirmed as the characteristic peak of this
compound class, retrieve two different groups of spectra. See Figure 79. The
first group of retrieved spectra shows compounds with a common
substructure that are involved in the first mechanism. See the spectra on the
left side of Figure 80. Similarly, the second group of spectra shows
compounds with a common substructure that are involved in the second
mechanism.
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Figure 80. Two groups of spectra showing compounds with common substructures that are involved in two different
mechanisms
Two separate substructure searches of 1,2,3,4-Tetrahydrocarbazole and
Piperazine result in the retrieval of two groups. You can see which group
yields a prominent peak at M-28.
These two groups of spectra prove that the peak m/z 198 in the spectra of
2,3,3a,4,5,6-Hexahydro-10-methyl-1H-pyrazino[3,2,1-j,k]carbazole is
formed by a Retro-Diels-Alder reaction (Mechanism 1).
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Bar Code Spectra
Fragments and Mechanisms
Bar Code Spectra
Mass Frontier can automatically predict possible fragments from a chemical
structure using general rules and library mechanisms, along with primary
determination of the structural plausibility of generated ions. The
prediction of mass spectra is hindered by the difficulty of predicting
thermochemical data, the thermodynamic stability of product ions, and
reaction rates. However, generated fragments and their mass-to-charge ratio
values can be used for creating bar code spectra. A bar code spectrum
contains peaks at predicted mass-to-charge ratio values with identical
(maximal) intensity. See Figure 81.
Figure 81. Experimental and Bar Code spectrum of
Pyrrolidine[2,1-c]-2H,5H-1,4-benzodiazepin-2,5-dione,1,3-dihydro(Electron Ionization mode)
With Mass Frontier, you can create bar code spectra from predicted
fragments.
To create a bar code spectrum
Click the Bar Code Spectrum
window.
button in any Fragments & Mechanisms
The created bar code spectrum is placed in a Spectra Manager window.
Bar code spectra are automatically linked with their original Fragments &
Mechanisms windows. If you click any bar code peak in Spectra Manager,
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the program displays corresponding fragments, along with its (their)
formation mechanisms. This link remains in place as long as the
corresponding Fragments & Mechanisms window exists.
Use bar code spectra in several strategies for investigating spectra-structure
relationships. The primary purpose of generating bar code spectra is that
they allow the possibility to identify spectral differences in structurally
similar compound classes, for which mass spectra are not available. To study
fragmentation dissimilarities between structurally related compounds, it can
be easier and quicker to compare two or more bar code in Spectra Manager
than to manually compare fragments and their mass-to-charge ratios
between Fragments & Mechanisms windows.
For example, if you are interpreting an unknown spectrum and you have
established two structural proposals, do the following:
1. Draw both structures separately in the Structure Editor.
2. Generate fragments for both structure to find out which structure
belongs to the unknown spectrum.
3. Create bar code spectra, and place them in the same Spectra Manager
window.
4. Compare the bar code spectra in the Compare Spectra page in Spectra
Manager. In the Difference Spectrum box, you see the specific peaks
that this pair of spectra do not have in common.
You can then compare these specific peaks with the unknown spectrum, and
take a closer look at the fragmentation mechanisms of these peaks. If a
specific peak is present in the unknown spectrum and the mechanism of
formation seems to be plausible, select the most likely structure. The
approach using bar codes, is superior to comparing explained peaks, because
it can be applied to a large number of structural proposals simultaneously.
The following example demonstrates how to identify the correct structure
for an unknown spectrum. Assume two structures are suggested:
1-Butanone, 1-phenyl- and 1-Propanone, 2-methyl,1-phenyl-. A
comparison of their bar code spectra shows that the peak at m/z 120 is
present in the unknown spectrum and only one of the bar code spectra. The
formation mechanism for the peak m/z 120 reveals a classical sequence of
McLafferty rearrangement and α-cleavage that is very common for
1-butanones or larger ketones. See Figure 82.
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Bar Code Spectra
Figure 82. Postulating structures from isobaric possibilities using Bar Code
spectra
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Chapter 7
Fragmentation Library
Use the Fragmentation Library module to create and manage fragmentation
mechanism databases. This module contains a graphical editor for entering
fragmentation reactions which can be stored in a database, together with
complementary information for the reaction. All fields of the database can
be queried, for example, authors, ionization method, or mass analyzer. All
library structures from the reactions are also fully searchable.
This module contains an expert system that automatically extracts a
decomposition mechanism for each fragmentation reaction in the database
and determines the compound class range that the mechanism can be
applied to. Mass Frontier uses this expert system to apply database
mechanisms to a user provided structure and automatically predicts the
fragmentation reactions for a given compound.
The current version of Mass Frontier comes with almost 5000
fragmentation schemes that contain around 19000 reactions collected from
mass spectrometry literature. The collected mechanisms are stored in the
HighChem database that automatically appears when the Fragmentation
Library module is opened.
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Fragmentation Library
Figure 83. Fragmentation Library window
This chapter contains the following sections:
• Fragmentation Library Toolbar
• Drawing Fragmentation Reactions
• Active Record
• Additional Information
• Saving Records
• Mechanism Extraction
• Reaction Symbols
• Using Library Reactions in Fragmentation Prediction
• Search Utilities
• HighChem Fragmentation Library
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Fragmentation Library Toolbar
Fragmentation Library
Toolbar
Figure 84 depicts the Fragmentation Library toolbar.
Undo (Ctrl+Z)
Draw Straight Arrow
Redo (SHIFT+Ctrl+Z)
Draw Arc Arrow
Cut (Ctrl+X)
Add Text
Copy (Ctrl+C)
Snap Arrow into Structure Box
Paste (Ctrl+P)
Snap Arrow into Structure Envelope
Delete
Check Reactions
Select All (Ctrl+A)
Align
Default
Add/Edit Structure
Print
Search
Library
Reaction Layout
New Record
Zoom In
Zoom Out
Figure 84. Fragmentation Library toolbar
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Drawing Fragmentation Reactions
Drawing
Fragmentation
Reactions
The Fragmentation Library module includes a graphical editor for entering
and editing fragmentation reactions. To open the Reaction Editor, select the
Reaction Editor tab in the top-left corner of the Fragmentation Library
module.
The Reaction Editor buttons on the module buttons bar can be detached
and placed anywhere in the program. Right-click to access some drawing
actions.
To add a new structure
To add a structure, use one of the following methods:
• Double-click on the drawing canvas.
• Click the Add/Edit Structure button on the button bar.
• Right-click on a blank canvas and choose New Structure.
The Structure Editor appears, where you can draw your fragment. This
Structure Editor is a dialog window, which means you cannot open another
window unless it is first closed. To place your fragment on the canvas, click
OK. The fragment can be moved anywhere on the canvas by dragging the
structure with the mouse.
To edit an existing structure
To add an existing structure, use one of the following methods:
• Double-click a fragment.
• Select the structure and click the Add/Edit Structure button.
• Point the mouse cursor at a structure, right-click and choose Edit
Structure. The Structure Editor appears.
• To confirm your changes, click OK. To reject the changes, click
Cancel.
To add a straight reaction arrow
To add a straight reaction arrow, use one of the following methods:
• Click the Draw Straight Arrow button and then click the cursor where
you want to place the arrow.
• Point the cursor at a structure, right-click, and choose New Arrow.
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Drawing Fragmentation Reactions
In order for the expert system to correctly extract mechanisms, the
structures in a reaction scheme must be properly connected by arrows. The
system considers stand-alone structures and disconnected arrows as errors
and ignores them. If a structure is connected with at least one arrow, the
selection squares are displayed in green. If a structure is not connected, the
selection squares are displayed in yellow. The same color-coding applies to
arrows. If an arrow is connected with a structure, this arrow end is shown in
green. If the arrow end is not connected, it appears in yellow.
To help you properly connect the drawing objects, small red selection
squares appear around the structure or arrow when you move them, if the
object is close enough to connect.
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Fragmentation Library
Active Record
Active Record
Mass Frontier applies the mechanisms of fragmentation reactions to a
structure you provide and automatically predicts fragmentation reactions for
the given compound. However, some mechanisms might be erroneous or
too general and the expert system might apply them even though they are
not desired. To exclude a particular record from the generation of
fragments, click to clear Active Record. See Figure 85.
This option is especially useful for stopping the use of library reactions
consisting of small fragments whose mechanisms can coincidently fit to
virtually any compound. This can cause the generation of an extremely large
number of fragments, which dramatically slows the generation process and
makes reviewing the predicted fragments difficult.
Record activity can be changed in the Data Editor’s Active Record box or in
column A of the record grid. These two boxes are linked and always work
together.
Note Changes to an Active Record box are immediately updated in the
database and do not need to be saved.
To activate or deactivate all the selected records, right-click the records and
choose Activate All Selected Records or Deactivate All Selected Records.
To sort active and inactive records in a library grid, click in the column
header. To restore the ID number ordering, click the ID header tab in the
library grid. It is possible to sort any column by clicking in its grid column
header. To display the reverse order, click again in the header.
By default, the Fragmentation Library window shows all the library records
in a corresponding library grid. If you need to list a subset of library records,
you can hide selected records. Right-click the selected records and select
Hide Selected Records. To restore the complete list of library records, click
the Reset button in the Search tab.
Note Hidden records are not deleted and are fully searchable.
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Fragmentation Library
Active Record
Active Record
Inactive Record
Activity check boxes
Figure 85. Fragmentation Library window showing active and inactive records
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Fragmentation Library
Additional Information
Additional
Information
In addition to a fragmentation scheme, text data can also be maintained in a
particular database record. To enter or edit text data, select the Data Editor
tab in the top-left corner of the Fragmentation Library module. There are
four available fields:
• Record activity
• Source of fragmentation scheme (journal, author, and so on)
• Experimental technique and instrument info
• Comment
Data Editor fields can be edited at any time, even for an existing record. To
keep the changes, the record must be saved. Changes to the Active Record
box are immediately updated in the database and do not need to be saved.
Note The Active Record field (labeled A) in the Data Editor is linked
with the box in column A in the record grid in the lower half of the
window. See Figure 86. Both of these controls always work together.
Figure 86. Data Editor page of the Fragmentation Library module
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Saving Records
Fragmentation Library
Saving Records
To make a change permanent in Fragmentation Library, it must be saved.
To save changes in Reaction Editor or Data Editor
1. Click any in the record grid.
2. Click the line up or line down key.
3. Click the Library button and choose Save Record To Library.
When saving a record, the software performs the following sequential
actions:
1. The fragmentation scheme in Reaction Editor is checked for formal
correctness. If the software detects an error, you are prompted to either
save the scheme as it is, or return to the record to correct the problem.
2. A fragmentation mechanism for every single unimolecular reaction in
the fragmentation scheme is extracted using advanced algorithms. This
process can take several seconds for complex reactions.
3. The reaction scheme, additional text, numerical data, and the extracted
mechanisms are stored in the database.
If the check for formal correctness encounters an error, you are prompted to
either save the record as it is, or to go back and make appropriate changes. If
you save a scheme with just a single error, the reaction mechanisms are not
extracted and the entire record is ignored in the prediction of fragments and
mechanism feature. You can return to an erroneous record at any time to fix
such a problem and enable the scheme for fragmentation prediction.
Reaction symbols appear only if a scheme is correctly saved.
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Fragmentation Library
Mechanism Extraction
Mechanism
Extraction
At the core of the automated prediction of fragmentation and
rearrangement pathways is a computer-based system for the extraction
of unimolecular decomposition mechanisms from reactions you
provide. This complex software system decodes the underlying principle
of fragmentation mechanisms from reaction drawings and builds a
knowledge base of fragmentation events. The system works
automatically and replaces the need for manual input of atom-atom
correspondence in precursor and product ion pairs. A computer learning
procedure has been developed to allow the processing of specific
fragmentation details. Similarly, in experimental mechanistic studies,
labeled or generic structures can be used to direct the desired
dissociation route. By means of deuteria or substituents (R)
participation, the decoding algorithm unambiguously extracts the
underlying mechanism.
Figure 87. Virtual isotope labeling can be used to specify the rearrangement course
Because many reactions stored in a fragmentation library follow general
fragmentation rules that can be predicted by the use of preprogrammed
unimolecular reactions, the library distinguishes between class specific
mechanisms and general fragmentation reactions. After a reaction has been
entered into the database, the system attempts to identify a general
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Fragmentation Library
Mechanism Extraction
fragmentation rule and assigns the relevant reaction symbol above the
arrow. See “Fragmentation, Rearrangement and Resonance Reactions” on
page 110.
In terms of fragmentation prediction, the determination of the preferred
ionization site is as important as detailed knowledge of the fragmentation
mechanism. The system supports virtual generation of charged molecules
based on library ionization reactions using the exact location of the positive
or negative charge, or the unspecified charge location symbol that can be
assigned to a structure drawing.
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Fragmentation Library
Reaction Symbols
Reaction Symbols
Mass Frontier’s expert system automatically extracts fragmentation
mechanisms from a reaction drawing after a scheme has been saved. If a
reaction follows one of the preprogrammed general fragmentation rules, the
arrow is captioned with the particular rule abbreviation. See
“Fragmentation, Rearrangement and Resonance Reactions” on page 110.
The abbreviations for general fragmentation rules are found in the
Options > Reaction Mechanism Overview main menu.
Even if a reaction is formally correct, it might not be possible to derive a
reaction mechanism from a drawing you provide. This can occur if you
enter an unfeasible fragmentation mechanism or because the unimolecular
reaction is incomprehensible to the expert system. If a mechanism cannot be
extracted, the reaction arrow has a cross through it. See Figure 88. In this
case, the mechanism is reduced to the exact precursor and product
structures and only the identical neutral or ionic precursor is matched with
the structure you provided in the fragmentation prediction process.
The software sometimes decodes a mechanism from a drawn reaction but
the atom-matching procedure is unable to find the corresponding atom
counterparts on both sides of the reaction leading to partially recognized
mechanisms. When this occurs, the reaction arrow appears with a small line
through it. This kind of reaction can be used only for fragment prediction
for some input structures according to the fragmentation algorithms
decision. A partially recognized reaction mechanism might not be selected
for fragments prediction, even when the input fragment looks similar to the
precursor in the library reaction.
To overcome such a problem with unrecognized or partially recognized
mechanisms, try to decompose complex one-step mechanisms into a
number of reaction steps and then save these in a fragmentation library.
Note Reaction symbols for an unrecognized or partially recognized
reaction do not appear immediately the reaction is drawn, but only after
the record has been correctly saved in a library.
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Fragmentation Library
Reaction Symbols
Figure 88. Reaction symbols used for partially recognized and unrecognized
mechanisms
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Fragmentation Library
Using Library Reactions in Fragmentation Prediction
Using Library
Reactions in
Fragmentation
Prediction
The primary purpose of library reactions is to create a database of
fragmentation and rearrangement mechanisms that can be applied to
structures you supply to predict the decomposition pathways occurring in a
mass spectrometer. The library mechanisms are a significant extension to
the general fragmentation rules which might not cover all the complex
processes for a broad spectrum of ionization and ion activation techniques.
The advantage of library mechanisms is the flexibility they give to alter
predicted fragmentation pathways and entering highly specific mechanisms
that apply for a limited class of compounds. Because the current library
contains around 19000 reactions, the fragmentation predictability is much
higher than if only general rules apply. This is especially true for
low-energetic experiments such as ESI or APCI that often yield complicated
skeletal rearrangements and unusual ring closures.
Any recognized and active mechanism that has been saved into a library
serves as a knowledge base for the prediction of fragmentation pathways.
After you have drawn and saved a reaction into any fragmentation library,
use the mechanism template for prediction fragmentation pathways for any
structure that the derived mechanism can be applied to. See Figure 89. The
range of applicability depends on many factors, but structurally similar
compounds with a common ring scaffold usually exhibit identical
fragmentation mechanisms.
Note Fragment stability and general ion energetics depend on many
thermodynamic parameters and even a slight structural dissimilarity
between two molecules can result in large differences in the course of
fragmentation pathways. For example, two identical structures with a
simple hydroxy group difference can occasionally exhibit completely
different spectra. Thus, the derived fragmentation analogy based on
library reactions might not always reflect the real fragmentation events
for structural analogues.
If a fragmentation reaction was predicted using a library reaction,
double-click the Lib arrow caption to see the corresponding mechanism.
Both template and generated fragmentation mechanisms are displayed in
red in the Fragmentation Library window and the Fragments &
Mechanisms window.
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Using Library Reactions in Fragmentation Prediction
Figure 89. Fragments & Mechanisms window showing predicted
rearrangement reaction based on mechanism template stored in
Fragmentation Library window
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Fragmentation Library
Search Utilities
Search Utilities
Fragmentation libraries are fully searchable by various criteria. Search
criteria can be combined to narrow your search. All searchable items are
found in the Fragmentation Library window in the Search page. To conduct
a search, type the desired query in relevant search box or select one of the
predefined options and click the Search button. The search results are listed
in the grid box in the lower part of the window.
Note To restore the complete list of library entries in the grid box after a
search has been performed, click the Reset button in the Search page.
Use a substructure search to select all the reactions in a library that match all
the reaction precursor or product structures with your query substructure.
The search results display the query substructure in red in the library
structure, in the same manner as for the Substructure Search procedure in
the Database Manager.
Because fragmentation libraries largely contain ionic structures that can
undergo resonance reactions, Mass Frontier offers a resonance substructure
search. This feature ensures the correct retrieval of all resonance structures,
even if the query structure is in a different resonance form than the library
structure. This feature works fully automatically, so you do not need to be
concerned about the particular resonance state of the ionic structures.
However, be aware of this functionality when reviewing search results, as the
query and library structures in positive search results might appear to be
different if the resonance reaction are possible. In this case, do not consider
this difference as an error, but rather be aware that there might be complex
resonance variations.
It is possible to ignore charges and radicals in substructure searches if their
location is ambiguous. When searching structures with an unspecified
charge location or substituents, be sure to review the search rules that apply.
See “Substructure Search Rules” on page 85.
Note It is possible to search in one library at a time.
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Fragmentation Library
Search Utilities
Figure 90. Search Page of the Fragmentation Library window
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Fragmentation Library
HighChem Fragmentation Library
HighChem
Fragmentation Library
HighChem has been collating fragmentation mechanisms published in all
the available printed media dedicated to mass spectrometry for a number of
years and these have been entered into the computer system. Each reaction,
along with the chemical structures, has been manually drawn in Reaction
Editor and saved in the HighChem Fragmentation Library, which currently
contains around 100000 individual reactions. Fragmentation pathways are
accompanied with complementary information such as the title, authors
and source of the information. This library collection serves as a knowledge
base for the prediction of fragmentation pathways from user provided
structures.
To ensure high-quality data, fragmentation mechanisms have been
evaluated in two stages: manual and automatic. The manual evaluation
included accuracy and plausibility assessments of reaction mechanisms and
consistency checking between fragment masses and peak m/z values, if the
spectrum was available. The automatic evaluation includes simple element,
charge, and radical consistency checks on both sides of the reaction, in
addition to newly developed algorithms for complex electron mapping that
has revealed formally erroneous mechanisms. Numerous problems and
errors regarding mechanisms were uncovered by both stages and either
appropriate corrections were made, or these mechanisms were excluded
from the library.
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Chapter 8
Fragments Comparator
Use the Fragments Comparator module to process and compare fragments
generated in Fragments & Mechanisms windows. This module supports
fragments generated using any ionization method or that originate from
different structures. Using fragment marks in the Fragments & Mechanisms
window, you can export only a selected set of fragments into the Fragments
Comparator module. The fragments are organized in columns where each
column represents a set of fragments provided by an associated Fragments &
Mechanisms window. The Fragments Comparator module can hold an
almost unlimited number of fragment sets, limited only by system resources.
The Fragments Comparator is an integral part of the Fragments &
Mechanisms module. If you double-click any fragment in the Fragments
Comparator module, the associated mechanism appears in the Fragments &
Mechanisms window.
Note The Fragments Comparator is able to recall only mechanisms that
are present in an open Fragments & Mechanisms window. If you close
the associated Fragments & Mechanisms window, the link is lost.
The comparison feature is especially useful when analyzing the
fragmentation products of structurally related compounds. Common
fragments point toward a common substructure in terms of fragmentation.
The most interesting are however the fragment differences. They can
indicate fragments along with corresponding peaks in a spectrum that are
characteristic for the distinction of structural details. Predicted m/z values of
fragments that are different for structurally related compounds will attract
your attention when examining spectral differences in the Database
Manager in the Compare Tab.
The Fragments Comparator window consists of Table and Structures views.
The Table view lists the numerical m/z values of fragments and Structures
view displays structural drawings of possible fragments. Because the
Fragments & Mechanisms module can generate several isobaric isomers for
a single m/z value, the Structures view imports only the first fragment for
each generated m/z value. Fragments are imported into these two views
simultaneously but the information is managed independently. If a column
in one view is moved or deleted, this action does not affect the other view.
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Fragments Comparator
In the Table view, you can select a column or a part of a column, and copy
the data. The m/z values can then be pasted into Excel or any spreadsheet
program.
In the Structure view, the cell can be resized by using the Cell Size track bar.
Columns can be moved in both views. The columns for imported fragments
(left part) can be deleted.
Note The Fragments Comparator can display structures of fragments
only if the associated Fragments & Mechanisms window is still open. If
you close the associated Fragments & Mechanisms window, the
corresponding column of fragments in the Structures tab are removed.
All Fragments in F & M: 1
and F & M: 2 windows
Fragments Common in F & M: 1
Fragments
and F & M: 2 windows
Originating from
Delete Column Fragments &
Different Fragments between
Mechanisms 1
Paste List of Fragments
F & M: 1 and F & M: 2 windows
and 2 windows
Copy Selected Cells
Change
Structure
Cell Size
Figure 91. The Fragments Comparator window showing Structure tab
Both the Table and Structures views in the Fragments Comparator window
are divided into two parts. The left part contains columns of imported
fragments for each Fragments & Mechanisms window. The right part
contains three columns that show three types of comparison results. The
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Fragments Comparator
first result column shows all available fragments (logical OR), the second
column shows all common fragments (AND), and third column shows
different fragments (NAND).
Note All fragments are compared by m/z values using resolution mass
settings. Choose Options > Mass Settings. The fragments are usually
predicted in several isomeric forms, therefore a structural comparison
would not be reasonable. Because the fragments are compared by
m/z values the calculated precision, defined in the mass settings, has a
significant influence on the comparison results.
Figure 92. The Fragments Comparator window
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Chapter 9
Mass Spectra Classification
Computer methods of analyzing mass spectral data center on three
fundamental methodologies: library search techniques, expert system
procedures, and classification methods. Classification is a powerful
enhancement of library search and fragmentation prediction methods. The
computer-oriented methods available in Mass Frontier complement each
other, but are based on different principles. This provides possibilities for
creating alternative strategies and for responsible data interpretation.
In contrast to statistical software packages, with Mass Frontier you can
directly apply classification methods to mass spectral data. All preprocessing
and data transformation procedures are carried out automatically which
ensures the classification methods are used correctly even by those with no
knowledge of multivariate statistics.
This chapter contains the following sections:
• Spectra Classification
• Principal Component Analysis (PCA))
• Neural Networks (Self-Organizing Maps)
• Fuzzy Clustering
• Spectra Transformation
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Mass Spectra Classification
Spectra Classification
Spectra Classification
The primary goal of spectra classification is to find correlations between the
properties of compounds and their mass spectra. Because physical and
chemical properties and biological activities of chemical compounds are, to
a large extent, functions of molecular structure, the results of classification
analysis reflect structural features that are determined by fragmentation ions
appearing in a mass spectrum. The advantage of classification methods is
that detailed knowledge of the complex spectra-structure relationship is not
required to get satisfactory results. Classification strategy in Mass Frontier is
based on a user-friendly, graphic presentation of the results, which you can
view on the screen.
Mass Frontier contains three classification methods: Principal Component
Analysis (PCA), Fuzzy Clustering (FC), and Self-Organizing Maps (SOM),
which are a special class of Neural Networks. These methods are based on
different principles and enable you to explore complex data from various
perspectives. PCA uses multivariate statistics, fuzzy clustering assigns data to
clusters, and SOM is based on competitive learning.
In the multivariate statistic, each spectrum can be considered as a single
point in an n-dimensional space, with the intensities being the coordinates
of this point. A dimension (axis) of that space represents a mass-to-charge
ratio (m/z) of the considered peak. Therefore, the dimensionality is
determined by the m/z value of the last peak in the spectrum. For example,
the EI spectrum of hydrogen exhibits two peaks at m/z =1 (intensity 2%)
and m/z = 2 (100%). This spectrum can be viewed as a point in a
two-dimensional space with the coordinates 2, 100. In reality, spectra have a
higher dimensionality than two. If the dimensionality is too high, or several
coordinates are equal to zero (usually a mass spectrum does not have peaks
at every m/z value), the classification methods might not provide the
required results. Therefore, a reduction of dimensionality is carried out
either before a spectrum is placed in n-dimensional space, or during the
classification process.
The basic hypothesis of multivariate statistical methods is the assumption
that the distance between points (spectra) in an n-dimensional space is
related to a relevant property of the compounds which represent these
points. If the points are close enough to form a cluster or a separated region,
assume that the compounds that correspond to these points exhibit
common or similar properties. To ensure the results of the classification
methods have statistical significance, place a large amount of spectra (usually
one or more groups, each with 10 - 1000 spectra) in the same
n-dimensional space. Then apply multivariate statistical methods, with
various parameters, to evaluate these points (spectra). The objective of a
classification process is to separate these points (spectra) into two or more
classes according to the desired structural or other properties.
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Mass Spectra Classification
Spectra Classification
m/z 3
m/z 2
Mass Spectrum 1
Mass Spectrum 2
m/z 1
Figure 93. Representation of two spectra as points in a 3-dimensional space
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Mass Spectra Classification
Principal Component Analysis (PCA)
Principal Component
Analysis (PCA)
Mass Frontier offers a classification method called principal component
analysis (PCA). The central idea of principal component analysis is to
reduce the dimensionality of a data set in which there are a large number of
interrelated (that is, correlated) variables, while retaining as much as possible
of the variation present in the data set. In the case of mass spectrometry, the
data set consists of the mass spectra of different compounds. The mass
spectra are expressed as the intensities of individual m/z ratios (variables).
Use PCA to find a new coordinate system that can be expressed as the
linear combination of the original variables (mass-to-charge ratios m/z) to
describe major trends in the data. Mathematically, PCA relies upon
eigenvalue/eigenvector decomposition of the covariance or the correlation
matrix of the original variables. PCA decomposes the data matrix X as the
multiplication of two matrices P (the matrix of new coordinates of data
points) and T’ (transposition of the coefficients matrix of the linear
combination of the original variables):
X = P ´ T’
Generally, data can be adequately described using far fewer coordinates, also
called principal components, than original variables. PCA also serves as a
data reduction method and a visualization tool. When the data points are
plotted in the new coordinate system, the relationships and clusters are often
more apparent than when the data points are plotted with the original
coordinates.
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Principal Component Analysis (PCA)
Figure 94. Geometrical interpretation of PCA
With geometrical interpretation of PCA, the axes of the new coordinate
system – principal components p1 and p2 – are created as the linear
combinations of the original axes. New coordinates (principal components)
are orthogonal (perpendicular) to each other. There is greater variation in
the direction of p1 than in either of the original variables, but very little
variation in the direction of p2. The first principal component describes the
direction of the greatest variation in the data set while the second principal
component describes the direction of the second greatest variation (and so
on, for data sets with more than two variables).
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Mass Spectra Classification
Neural Networks (Self-Organizing Maps)
Neural Networks
(Self-Organizing
Maps)
Self-organizing maps (SOM), sometimes called Kohonen networks, are a
special class of neural networks. A self-organizing map consists of neurons
placed at the nodes of a two-dimensional lattice. The neurons become
selectively activated to various input mass spectra or classes of spectra in the
course of a competitive learning process. The neurons compete among
themselves to be activated or excluded. SOM can be considered as a
nonlinear generalization of PCA.
Use self-organizing maps to transform a set of n-dimensional input spectra
into a discrete two-dimensional map, and to display this transformation.
Each input spectrum presented to the network activates a neuron according
to a complex set of interrelationships between spectra. In SOM, each mass
spectrum must always activate a neuron and this spectrum is shown on the
particular neuron. Spectra that activate the same neuron belong, in terms of
classification, to the same pattern. To ensure that the self-organizing process
has a chance to develop properly, expose the networks to a certain number
of different spectra. Therefore, with Mass Frontier, use a minimum of 10
spectra in a self-organizing process.
Figure 95. SOM Architecture used in Mass Frontier. Each spectrum activates a
neuron in the map
Note Different results from the SOM classification method are
produced for an identical data set if the input data is processed in a
different order. This order sensibility is an inherent feature of neural
networks and is not a result of faulty algorithms.
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Fuzzy Clustering
Mass Spectra Classification
Fuzzy Clustering
Cluster analysis is a technique for grouping data into clusters to find
common structural features in spectral data. Membership degrees between
zero and one are used in fuzzy clustering instead of crisp assignments of the
data to clusters. Fuzzy clustering is based on the dot product distance
between the center of clusters and experimental spectral points. The dot
product is calculated from mass spectral n-dimensional space with the
intensities being the coordinates and m/z values dimensions. The
dimensionality of spectral space is determined by the number of peaks
whose intensities are above the predefined threshold. The number of
clusters is usually defined a priori.
Figure 96. Transformation of two-dimensional spectral space into
one-dimensional Fuzzy Clustering model
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Mass Spectra Classification
Spectra Transformation
Spectra
Transformation
Various mathematical transformations of mass spectra can increase
classification efficiency. Better separation of classes can be achieved in some
cases if transformed, instead of original spectra, are submitted to
classification. In addition, some transformation procedures reduce the
number of variables and lower the dimensionality of the spectral space,
which shortens the computing time.
Because the most common neutral loss is 14 (loss of CH2), the logical
spectra transformation is into modulo-14 spectra, which thereafter can be
used as input data for PCA. Modulo-14 spectra are calculated as the sum of
peak heights at mass-to-charge ratio values shifted by 14. Each modulo-14
spectrum has 14 dimensions (transformed mass-to-charge ratio values) that
are significantly lower than regular spectra. Mass Frontier offers modulo-14
transformation with (A1) or without (A2) normalization of such spectra.
Classification of mass spectra assists the interpretation of structurally related
compounds. Because the characteristic peaks in spectra of structurally
related compounds can be shifted due to various substituents, it can be
difficult for classification methods to recognize structural similarity.
Overcome this difficulty by transforming spectral data into auto-correlation
spectra.
The auto-correlation function:
is suitable for detecting periodicity in a series of spectra. In Mass Frontier,
you can choose auto-correlation transformation with (B1) or without (B2)
normalization of mass spectra. Because auto-correlation does not reduce the
space dimensionality and requires computing time to be calculated, a
classification that uses this transformation is the most time-consuming
procedure among the transformation methods.
With Mass Frontier, you can submit original (not transformed) spectra (C1)
to classification as well.
Mass Frontier offers the following spectra transformations:
• Modulo-14 spectra normalized
A1
• Modulo-14 spectra not normalized
A2
• Auto-correlation spectra normalized
B1
• Auto-correlation spectra not normalized B2
• Original spectra (not transformed)
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Mass Spectra Classification
Spectra Transformation
No general rule exists concerning the selection of an appropriate mass
spectrum transformation. Classification methods can be used for a broad
range of problems, and each of them might need a different spectrum
transformation. To reliably find the transformation that provides the best
separation of classes for particular groups of spectra, experiment with all of
them. Subsequently, use the transformation which provides you with the
most information when dealing with comparable data.
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Chapter 10
Spectra Classifier
You can use Mass Frontier to classify spectra according to physical or
chemical properties such as point of origin, toxicity, aromaticity, and so on.
For spectra that cannot be found in a library, classification can involve
identification of substructure types or compound classes (structure
elucidation) in order to establish and confirm structural proposals. Use
classification in cases when only structurally related compounds need to be
retrieved from a complex chromatogram (metabolite research).
Use the Spectra Classifier module to retrieve and organize spectra, which
can then be submitted for Principal Component Analysis (PCA),
Self-Organizing Maps (SOM) and Fuzzy Clustering classification. Spectra
are organized into groups with their own names and graphic
representations. When spectra are organized into groups, it is easier to
distinguish between the classes of spectra that develop from the submitted
groups of spectra after the classification process.
In classification analysis, it is important to distinguish between groups and
classes. It does not make sense to speak about classes prior to the
classification process. Classes can be assigned only after the selected
classification method clearly shows the occurrence of clusters or regions that
consist of spectra with the desired properties. Prior to classification, spectra
are allocated only to groups according to user-defined criteria; that is,
a priori information. Often the objective of the classification process is to
obtain classes that closely resemble groups of spectra submitted to
classification
This chapter contains the following sections:
• Spectra Classifier Window
• Classifying Mass Spectra
• Maintaining Groups of Spectra
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Spectra Classifier
Spectra Classifier Window
Spectra Classifier
Window
The Spectra Classifier window is the gateway to PCA, SOM and Fuzzy
Clustering. Any spectra that you want to classify must first be sent or pasted
to the Spectra Classifier window. A spectrum or spectra is automatically
assigned to a group. Mass Frontier automatically assigns a name to a new
group, which you can rename. The program also assigns a graphic
representation to each group that is prepared for classification. Up to 255
groups of spectra can be added to the Spectra Classifier and each group can
consist of an unlimited number of spectra. Conversely, it is also possible for
a group to contain only a single spectrum. The points are assigned symbol
types and colors according to settings in Classification Layout. These
settings only show group membership and have no influence on the results
of the analysis.
To open Spectra Classifier window
Click the Spectra Classifier
button on the tool bar in the main window
or choose Tools > Spectra Classifier. An empty Spectra Classifier window
opens. See Figure 97.
Only one Spectra Classifier window can be opened at a time. If you click the
Spectra Classifier button or choose Tools > Spectra Classifier, and the
Spectra Classifier is already open, this window becomes active.
To choose spectra for classification
Use one of the following methods to choose spectra for classification:
• Select one or more records in a Database Manager window that you
want to classify and click the Add Selected Records to Spectra
Classifier
button.
• Select one or more scans or components in Chromatogram Processor
window that you want to classify and click the Add Selected Scans or
Components to Spectra Classifier
button.
• Use the Copy (Database Manager, Chromatogram Processor) and Paste
commands to add spectra to the Spectra Classifier module.
There are two panes in the upper half of the Spectra Classifier window. The
left one is a container of groups of spectra that have been sent from a
Database Manager window. You fill this pane successively with the spectra
you have chosen for classification. Each group of spectra is visually
represented as a single line in the left or right pane. The right pane contains
the groups of spectra that are actually classified if you click the Classify
Now button. Move a group from one pane to another by clicking Add or
Remove. With these two panes, you can have spectra available in the left
pane for inclusion in future classification runs.
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Spectra Classifier Window
Group Up
Group Down
Paste Spectra
Show Spectra Source
Show Spectra Source
Classification Layout
A1:
Modulo 14 Spectra
(Normalized)
A2:
Modulo 14 Spectra
B1:
Autocorrelation Spectra
(Normalized)
B2:
Autocorrelation Spectra
C1:
Original Spectra
(Normalized)
Figure 97. Spectra Classifier window
If you select a line (group) in either of the two panes, all corresponding
records are previewed in the grid that displays spectra, structures, and
additional information. If you double-click a line (group) in either of the
two panes, the Database Manager window where the corresponding spectra
originate, opens automatically and these records are selected.
Note Spectra Classifier can hold only spectra that are still present in the
original Database Manager or Chromatogram Processor windows. In
addition, once you have created a group of spectra in Spectra Classifier,
do not move the spectra from the original Database Manager window or
clear the component spectra from a chromatogram in Chromatogram
Processor. If you violate either of these conditions, this group of spectra
is removed from Spectra Classifier.
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Spectra Classifier
Spectra Classifier Window
Spectra Classifier contains five buttons for selecting the transformation to be
applied to the spectra. See Figure 97. Due to the long names of
transformation techniques, only short code names are displayed. However,
if you point the cursor at a code name, the full name appears.
You can choose from three classification methods: Principal Component
Analysis, Neural Networks, and Fuzzy Clustering. If you choose Principal
Component Analysis, you can set the number of principal components that
are calculated. If you choose Neural Networks, the lattice size can be set in
two ways; either you assign the x and y size, or the program sets the optimal
size automatically (recommended method). The Fuzzy Clustering method
has no options.
Once the data has been prepared, start the classification by clicking the
Classify Now button. All groups of spectra listed in the right pane are
classified according to the chosen options (transformation and classification
method). After the classification is completed, the results are displayed
either in a Spectra Projector window or in a Neural Networks window.
While a classification is being processed, you can continue working with the
program.
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10 Spectra Classifier
Classifying Mass Spectra
Classifying Mass
Spectra
The following example clarifies how the classification of mass spectra can be
carried out. Suppose you want to classify the derivatives of nicotine and
caffeine. You have an EI spectrum of an unknown compound that is either a
metabolite of nicotine or caffeine, and you need to find out to which of
these two compound classes it belongs. To complete this task, retrieve
sufficient amount of spectra for each group (nicotines and caffeines) which
are then submitted to a Principal Component Analysis, to form clusters of
each group. Do this by performing a substructure search, where the search
query is the structure of nicotine and caffeine. If sufficient amounts of
spectra (more than 10 spectra) for each group are found, select these records
and add them, separately for each group, to the Spectra Classifier. Then
assign an appropriate name to each group by using the Selected Group of
Spectra box. You can use the copy and paste commands. Then, select a line
in the left pane of Spectra Classifier and click Add. Repeat this for the next
group.
Mass Frontier automatically assigns a graphic representation to each group.
This is shown in the right pane for each line. Change the graphic
representations of groups by choosing Menu > Projection Layout. Note
that the colors and type of graphic representation are only used to
distinguish the groups in the plots and have no influence on the results of
the analysis. See Figure 98.
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Spectra Classifier
Classifying Mass Spectra
Figure 98. Retrieving data for a classification task
When all the groups of spectra you want to classify are in the right window,
click the Classify Now button to launch the classification process. After the
generation is finished, a new Spectra Projector window appears.
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10 Spectra Classifier
Maintaining Groups of Spectra
Maintaining Groups
of Spectra
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With Mass Frontier, you can save and open references to records with the
extension .ref that are present in the Database Manager spreadsheet. Use
this feature to maintain groups of spectra that are sent from the Database
Manager to the Spectra Classifier window. If you have retrieved spectra for
classification, save these records as references, rather then save the spectra as
ASCII files (.jcamp or .msp). In contrast to .jcamp or .msp files, a reference
file stores the locations, where all relevant information about each spectrum
is saved (spectrum, structure, experimental conditions, and so on). In
addition, reference files are easy to manipulate which means you can add or
remove one or more records to them. Deconvoluted spectra from the
Chromatogram Processor cannot be saved as references.
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Chapter 11
Spectra Projector
Use the Spectra Projector module to display the results of Principal
Component Analysis (PCA) and Fuzzy Clustering (FC). In Spectra
Projector, mass spectra are projected as points on a two-dimensional plane
or a three-dimensional twistable space, according to principal components
or cluster combinations you define. Use classification analysis to find classes
of spectra on the projection that exhibit common or similar properties.
With the Spectra Projector, you can place an external spectrum onto a
projection in order to examine its class membership.
Classification can also be applied to spectra that cannot be distributed
a priori into groups. In this case, classify only one group of spectra, and
attempt to find points on the projection that represent compounds with the
desired properties. Use the Spectra Projector to select such points in order to
examine their spectra and structures.
This chapter contains the following sections:
• Generating Spectra Projector Window
• Spectra Projector Window
• 3-D Projection Mode
• Opening and Saving of Classification Results
• Accessing Spectra from Spectra Projector
• Adding An External Spectrum
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Spectra Projector
Generating Spectra Projector Window
Generating Spectra
Projector Window
A Spectra Projector window cannot be directly opened from the program
desktop. It must be generated from the Spectra Classifier module, by
selecting the Principal Component Analysis or Fuzzy Clustering options
and clicking the Classify Now button. See You cannot alter classification
results once they have been generated. If you want to remove a spectrum
(point) from a projection plane, you must remove this spectrum from the
input data, and then launch a new classification process. An unlimited
number of Spectra Projector windows can be opened at any given time in
the program.
Figure 99. Generating Spectra Projector windows from Spectra Classifier using PCA and FC
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11 Spectra Projector
Spectra Projector Window
Spectra Projector
Window
The Spectra Projector displays spectra as points on a two-dimensional plane
or in a three-dimensional space. A combination of two principal
components, which make up the 2-D projection plane, can be selected in
the tab control. If you are viewing PCA results using the 3-D projection
mode, you can select a combination of the three associated principal
components.
For example, if you click the 2-D projection tab captioned 2-5, the spectra
are projected onto the plane of the 2nd and 5th principal components. The
number of tabs is determined by the number of principal components that
have been selected in the Spectra Classifier module. The Spectra Projector
displays a tab for every possible combination of the selected principal
components. For example, if you have generated Spectra Projector with
3 principal components, 3 tabs are available (1-2, 2-3, and 1-3). For
the 5 principal components (which is the default), 10 tabs are available (1-2,
1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5). The same principle applies
to the 3-D projection mode. Note that in the 3-D mode, the number of
combinations of principal components for more than five components is
significantly higher in comparison to 2-D. Therefore, to analyze your
spectra in the 3-D mode, do not use more than five principal components.
Set the number of principal components in the Spectra Classifier window
prior to the PCA calculation.
The Status bar in a Spectra Projector window is divided into three parts. See
Figure 100. The left part informs you which principal components have
been selected. The middle part displays the type of spectra transformation
that has been used for classification. The right part shows how many spectra
have been classified (the total number of spectra from all groups).
Use the Spectra Projector to enlarge any region of the projection plane
independently, for each combination of principal components, by using the
left mouse button. To get the original scale, click the Zoom Out button,
which appears in the top-left corner of the projection plane after any change
of scale. In the 3-D mode, you cannot enlarge the projection.
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Spectra Projector
Spectra Projector Window
Open Spectra Projections Open Spectrum
Save Spectra Projections Paste Spectrum or Tree
Print Projection
3D Projection
Copy Projection
Show Axis
Select Spectra and Show their Origin
Classification
Layout
Show Legend
Figure 100. Spectra Projector window
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3-D Projection Mode
Spectra Projector
3-D Projection Mode
Use Principal Component Analysis and Fuzzy Clustering to reduce the
dimensionality of a spectral space to a level comprehensible to the human
eye. Because a paper or screen plot is inherently 2-D, the method of choice
is usually 2-D Principal Component Analysis or Fuzzy Clustering.
However, if a 3-D projection on a 2-D computer screen is accompanied
with motion, you might perceive this simulation as 3-D space. To increase
the space feeling, the 3-D plots are interactive; that is, the plot can be
rotated with the mouse. Spectra Projector enables interactive 3-D viewing of
your data.
The 3-D projection mode used in Mass Frontier stems from the idea that
spectra classification of complex data set in a 3-D space can be more
effective and reliable than a 2-D plot. This idea is based on the fact that the
human brain recognizes visual patterns in 3-D space efficiently.
When analyzing Principal Component Analysis or Fuzzy Clustering results,
do not rely entirely on 2-D plots. Two or more clusters which overlap in a
2-D plot might be separated in 3-D space, or two seemingly separate
clusters discernible in a 2-D plot might appear to be too close together in
3-D mode to be considered as two different objects.
To view PCA or FC results in 3-D mode
• Click the 3-D Projection
button.
• Left-click anywhere in the classification plot and move the mouse to
rotate the plot interactively. Note that the arrow cursor is replaced by a
circle during the dragging step.
To better appreciate the three-dimensionality of the space, display the x-, y-,
and z- axes by clicking the Show Axes
button, which is next to the
3-D Projection button.
Note It is not possible to enlarge a part of the plot in 3-D projection
mode. However, as in the 2-D mode, the selection of particular spectra
and the display of their origins is possible.
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Spectra Projector
Opening and Saving of Classification Results
Opening and Saving
of Classification
Results
With Mass Frontier, you can save and open projection planes that have been
generated with particular initial settings (transformation method and
number of principal components).
To open spectra projection planes
Do one of the following:
• Click the Open Spectra Projections
Projector window.
button in any Spectra
• Choose File > Open > Spectra Projections.
To save spectra projection planes
Do one of the following:
• Click the Save Spectra Projections
window.
button in any Spectra Projector
• Choose File > Save > Spectra Projections.
Projection planes are saved as graphics, without the possibility of recalling
the original spectra, which are displayed as points. If you need to access the
spectra with structures from projection planes, you must save the references
to records in the Database Manager, as described in Chapter 10. In this
case, the classification must be regenerated from the original data set that
was saved as references. However, it is possible to add an external spectrum.
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Accessing Spectra from Spectra Projector
Accessing Spectra
from Spectra
Projector
It is an important part of classification analysis to know which spectrum is
represented by a point on a projection plane. Because Mass Frontier links
corresponding modules, spectra with structures or chromatographic
components can be recalled from any projection plane.
To access a single spectrum, scan, or deconvoluted component
Double-click a point on the projection. The linked module appears on top
of all other windows, the corresponding record or component is selected,
and the spectrum is shown.
To access several spectra in a region
Do one of the following:
• Select a region with the mouse cursor while holding down the right
mouse button.
• Click the Select Spectra And Show Their Origin
button and then
select a region with the cursor while holding down the left mouse
button.
If the selected spectra originate from the Database Manager, you are
prompted about whether to copy the records with spectra and structures to
the last active Database Manager window or to a new one. If your spectra
originate from a chromatogram (Chromatogram Processor), the
corresponding scans or components appears in the same color as they appear
in the Principal Component Analysis or Fuzzy Clustering plot.
Note Spectra Projector is able to recall only those records that are
present in a Database Manager window. If you close the Database
Manager window that is the source of the input data for classification,
the link between a point and its spectrum is interrupted. The program
automatically warns you if you try to close a Database Manager window
that is linked with one or more Spectra Projector windows. In addition,
to keep the links between points and spectra intact, you must not move
records in, or between, Database Manager windows by using the cut and
paste commands.
If the data originated from Chromatogram Processor, the window must
still be open.
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Spectra Projector
Adding An External Spectrum
Adding An External
Spectrum
Use principal component projections of mass spectra to find classes of
compounds with common or similar properties. Sufficiently separated
classes can then be used to investigate the class membership of an unknown
spectrum. Use the Spectra Projector to add an external spectrum, which was
not used for classification, to the projection plane. If the added spectrum is
clearly projected into a particular class region, assume that this compound
has similar, usually structural, properties.
Figure 101 shows that the two groups are separated into clusters. You can
add the unknown spectrum to the projection plane by pasting or opening its
spectrum into the Spectra Projector. The projection of this external
spectrum clearly shows that it belongs to the nicotine class (squares are PCA
and triangles are FC). This result can be confirmed or rejected by using the
fragmentation pattern of nicotine.
Figure 101. Projection of unknown metabolite into the nicotine class using PCA and Fuzzy Clustering
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Chapter 12
Neural Networks
Use the Neural Networks module to display classification results from
Self-Organizing Maps (SOM), which are a class of neural network.
A self-organizing map is a network of neurons, arranged in the form of a
two-dimensional lattice. The size of a lattice can either be calculated
automatically or user-defined. During classification, neurons become
selectively activated to various input spectra as a result of a competitive
learning process.
Use classification analysis using SOM to find classes of spectra on the map
that exhibit common or similar properties. If one or more spectra activate
the same neuron, assume the spectra belong to a common class. In this case,
the spectra exhibit certain similarities. In addition, spectra that activate
neighboring neurons, and those neurons that have low Euclidian distance
between each other (shown by border line thickness), can also be considered
as related.
In neural networks, each mass spectrum must always activate a neuron and
this spectrum is shown on the particular neuron. Neurons are displayed as
rectangles on the screen. Spectra are represented as symbols or numbers and
are placed onto neurons. Because the spectra are located in discrete objects
the interpretation of SOM is relatively easy, in contrast to PCA and FC
where you do not have to deal with diffuse clusters. However, it might
happen that larger numbers of neurons are activated by a single spectra and
this advantage is lost.
Spectra that activate the same neuron belong, in terms of classification, to
the same pattern. Therefore, all spectra drawn inside a neuron box are equal
for classification purposes and their graphical positions within a neuron are
irrelevant.
Note Different results from the SOM classification method are
produced for an identical data set if the input data is processed in a
different order. This order sensibility is an inherent feature of neural
networks and not a result of faulty algorithms.
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Neural Networks
This chapter contains the following sections:
• Generating Neural Networks Window
• Neural Networks Window
• Working with Neural Networks
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12 Neural Networks
Generating Neural Networks Window
Generating Neural
Networks Window
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Neural Networks are generated from the Spectra Classifier module. As in
the Spectra Projector, the Neural Network module cannot be opened
directly from the program desktop. Once the network has been generated,
classification results cannot be altered. If you want to remove a spectrum
(symbol) from a network, you must remove this spectrum from the input
data, and then launch a new classification process. See Figure 102. An
unlimited number of Neural Networks windows can be opened at any given
time in the program.
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Neural Networks
Generating Neural Networks Window
Figure 102. Generating a Neural Networks window from Spectra Classifier
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12 Neural Networks
Neural Networks Window
Neural Networks
Window
A Neural Networks window displays a topological map of neurons that are
drawn as rectangle. Every neuron has a minimum of two and maximum of
six neighboring neurons. Spectra are displayed as symbols or numbers
within neurons.
The Status bar in a Neural Network window is divided into three parts. The
left part informs you about the lattice dimension. The middle part displays
the type of spectra transformation that has been utilized for classification.
The right part shows how many spectra have been classified (the total
number of spectra from all groups).
Use the Neural Networks module to enlarge any region of the lattice, by
using the left mouse button. To return to the original scale, click the
Zoom Out button, which appears in the top-left corner of the projection
plane after any change of scale.
Open Neural Networks
Save Neural Networks
Print Neural Networks
Copy Neural Networks
Classification Layout
Open External Spectrum
Paste External Spectrum
Show Distance between Neurons
Show Legend
Select Spectra and show their origin
Spectrum
Distance between neurons
Neuron
Neuron activated by
external spectrum
Figure 103. Neural Networks window
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Neural Networks
Neural Networks Window
Although neurons are displayed in a regular 2-D lattice, the actual Euclidian
distances between neurons vary. To show the approximate distances
between neighboring neurons, the borders of the neuron are shown using
different thickness. The line thickness is proportional to the distance
between immediate neighbors. The thinner the line, the closer together are
the neurons.
To show neuron distances
Click the Show Distances between Neurons
button. See Figure 103.
Use information about neuron distances when classifying an unknown
spectrum. The distances show how a neuron that has been activated by an
unknown correlates with neighboring neurons. The distances can also
suggest that spectra from a common might have been activated by multiple
close neurons.
Note Spectra that activate the same neuron belong, in terms of
classification, to the same pattern. Therefore, all spectra drawn inside a
neuron box are equal for classification purposes and their graphical
positions within a neuron are irrelevant.
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12 Neural Networks
Working with Neural Networks
Working with Neural
Networks
With Mass Frontier, you can save and open neural networks that have been
generated with particular initial settings such as transformation method and
lattice dimension.
To open or save neural networks
Do one of the following:
• Click the Open Neural Networks
window.
in any Neural Networks
• Choose File > Open > Neural Networks.
To save neural networks
Do one of the following:
• Click the Save Neural Networks
window.
button in any Neural Networks
• Choose File > Save > Neural Networks.
Neural Networks are saved as graphics, without the possibility of recalling
the original spectra, which are displayed as symbols. If you need to access
the spectra from lattice, you must save the references to records in the
Database Manager, as described in Chapter 10, “Spectra Classifier.” In this
case, the classification must be regenerated from the original data set that
was saved as a reference.
It is an important part of classification analysis to know which spectrum is
represented by a symbol on a lattice. As Mass Frontier links corresponding
modules, spectra with structures or chromatographic components can be
recalled from any neural network.
To access a single spectrum, scan, or deconvoluted component
Double-click a symbol on the lattice. The linked module appears on top of
all other windows, the corresponding record or component is selected, and
the spectrum is shown.
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Neural Networks
Working with Neural Networks
To access several spectra in a region
Do one of the following:
• Select a region with the mouse cursor while holding down the right
mouse button.
• Click the Select Spectra And Show Their Origin
button and then
select a region with the mouse cursor while holding down the left mouse
button.
If the selected spectra originate from the Database Manager, you are
prompted whether to copy the records with spectra and structures to the last
active Database Manager window or to a new one. If your spectra originate
from a chromatogram, the corresponding scans or components appear in
the same color as they appear in the Neural Networks window.
Note The Neural networks module is only able to recall records that are
present in a Database Manager window. If you close the Database
Manager window that is the source of the input data for classification,
the link between a symbol and its spectrum is interrupted. The program
automatically warns you if you try to close a Database Manager window
that is linked with one or more Neural Networks windows. In addition,
to keep the links between symbols and spectra intact, you must not
move records in, or between windows. The Database Manager must still
be open.
If the data originated from the Chromatogram Processor, the window
must still be open.
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Chapter 13
Chromatogram Processor
Use the Chromatogram Processor module for extraction and processing of
mass spectral scans from GC/MS or LC/MS files and from MS data.
Component detection and spectra deconvolution techniques are available.
This module also offers spectral averaging and background subtraction
features. Three types of chromatograms can be displayed: Total Ion
Chromatogram (TIC) of MS and MS/MS, and Selected Ion Chromatogram
(SIC). Use the SIC feature to display individual mass chromatograms of
ions that are characteristic for a specific compound of interest.
Mass spectral scans, deconvoluted components, and various types of MS
data can be classified using Principal Component Analysis (PCA),
Self-Organizing Maps (SOM), or Fuzzy Clustering (FC) methods. Spectra
can be searched in a library for positive compound identification, or the
spectra can be copied to the Database Manager module for further
processing and archiving. Several chromatograms can be opened
simultaneously. Fully customizable chromatogram and mass spectrum
layouts are available. Use the Chromatogram Processor to copy
chromatograms with extracted spectra import into reports, spreadsheets, or
other Windows programs. MS data from Xcalibur is displayed in tree
structures allowing the user to clearly view the dependencies. Using text
boxes, you can annotate chromatographic scans or components. This
module does not include target analysis, and automatic quantitation of ions
is not available.
This chapter contains the following sections:
• Chromatogram Processor Window
• Data File Formats
• Opening Chromatograms
• TIC Page
• Info Page
• Spectra Averaging
• Background Subtraction
• Processing Extracted Spectra
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Chromatogram Processor
• Selected Ion Chromatogram
• Thresholding, Baseline Correction and Smoothing
• Automated Component Detection and Spectra Deconvolution
• Processing Xcalibur MSn Data
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13 Chromatogram Processor
Chromatogram Processor Window
Chromatogram
Processor Window
Apply m/z value
Show Tree View
Text Box
Add Scans or Components to Spectra Classifier
Search Spectrum
Spectrum Layout
Chromatogram Layout
Delete
Copy
Print
Save Selected Scans
or Components
Open Chromatogram
Filters applied to scan
Baseline Correction & Chromatogram Smoothing
Components Detection & Spectra Disconsolation
Selected Ion Chromatogram (SIC)
Set Manual Background Subtraction Scan
Cancel Manual Background Subtraction
Scans Average
Clear Chromatogram
Select Scans or
Components
Edit Components
Show Scan Points
Next
Scan
Previous
Scan
Selected
Component
Active parallel spectrum for selected component tree
Next parallel spectrum for selected component tree
Parallel spectra for selected component tree
Selected component tree
Previous parallel spectrum for selected component tree
Figure 104. Chromatogram Processor window
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Chromatogram Processor
Data File Formats
Data File Formats
Mass Frontier supports the following data file formats for importing
GC/MS and LC/MS files:
• Xcalibur .raw files (MS and MSn)
• Finnigan LCQ, GCQ, ITS40, and Magnum
• Varian Saturn
• ChemStation .hp
• netCDF
• DOS, Windows, and UNIX .jcamp
These files can be imported to the Chromatogram Processor but cannot be
exported. Single scans can be saved in .jcamp or .mps format.
Mass Frontier supports centroid-type data for mass spectra. Centroid mass
spectra are displayed as a bar graph. Profile-type data is not supported.
Due to various netCDF standards implementation, some .cdf files might
not be readable in Mass Frontier.
Note Features connected with chromatographic spectral trees are
supported for Xcalibur .raw files only (Data Dependent files).
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Opening
Chromatograms
Chromatogram Processor
Opening Chromatograms
To load a GC/MS or LC/MS file to a Chromatogram Processor window
1. Click the Chromatogram Processor
choose File > Open > GC/LC/MS.
button on the speed bar or
2. After an Open GC/LC/MS File window appears, select the appropriate
file type and the extension in the Files of type box.
3. Choose the file to open and click the Open button.
With Mass Frontier, you can open chromatograms with up to 30000 scans.
If you open a file that contains more than 30000 scans, the chromatogram
is displayed up to the retention time corresponding to scan number 30000.
The rest are ignored and are displayed. The program does not allow the
opening of GC/MS or LC/MS files from a CD-ROM. If you have files on a
CD-ROM, copy these files to the hard drive to make them accessible for
importing. In addition, disable the Read Only file attribute.
To change the file attribute
1. Select all the files whose attributes to change in Windows Explorer.
2. Right-click the selected files and a pop-up menu appears.
3. Select Properties in the pop-up menu.
4. Check the Read Only attribute box that is located at the bottom of the
Properties dialog window.
Mass Frontier comes with GC/MS and Xcalibur MS demonstration files
which are located in the /Chromatograms directory.
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Chromatogram Processor
TIC Page
TIC Page
Total Ion Current (TIC) chromatograms are displayed in the TIC tab in the
Chromatogram Processor. Both the retention time and the intensity axis can
be zoomed by holding down the left mouse button and dragging a rectangle
around the region you want to rescale. To unzoom the TIC, click the
Zoom Out button in the top-right corner.
To select a scan from the TIC, click anywhere in the chromatogram pane
and the scan corresponding to the selected retention time appears in the
Scan tab. The active scan is indicated by a vertical line, which is purple by
default. To move the scan point to the next or previous scan, click the arrow
button of the direction you want to move the scan point. Retention times
(tR) and scan numbers of active scans are displayed in the status bar.
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Info Page
Chromatogram Processor
Info Page
The Info tab in the Chromatogram Processor window shows additional
information saved in the data file. Each GC/MS or LC/MS file contains a
different list of items describing sample, instrument, experimental
conditions and other information. See Figure 105. Mass Frontier extracts all
additional information from the file and puts them into the Info tab.
Figure 105. Info page of the Chromatogram Processor window
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Chromatogram Processor
Spectra Averaging
Spectra Averaging
With Mass Frontier, you can extract the average mass spectrum from several
scans. Click the Spectra Average
button and mark off the region of the
scans to be averaged by dragging a rectangle around the region. The marked
region is displayed in the same color as the active scan line (the default is
purple). The average spectrum is displayed in the Scan tab. See Figure 106.
To cancel the spectra average mode, click anywhere in the chromatogram
pane and the single scan mode is restored.
Figure 106. TIC page showing a selected region of several scans to be averaged
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Background
Subtraction
Chromatogram Processor
Background Subtraction
To eliminate the background signal from an active scan or from the average
of spectral scans, perform a manual background subtraction. Set the
location of two representative background scans by clicking the Manual
Background Subtraction
button and then left-clicking the scan point
in the chromatogram pane. The background scans are indicated by a vertical
line, which is depicted in a different color (the default is green) from the
scan line. The resulting spectrum is displayed in the Scan tab. To cancel a
background subtraction, click the Cancel Background Subtraction
button. Use the Selected Ion Chromatogram feature to choose the two most
representative background scans. Such background scans can be used in the
Manual background subtraction option in the Automated Components
Detection and Spectra Deconvolution procedures.
To see the effects of the background subtraction on a scan, copy both the
extracted and original spectra to the Database Manager and compare them
by using the spectra comparison routine in the Compare Spectra tab.
Background subtraction can be used in conjunction with the Selected Ion
Chromatogram feature. In this case, the Selected Ion Chromatogram
profiles are extracted from background subtracted scans.
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Processing Extracted Spectra
Processing Extracted
Spectra
A spectrum obtained from the average of scans, from scans with a subtracted
background or from component deconvolution can be further processed in
the Database Manager, the Spectra Classifier, or can be directly searched for
in a library from the Chromatogram Processor. To transfer the extracted
spectrum to the Database Manager or Spectra Classifier, use the Copy
button. After clicking the Copy button in the Chromatogram Processor
window, paste the mass spectrum to a Database Manager or Spectra
Classifier window. In addition, when you click the Copy button in the
Chromatogram Processor, the chromatogram graphic and the spectrum
from the Scan tab are copied to the Windows Clipboard and can be used for
creating reports in any Windows application.
The Chromatogram Processor window contains a Search button for the
direct searching of spectrum from the Scan tab in libraries. To search the
extracted spectrum in libraries, click the Search button in the
Chromatogram Processor window and the Spectra Search dialog window
appears.
Mass Spectra obtained in Chromatogram Processor can be classified by
using Principal Component Analysis and Neural Networks.
To choose scans or deconvoluted spectra for classification
Select scans or components that you want to classify and click the Add
Selected Scans or Components to Spectra Classifier
button.
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Selected Ion
Chromatogram
Chromatogram Processor
Selected Ion Chromatogram
With Mass Frontier, you can display a chromatogram for a selected ion in a
different color. In Xcalibur, the selected ion chromatogram (SIC) is
sometimes called an individual or single ion chromatogram, ion profile, or
selected ion monitoring (SIM). The program can display up to three
selected ion chromatograms per window.
To display an SIC for a particular mass-to-charge ratio
Do one of the following:
• Click on the spectral peak of interest on the Scan page.
• Select the Data tab and click the m/z value in the mass-to-charge ratio
table.
• Click the Selected Ion Chromatogram
button.
In either case, a dialog window appears where you can change or add the
mass-to-charge ratio and confirm your choice. Use this dialog window to
select a color for a particular m/z value.
Mass Frontier extracts the SIC from the original file. This process can be
time-consuming for chromatograms with a large number of scans. However,
because Mass Frontier is a multithreading application, you can still use
other windows during the SIC extraction.
Use a selected ion chromatogram to verify automated component detection
and spectra deconvolution results. An SIC helps you to quickly recognize
mixture components in a peak region. Examine an SIC of model peaks for
any component you want to use in further analysis. Remember that some
structural (alkanes) or optical isomers produce almost identical mass spectra
and even if you can clearly see two or more maxima in a peak region, an SIC
might not reveal a multicomponent profile.
Use an SIC to determine whether the composition of the background
changes over the course of a run. To view the background profile, extract
the SIC of a base peak, or prominent peak, from a scan which is clearly in a
non-peak region. If the SIC of a background peak has a variable profile
around the peak you are focusing on, choose two scans with different SIC
profiles for background subtraction.
Background subtraction can be used in conjunction with the SIC option. In
this case, the SIC profiles are extracted from background subtracted scans.
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Selected Ion Chromatogram
Note SIC peaks can appear larger on the screen than the corresponding
TIC peaks do. In reality, SIC signals are disproportionally lower than
TIC signals, especially if the data was not acquired using the selective ion
monitoring mode. See Figure 107. It would be impractical to examine
minute SIC peaks displayed together with TIC on the screen. Therefore,
SIC data are normalized to the maximum of the TIC signal and visually
enlarged for display purposes.
Figure 107. Chromatogram Processor, showing selected ion chromatograms of
ions with m/z 240, 228, and 227
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Thresholding, Baseline Correction and Smoothing
Thresholding,
Baseline Correction
and Smoothing
The interfacing of gas and liquid chromatography with mass spectrometry
often produces a signal that is not associated with information of interest.
Mass Frontier provides tools for GC/MS and LC/MS data processing to
improve the useful signal using a variety of advanced algorithms. The
following types of data processing techniques are used in Mass Frontier:
• Thresholding
• Baseline Correction and Noise Elimination
• Smoothing
The main purpose of chromatographic data processing is to prepare data to
be valid for the automated detection and deconvolution of chromatographic
components. There are two different ways of combining data processing
and component detection. You can preprocess data in advance by using a
variety of methods for every processing type separately with number of
customizable options and then begin component detection. You can also
perform data processing at the same time as component detection by using
predefined methods and options optimized for common chromatograms in
the same window used for component detection.
Thresholding
Thresholding is a data processing method that analyzes every scan to reduce
ion intensities or delete spectral peaks if algorithmic criteria have been met.
The main purpose of thresholding is to eliminate noise or impurity (column
bleeding) peaks or peaks originating from minor components that are not of
interest.
To apply thresholding to your data, click the Chromatogram Processing
button and choose Thresholding. The dialog window appears with the
following parameters:
• Apply Threshold to Top Stage Only
If checked, only MS1 (full scan) scans are processed.
• Minimal Remaining Peak Count
If checked, the number of the most intense peaks specified in the
Minimal box are affected by thresholding.
• Algorithmic
If checked, one of the following algorithms is used: Linear Fit,
Histogram or Median.
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• Apply Threshold to Spectra with Peak Count Higher than
If checked, algorithmic thresholding is applied to spectra with a number
of peaks higher than the specified value.
• Maximal Allowed Threshold
If checked, the algorithmic threshold is applied to spectral peaks with an
intensity lower than the specified value.
• Manual
If checked, all peaks that exhibit an intensity lower than the value given
in the Threshold box are deleted.
Baseline Correction and
Noise Elimination
Baseline correction and noise elimination algorithms analyze ion profiles
(selected ion chromatograms) of all ions appearing in spectra over the entire
region of a chromatogram. In contrast to thresholding, where the individual
scans and their spectral peaks are independently analyzed and modified,
baseline correction and noise elimination analyze and modify spectral peaks
in conjunction with identical peaks in a given retention time range.
To apply baseline correction and noise elimination to your data, click the
Chromatogram Processing button and choose Baseline. The dialog
window appears with the following parameters:
• Process Top Stages Only
If checked, spectra in MS1 stage (full scan) are processed.
• Segmentation
If checked, the chromatogram is divided into discrete parts to which the
algorithms are applied separately. This option can provide better results
if a chromatogram exhibits diverse shape, peak density and baseline
characteristics over the retention time scale. Avoid segmentation if a
Loess derivative filter is used.
• Use Baseline Correction
If checked, one of the following methods is applied to your
chromatogram: Top-Hat filter, Savitzky-Golay derivative filter, or Loess
derivative filter.
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• Use Noise Elimination
If checked, apply the following methods individually or simultaneously:
Counter filter to reduce chemical noise, and Quantile filter to reduce
electronic noise.
Smoothing
Smoothing is a process by which ion profiles (not total ion chromatogram)
for every ion (m/z value) found in the data file are averaged with their
neighbors in a time series. Smoothing can increase correct component
detection and can eliminate spikes that cause false positive results.
To apply smoothing to your data, click the Chromatogram Processing
button and choose Smoothing. The dialog window appears with the
following parameters:
• Smooth Top Stages only
If checked, smoothing is applied only to MS1 stage (full scan).
• Segmentation
If checked, the chromatogram is divided into discrete parts to which the
algorithms are applied separately. This option can provide better results
if the chromatogram exhibits diverse shape, peak density and baseline
characteristic retention time scale.
• Method
This box includes the Savitzky-Golay, Median and Loess smoothing
methods. In contrast to Median and Loess methods, the Savitzky-Golay
method requires time-equidistant scans.
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Automated Component Detection and Spectra Deconvolution
Automated
Component Detection
and Spectra
Deconvolution
Mass Frontier incorporates an advanced automated system for detecting
chromatographic components in complex GC/MS or LC/MS runs and
extracting mass spectral signals from closely coeluting components
(deconvolution). Individual mass spectra or spectral trees obtained after
deconvolution can be searched in libraries or classified using Principal
Component Analysis and Neural Networks.
Mass Frontier identifies components using the following algorithms:
• Rapid Component Detection (RCD) Algorithm
• Joint Component Detection (JCD) Algorithm
• Total Extraction Component Detection (TECD) Algorithm
• Direct Infusion Algorithm
These algorithms involve the combined use of the following procedures:
1. Noise examination and signal filtering.
2. Baseline definition and demarcation of chromatographic peaks.
3. Background scan determination and background subtraction.
4. Component candidate detection and model ion selection (m/z).
5. Component candidate confirmation or rejection.
6. Spike elimination
7. Calculation of exact component retention time.
8. Spectra deconvolution using linear algebra.
The Components Detection and Spectra Deconvolution system works
automatically. Use the system for broad types of chromatographic runs, for
both GC/MS and LC/MC analyses, for clean and noisy signals, and for
simple and more complex chromatograms. However, some parameter
changes might be needed to optimize the system for specific applications.
Use this automated procedure for small and medium size organic
compounds and not for the processing of proteins, peptides,
oligonucleotides, or other biomolecules.
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Figure 108. Deconvoluted data dependent chromatogram, showing spectral
Detected components are marked with a triangle in the original
chromatogram. As the program calculates the precise retention time (tR) of
each component, the component triangle might appear between scan
points.
Note To display a deconvoluted spectrum of a component rather than
of a scan, click the triangle that marks the detected component in the
chromatogram pane. To display an original scan at the position of a
detected component, click above the triangle.
If you select a component by clicking its triangle, a deconvoluted spectrum
appears in the spectrum pane. This spectrum can be processed in the same
way as any spectra in the program, for example, searched in a library or
copied to the Database Manager.
If you move the mouse cursor over a component triangle, a tool tip informs
you about the component number, precise retention time and the model
ion m/z value that were used in the automated detection and deconvolution
processes.
To copy or process more than one detected component in the Database
Manager or in Spectra Classifier modules, you must first select them.
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To select one or more components
Click the Select Scans or Components
button and select the desired
components with the mouse cursor while holding down the left mouse
button.
To select all detected components in a run
1. Right-click anywhere in the chromatogram pane. A pop-up menu
appears.
2. Click Select All.
The program makes a strict distinction between original scans and detected
components and does not mix them. When selecting a chromatographic
region, components are preferred to scans. If you select a chromatographic
region that contains components, only these components are selected. If you
select a region that does not contain any components, all scans in that
region are selected.
Figure 109. Chromatogram Processor window, showing items connected with
selected component generated from LC/MS/MS data
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RCD Algorithm
The Rapid Component Detection (RCD) algorithm is based on a “model
ion” that represents a characteristic ion (m/z value in MS1 spectra) for every
detected component. The algorithm starts with component candidate
detection and model ion selection and continues with the correlation of
model ion profiles to confirm or reject a candidate.
To start automated component detection and the spectra deconvolution
procedure using RCD algorithm
1. Click the Components Detection and Spectra Deconvolution button
and choose RCD.
2. When the parameters setup dialog window appears, change the settings
as needed, and then click Calculate.
The RCD algorithm dialog window contains various parameters that can be
optimized for specific types of analysis. Note that these parameters are
interdependent, so a change of one parameter can also affect algorithms
linked with other parameters.
• Threshold of Total Signal
The program uses a different threshold level from the one given in the
data file. In especially noisy chromatograms, setting the threshold higher
can reduce the number of false positive results. However, if the
algorithm is too restrictive and is missing some chromatographic peaks,
lower the default value.
• Minimum Model Ion Abundance
The algorithms search for spectral peaks that have the most rapid rise
and fall of signal in a peak region. This peak is called a model ion. To
eliminate random fluctuations, a model ion must exhibit a minimum
abundance value.
• Smoothing
Use this option when noisy data needs to be analyzed. Mass Frontier
automatically determines the smoothing factor according to the
signal-to-noise ratio. You can change this value or switch off smoothing.
The program uses an exponential filter similar to the analogue RC filter.
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• Spectra Difference Factor
To eliminate false positive component detection, the adjacent
components must show some degree of spectral dissimilarity that is
represented as the match factor used in library searching. The spectra
Difference Factor value is the minimal match factor between spectra
that detected components might exhibit.
• Background Subtraction
If you choose the Automatic option, the program attempts to find a
region of relatively constant signal intensity before and after every peak
and sets two background scans there. If you want to use the Manual
option, set the background scans by using the Set Background
Subtraction Scan button before starting the detection and
deconvolution process. Two manual background scans can be set
anywhere in the chromatogram. If you choose None, background
subtraction is not performed.
• Precursor Ion Subtraction
If a product ion chromatogram is being deconvoluted, the selected
precursor ion can be subtracted from all scans to improve component
detection. However, if a component does not fragment and only a
precursor ion can be observed, do not apply subtraction because this
component might be overlooked. Note that Mass Frontier does not
support scan events that are often used in connection with product ion
scanning.
• Retention Time Range
Detection and deconvolution calculations are time-consuming
processes. The computing time needed depends on a number of factors,
the most significant of which are the number of scans and the number
of mixture peaks. To speed up your work, select only a part of a
chromatogram to be analyzed. Other regions are ignored.
• Spectra Deconvolution
Two extraction algorithms can be applied depending on the intended
use of the component spectra. If components are intended for a library
search, use Sharp spectra deconvolution. If the purpose of component
detection is target analysis, use Soft deconvolution. Generally, Sharp
deconvolution subtracts peaks from coeluting components with a higher
multiplication factor and thus produces spectra with fewer peaks and
lower intensities of isobaric peaks than Soft deconvolution.
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JCD Algorithm
The Joint Component Detection (JCD) algorithm is based on the statistical
analysis of all ion profile maxima. Ion profiles (ion chromatograms) with
comparable shapes and maxima belonging to a limited time range are
considered as a single component. The algorithm extracts individual mass
spectral peak abundance profiles to produce a purified spectrum or spectral
trees and generates the peak shape of a representative component. The JCD
algorithm is recommended, but this requires significant computer
processing resources.
To start automated component detection and the spectra deconvolution
procedure using the JCD algorithm
1. Click the Components Detection and Spectra Deconvolution button
and choose JCD.
2. When the parameters setup dialog window appears, change the settings
as needed, and then click Calculate.
The RCD algorithm dialog window contains the following parameters that
can be optimized for specific types of analysis. Note that these parameters
are interdependent, so a change of one parameter might also affect
algorithms linked with other parameters.
• Mass Merge Power
Specifies the mass difference within which the algorithm merges spectral
peaks into one m/z value. A low value might result in more components
(oscillating ions) and a high value can result in fewer components
(merging ions).
• Average Peak Width
Specifies the chromatographic peak width in scans that the algorithm
uses to identify a potential component. If a value is too high, this can
result in the loss of narrow peaks. If a value is too low, it might split a
real component into two different components.
• Baseline Correction
If checked, an automated baseline correction for each ion profile is
applied using the Top-Hat algorithm. Use this option only for
chromatograms with an elevated baseline.
• Smoothing
If checked, the smoothing of each ion profile is performed using the
Average Peak Width value and the Loess algorithm
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• Noise Modification
Determines how to adjust the intensity values of spectral peaks where
abundances are comparable to noise level. Choose one of the following
methods:
− None
Spectral peak intensities are not altered
− Elimination
Spectral peaks with intensities lower than the specified value are
eliminated. Use this method if low abundant peaks are not of
interest.
− Transition (default)
Artificial noise is added to replace random spikes with constant
noise for better detection of low abundant components.
• Analyze MS Stages
Determines which MS stages are considered in the analysis of ion
profiles and for detecting components. The remaining stages are used
only for spectral tree reconstruction. The choices are as follows:
− Top Stages (default)
Analyzes only the top-stage ions present in the data, where top
means MS1, or MS2 if MS1 is not present. The algorithm builds the
component tree by joining the corresponding lower stage spectra
that meet the following criteria: they occur within the component
envelope and the software detected the precursor m/z in the top
stage as a component ion. (If the deconvoluted MS1 spectrum
contains peaks that have been further isolated, the corresponding
MS2 spectra are assigned to the spectral tree)
− Lower Stages
Analyzes all ions except those from the top MS stages. The
algorithm calculates the spectrum of the top stage from the total ion
abundance of the top stage. The resulting spectral trees do not have
as much depth as do those from the Top Stages option.
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− All Stages
Analyzes all ions regardless of the MS stage. The resulting spectral
trees do not have as much depth as do those from the Top Stages
option.
Use the Top Stages level in most cases. Use the Lower Stages level if a
Data Dependent experiment is set in such a way that ions are isolated
according to a predefined list of m/z values and top-stage spectra are
noisy. Use the All Stages level for the preliminary analysis of complex
data.
• Eliminate non-model ion stages
If checked, the product ion spectrum along with subsequent stages is
deleted if its precursor ion is not a model ion (usually the most intense)
in the top stage.
• Retention Time Range
Enables you to specify only a part of a chromatogram to be analyzed
(time range), which can speed up your work. In this case, the algorithm
ignores regions outside the specified range.
• m/z Range
Enables you to specify only a part of a chromatogram to be analyzed
(m/z range), which can speed up your work. In this case, the algorithm
ignores regions outside of the specified range.
• Baseline Threshold
Specifies the peak baseline as a percentage of base peak height.
• Merging Factor
Specifies a limit for the time difference of the ion profiles maxima. Too
high a value can cause the merging of randomly coeluting components.
Too low a value can split a component into more false-positive
components.
• Sharpness Tolerance
Specifies the degree (percent) of similarity of ion profile shapes. If two
ion shapes meet the specified percentage for the sharpness tolerance, as
well as other parameters, the algorithm merges the ions into a single
component.
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• Wide Component Merge Mode
Specifies whether a limit for the time difference of the ion profile
maxima is used in a combination of the following parameters: Average
Peak Width and Merging Factor. Select this box (default) to avoid
splitting a component into ion peaks that are detected as redundant
components in chromatograms with wide peaks. Clear this box if an
incorrect component merge occurs.
TECD Algorithm
The Total Extraction Component Detection (TECD) algorithm creates
spectral trees for every section of a chromatogram that starts with an MS1
scan. Each generated tree is then divided into subtrees based on the value of
the Minimal Tree Depth parameter. Generated subtrees are merged into
components based on the precursor ion m/z value identity and the spectral
tree match factor similarity.
To start automated component detection and the spectra deconvolution
procedure using the TECD algorithm
1. Click the Components Detection and Spectra Deconvolution button
and choose TECD.
2. When the parameters setup dialog window appears, change the settings
as needed, and then click Calculate.
The TECD algorithm dialog window contains the following parameters
that can be optimized for specific types of analysis. Note that these
parameters are interdependent, so a change of one parameter can also affect
algorithms linked with other parameters.
• Minimal Tree Depth
Specifies the minimum number of tree sections the algorithm creates
from the initial spectral tree. The value determines the MS stage where a
division takes place.
• Tree Match Factor
Specifies the minimum percentage that two spectral trees within
adjacent tree sections must match before the algorithm considers the
two spectral trees as the same component. Matching spectral trees are
defined as having identical precursors up to the level specified by the
Minimal Tree Depth value and a Tree Match Factor value that exceeds
the specified value.
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• Wide Component Merge Mode
Enables a comparison of the spectral trees for potential matching and
merging, not only in adjacent sections, but also in sections up to the
distance specified by the Allowed Gap value.
• Allowed Gap
Specifies the maximum distance between nonadjacent tree sections over
which to compare the spectral trees for potential merging.
• Retention Time Range
Enables you to specify only a part of a chromatogram to be analyzed
(time range), which can speed up your work. In this case, the algorithm
ignores regions outside of the specified range.
• m/z Range
Enables you to specify only a part of a chromatogram to be analyzed
(m/z range), which can speed up your work. In this case, the algorithm
ignores regions outside of the specified range.
Direct Infusion Algorithm
The Direct Infusion algorithm is a spectral tree construction utility rather
than a component detection method. It creates one or more spectral trees
from a single raw file by reading of various MSn scans acquired in one run
and constructing a tree according to their MS stage and precursor ion m/z
values.
To start the spectral tree construction utility using the Direct Infusion
algorithm
1. Click the Components Detection and Spectra Deconvolution button
and choose Direct Infusion.
2. When the parameters setup dialog window appears, change the settings
as needed, and then click Calculate.
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The Direct Infusion algorithm dialog window contains various parameters
that can be optimized for specific types of analysis. Note that these
parameters are interdependent, so a change of one parameter can also affect
algorithms linked with other parameters.
• Minimal Tree Depth
Specifies the MS stage that has only a single precursor. The value
determines the MS stage where a tree branch division takes place. If the
default value 1 is used, the algorithm creates a single tree from all the
scans in a run (file). If the value 2 is used and the analyzed run contains
MS2 spectra with different precursor m/z values, the algorithm creates
separate trees for each MS2 scan with a unique precursor ion m/z value.
• Include Upper Spectra
If checked, the resulting spectral trees contain actual scans in the stage
above the level set in Minimal Tree Depth. If unchecked, the spectra
above the stage set in Minimal Tree Depth. contain spectra with a single
peak equal to the precursor ion of the product spectra.
• Average Scan
If checked, every tree node contains only average spectra.
• Calculate Envelope
If checked, the ion profile (envelope) for each tree is calculated. Use to
preview scans that belongs to a particular tree.
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n
Processing Xcalibur MS Data
Processing Xcalibur
MSn Data
A spectrum obtained from the average of scans, from scans with a subtracted
background or from component deconvolution can be further processed in
the Database Manager, Spectra Classifier, or can be directly searched for in a
library from Chromatogram Processor. To transfer the extracted spectrum
or tree to the Database Manager or Spectra Classifier, use the Copy button.
After clicking the Copy button in the Chromatogram Processor window,
paste the mass spectrum to a Database Manager or Spectra Classifier
window. In addition, when you click the Copy button in the
Chromatogram Processor, the chromatogram graphic and the spectrum
from the Scan tab are copied to the Windows Clipboard and can be used for
creating reports in any Windows application.
The Chromatogram Processor window contains a Search button to directly
search spectrum from the Scan tab in libraries. To search the extracted
spectrum in libraries, click the Search button in the Chromatogram
Processor window and the Spectra Search dialog window appears.
Classify mass spectra or spectral trees obtained in the Chromatogram
Processor by using the Principal Component Analysis, Neural Networks,
and Fuzzy Clustering methods.
To choose scans or deconvoluted spectra for classification
Select scans or components that you want to classify and click the
Add Selected Scans or Components to Spectra Classifier
button.
Use the Chromatogram Processor to view and process Xcalibur Data
Dependent experiments and product ion scanning .raw files and extract
spectral MSn trees from chromatograms. If Xcalibur data is opened in
Chromatogram Processor, a tree view control appears on the right side of
the window. Use this tree view control to select one or more product scans
at any MSn stage. If deconvoluted components are present, they are listed at
the bottom of the tree control.
If component detection and spectra deconvolution procedures are applied
to data-dependent chromatograms, the program generates components as
spectral trees. The tree components can be processed in the same way as
regular spectra. They can be edited in Components Editor, searched in
spectral libraries or classified by using the Spectra Classifier. Spectral trees in
the Chromatogram Processor can only be viewed. To edit them, copy and
paste them into the Database Manager, where all editing utilities are
available.
Note that the spectrum in the bottom right of the Chromatogram Processor
window is displayed for the selected tree node spectrum.
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Figure 110. Chromatogram Processor window showing selection cascade for
deconvoluted spectral tree
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Chapter 14
Components Editor
Use the Components Editor to edit, search and organize chromatographic
components stored in a library or generated by the Components Detection
and Spectra Deconvolution features. This module has a complete set of
management tools to delete unrelated components, add chemical structures,
edit extensive data fields, process spectral trees, annotate spectral peaks, and
sort search match lists for every component in a processed chromatogram.
The Components Editor modal window is accessible from the Database
Manager or Chromatogram Processor window. The modal window is a
program module that pops up over an applications frame window. When
the modal window is present, no other application window can be used. To
close this module, click either the OK or Cancel button.
The Components Editor closely resembles visually the Database Manager
module; however, the processing item is a chromatographic component
rather than a database record. To make the modules easy to distinguish, the
Components Editor has a light blue bar on the left site of the window. Both
modules handle almost identically. See Chapter 4, “Database Manager,” for
an explanation of module functions.
Each chromatographic component is represented by a single row. The
columns contain supplementary component information. One of the
columns lists Model Ion values that have been used for component
detection. These values help you to orient yourself and find components of
interest. In most cases, the model ion is the base peak in the full scan
spectrum; however, if closely coeluting components have isobaric base
peaks, the algorithms select different model ions to distinguish the
components.
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Components Editor
Figure 111. Components Editor window
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Components Editor
Searching Components
Searching
Components
Use the Components Editor to search one selected subset or all the
chromatographic components in the libraries at once. To search selected
components, select components in the spreadsheet and click the Search
button in the tool bar and then choose the Search Selected Components
item from the pop-up menu. To search all the components from the
processed chromatogram, click the Search button and choose
Search All Components.
Note Searching a large number of components might take a
considerable amount of time depending on the library size.
After the search is completed, the highest match factor for each component
search is listed in the Match column in Spreadsheet. If the search procedure
did not lead to any plausible matches, the match factor is not displayed. If at
least one match was found, the text in the name filed of the component row
appears in red.
To process the match list of a component, select the component row and
open the Hit Selector window by clicking the Hit Selector
button. In
the Hit Selector, you can review the match list and accept a library record
that correspond to the component by selecting the match and clicking OK.
If you accept a library record for a component, all relevant information
(structure, name, molecular mass, ion types and so on) is adopted and
entered in the component fields. If you decide to reject the match list, click
Cancel. You can undo an accepted match by clicking the Reject Library
Hit
button that appears next to the Hit Selector button.
Note The Hit Selector window lists the best matches found during the
library search. The match (hit) factor describes the exactness of the
match to your component.
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Components Editor
Searching Components
Figure 112. Components Editor and Hit Selector windows showing assignment
of a library search match to a chromatographic component
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Chapter 15
Mass Settings
All calculated masses displayed in Mass Frontier are monoisotopic masses.
The monoisotopic mass of an ion is the mass of the isotopic peak which is
composed of the most abundant isotopes of its elements. Because the
program’s calculations support only single-charged ions (z = 1), the
calculated—not measured—exact masses in structure-based modules are
equal to their m/z value.
This chapter contains the following sections:
• Resolution
• Precision
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Mass Settings
Resolution
Resolution
Resolution settings are used for the differentiation of adjacent peaks
(spectra) and m/z values (calculations). Because Mass Frontier processes only
centroid spectra, the differentiation is defined here as the spacing between
resolved peaks (spectra) or as the smallest difference in m/z values that is
distinguished (calculations). No additional parameters, such as peak width,
are taken into account.
Mass frontier supports the following resolution types:
• Unit Resolution
• Resolving power M/ΔM
• Mass Resolution ΔM
where,
M = m/z (mass-to-charge ratio)
ΔM = M2 - M1 (M1, M2 are two adjacent peaks)
Peaks (m/z values) that fall into the ΔM band are merged into a
single peak (m/z values).
To change resolution settings
Choose Option > Mass Settings from the main menu.
To set the Resolution setting for all calculations in Mass Frontier, you must
define Resolution type in the User Defined Type box and set the
appropriate value and unit in the Unit and Value boxes.
For imported spectra or GC/LC/MS chromatograms from a file, there are
two options for the setting of the resolution (Resolution & Precision of
Experimental Spectra box). You can either adopt the resolution saved in a
file (Acquired from Source box), or set your own resolution settings to be
used for calculations (User Defined Type box). Note that it is not possible
to improve the resolution of experimental data by using User Defined Type.
Do not set resolution values that are better than the resolution of the mass
spectrometer actually used for data acquisition. Use this option to artificially
reduce the resolution of experimental data when working with low- and
high-resolution spectra in the same data set (spectra search, target analysis,
or classification).
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Mass Settings
Resolution
Note Changing the Resolution in Mass Settings dialog window does not
affect spectra that are opened (with the Chromatogram Processor) and
fragments that have already been calculated with the (Fragments &
Mechanisms and Fragments Comparator modules). If you require
spectra or fragments with a new setting, you must reload these spectra or
regenerate these fragments.
Figure 113. Resolution page of the Mass Settings dialog window
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Mass Settings
Precision
Precision
Precision refers to m/z values and is defined as the position of the last digit
relative to the decimal point that is displayed. Precision settings are used
only for the display of m/z values and peak positions on the m/z scale. In
contrast to resolution, precision settings are ignored in all calculations as the
highest possible precision is always used. To assure the correct display of
m/z values, set the precision to the same or a higher order than the
resolution.
Note When working with experimental spectra, some of the digits of the
displayed m/z values might not be significant.
Figure 114. Precision page of the Mass Settings dialog window
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Chapter 16
Microsoft Office in
Mass Frontier
Mass Frontier supports the Object Linking and Embedding (OLE) features
of Microsoft Excel and Word documents; that is, these programs and their
native data files can be opened inside the Mass Frontier desktop. You do not
need to leave Mass Frontier to work with Excel and Word documents
because they become part of Mass Frontier. Use this feature with embedded
Excel documents for data exchange between Mass Frontier modules and
Excel worksheets.
Note The Mass Frontier installation package does not include the
Microsoft Office software. In order to use the Object Linking and
Embedding features of Excel and Word, these programs must be
purchased separately. Install Microsoft Office prior to installing
Mass Frontier.
This chapter contains the following sections:
• Data Exchange between Excel and Mass Frontier
• Exporting Data to Excel
• Importing Spectra from Excel
• Using Excel as Spectrum Editor
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Microsoft Office in Mass Frontier
Figure 115. An Excel worksheet embedded into Mass Frontier
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16 Microsoft Office in Mass Frontier
Data Exchange between Excel and Mass Frontier
Data Exchange
between Excel and
Mass Frontier
There are three basic types of data you can exchange between Excel and
Mass Frontier: text tables, graphics, and mass spectra in table format.
The data can be exchanged by using the copy and paste commands.
Mass Frontier does not support the import or export of native Excel .xls files
directly into the Database Manager. However, Excel documents can be
embedded into Mass Frontier and data can be exchanged by using the
Clipboard. Choose Microsoft Office > Open Microsoft Excel Document.
In this case, a new window opens and the Excel menu and toolbar appear.
Note Embedded Excel or Word windows have their own main menus
(displayed above the Mass Frontier menus) and buttons (displayed
below Mass Frontier buttons). Microsoft Office controls (menus and
buttons) are visible only if an Excel or Word window is active. These
controls do not contain Open and Save commands. If you want to open
or save an Office document, you must choose Microsoft Office on the
Mass Frontier main menu.
Mass Frontier Controls
Microsoft Excel Controls
Figure 116. Locations of Excel and Mass Frontier controls
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Microsoft Office in Mass Frontier
Exporting Data to Excel
Exporting Data to
Excel
You can export four types of data from the Database Manager to Excel: text
tables, graphics, mass spectra in numerical table format, and spectral trees.
Because all these types can be accessed at the same time, you must specify
the type of data you want to export to Excel.
To export additional information associated with a record
1. Choose the Info tab (Info tab data must be visible).
2. Click the small Copy Selected Tab button in the top-right corner of the
Info tab control.
3. Paste the data by clicking the Paste button in Excel.
To export mass spectrum or mass differences graphics
1. Choose the Mass Spectrum or Mass Differences tab (a Mass Spectrum
or Mass Differences spectrum must be visible).
2. Click the small Copy Selected Tab button in the top-right corner of the
Mass Spectrum tab control.
3. Paste the graphic into Excel by clicking the Paste button.
To export a mass spectrum in numerical table format
1. Choose the Data tab (an m/z and abundance table must be visible).
2. Click the small Copy Selected Tab button in the top-right corner of the
Data tab control.
3. Paste the table into Excel by clicking the Paste button.
To export records data in the spreadsheet
1. Select one or more records in the spreadsheet.
2. Click the Copy Selected Rows button to the right of the Spreadsheet
tab control.
3. Paste the data into Excel by clicking the Paste button.
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Importing Spectra
from Excel
Microsoft Office in Mass Frontier
Importing Spectra from Excel
Mass spectra in table format or spectral trees stored in Excel can be
imported to the Database Manager by using the Clipboard. Spectral tables
can be organized horizontally or vertically. In order to correctly interpret
m/z values and abundance, follow one of these conventions:
1. If the spectral table is vertical, the first column must be the m/z value
and the second must be abundance.
2. If the spectra are oriented vertically and the first column is abundance
and the second column is the m/z value, caption the first row of the first
column Abundance and the first row of the second column m/z.
3. If the spectral table is oriented horizontally, the first row must be the
m/z value and the second row must be Abundance.
4. If your spectra are oriented horizontally and your first row is abundance
and the second row is the m/z value, caption the first column of the first
row Abundance and the first column of the second row m/z.
5. More than one spectrum can be imported at a time. In this case, your
first column or row, depending on the orientation, must be the
m/z values and all other columns (or rows) must be Abundance.
To import spectra from Excel to Mass Frontier
1. Verify that your table actually contains mass spectra.
2. Select the table you want to export to Mass Frontier.
3. Click the Copy button in Excel or, if your document is embedded, click
the Copy button on the Excel toolbar.
4. Click the Paste button in the Spectra Manager window.
Mass Frontier supports standard tables with separated numbers, so spectra
can also be imported from other programs.
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Microsoft Office in Mass Frontier
Using Excel as Spectrum Editor
Using Excel as
Spectrum Editor
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Excel can be used as a spectrum editor for Mass Frontier by using the export
and import features. Use this tool if, for example, you have an experimental
spectrum with a number of noise peaks at high m/z values that you want to
delete, or you want to extract part of a spectrum that is important to your
report or presentation. Do not add, delete, or alter prominent peaks.
Thermo Electron Corporation
Chapter 17
Formula Generator
Use the Formula Generator to calculate a list of theoretical molecular
formulas that best fit an m/z value. Because the number of possible
molecular formulas for a given m/z value is closely related to the mass
tolerance, elements used, and the maximum allowed number of atoms for
each isotope, always carefully evaluate these parameters before a generation
starts. The m/z value can either be manually entered or automatically taken
from any spectral peak in the Database Manager or the Chromatogram
Processor. You can also use the Formula Generator to calculate the isotopic
pattern for any generated formula.
This chapter contains the following sections:
• Formula Generation from a Peak
• Formula Generator Options
To open a Formula Generator window
• Click the Formula Generator button on the main tool bar.
• Or choose Tools > Formula Generator from the main menu.
Figure 117. Formula Generator window
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Formula Generator
Formula Generation from a Peak
Formula Generation
from a Peak
With Mass Frontier, you can calculate the possible elemental composition
for any spectral peak displayed in the Database Manager and
Chromatogram Processor windows by simple peak picking. The advantage
of this method compared to manual entering of the m/z value is that all
the decimal places are accurately transferred to Formula Generator and the
m/z tolerance value is automatically calculated from the source spectra if this
option is enabled in the Acquired from Source box.
To generate possible formulas for a peak
1. Click the Option button and review the predefined settings. Give
special attention to the Charge setting (Limits tab) according to the
polarity of the MS mode used for spectra acquisition. Be sure to check
the maximum number of elements used for calculation in the Elements
in Use tab.
2. Click the Pick a Peak
minimizes to a bar.
button. The Formula Generator window
3. Click on a spectral peak in the Database Manager or the Chromatogram
Processor window.
A list of possible formulas appears.
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17 Formula Generator
Formula Generation from a Peak
Figure 118. Formula Generator window
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Formula Generator
Formula Generator Options
Formula Generator
Options
Use Formula Generator options to speed up calculations, restrict the
number of possible formulas, and set the charge state and polarity.
Available parameter settings include the following:
• Charge
Charge state and polarity.
• Nitrogen rule
Decide whether to use the nitrogen rule in the elemental composition
calculation. If yes, the choices include even-electron (radical cation) or
odd-electron (protonated) ion.
• Hydrogen Count
Use to specify whether to use an algorithm for the exclusion of
implausible formulas with improbable high numbers of hydrogen.
• Valence Test
Use to specify whether to exclude formulas if the atoms cannot be
connected in any way using valences common in organic chemistry.
• Ring plus Double Bond Equivalent (RDBE)
Only formulas are displayed for which the ring plus a double bond
equivalent is within the range From – To. The RDBE limits the
calculated formulas to those which make sense chemically.
• Elements in Use
Use this tab to specify isotopes of particular elements with a maximum
number that is considered for the calculation of formulas.
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17 Formula Generator
Formula Generator Options
Figure 119. Molecular Formula Settings window
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Chapter 18
Report Creator
Use the Report Creator to create customizable reports. You can create
reports by extracting any information from the modules on the screen. Both
text and graphics can be extracted and you can specify the order in which
objects from the module appear in the report. Reports can be created from
the following modules: Structure Editor, Database Manager, Fragmentation
Library, Chromatogram Processor, Spectra Projector, Neural Networks, and
Fragments & Mechanisms. Reports can be printed or exported to PDF files
and the report layout saved.
This chapter contains the following sections:
• Report Creator Window
• Creating Reports
Figure 120. Report Creator window
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Report Creator
Report Creator Window
Report Creator
Window
The Report Creator window is divided into two parts. See Figure 121.
Use the left part to manage the modules from which you want to create a
report. Select a module by highlighting its name and its corresponding
customizable items are displayed on the right side. Use the right part to
select and annotate a number of text or graphical objects for the selected
module.
Use the left part of the Report Creator window to add or remove a module
from reporting or to change the order in which they appear in the report.
Note A module can be listed only once on the left side of the Report
Creator.
Add Module
Insert Module
Remove Module
Move Down Module
Move Up Module
Generate and Preview Report
Automated Save/Reload by
closing/opening Report Creator window
Save current report settings
Load report settings
Figure 121. Report Creator window
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Creating Reports
Report Creator
Creating Reports
Create Reports by using the Report Creator only from windows that are
open. You cannot report data that is stored in Mass Frontier but does not
appear on the screen when Report Creator is launched.
To create a report
1. Open one or more modules with the corresponding data you want to
report.
2. Click the Report Creator
button on the main tool bar, or choose
Tools > Report Creator from the main menu
When the Report Creator opens, all modules available for reporting are
listed on the left side. If you click on a module name, options and
annotation fields specific to the selected module appear on the right side.
To change the general report settings such as header, footer, separation
lines, page breaks, or orientation, click the Report Layout
button in
the Report Creator window.
Once the modules and their objects have been selected and annotated,
generate a report preview by clicking the Preview
button. See
Figure 122.
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Report Creator
Creating Reports
Figure 122. Report Preview window
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Index
A
activating Mass Frontier 5
algorithms
Direct Infusion 217
Joint Component Detection (JCD) 213
Rapid Component Detection (RCD) 211
Total Extraction Component Detection (TECD) 216
atom properties 36
atoms and bonds 34
B
bar code spectra 133, 133
baseline correction 206
bond properties, structure 38
bonds and atoms 34
bonds, unspecified location 46
C
calculations, MS 45
charge site, structure 47
Chromatogram Processor 193–220
Chromatogram Processor window, illustrated 195, 220
chromatograms, spectral tree 59
cleaning structures 43
component detection and spectra deconvolution 208–218
components
searching 223
Components Editor 221–224
Components Editor window, illustrated 222
copying structures 39
creating
libraries 96
reports 243
spectral trees 54–59
D
data exchange between modules 89
data exchange, Excel 231
Thermo Electron Corporation
Database Manager
editing records 70, 71
fragments 90
introduction 61
mass differences, displaying 68
mass spectral data, exchanging between modules 89
opening 63
records 65–67
spectra, comparing 69
spreadsheets 75
structures 72–74
Database Manager window, illustrated 64
databases, mass spectral 93
Direct Infusion algorithm 217
E
editing records in Database Manager 70, 71
Excel
exporting data to 231, 232
importing data from 231, 233
F
features, Mass Frontier 11
Formula Generator 235–239
Formula Generator window, illustrated 237
formulas
generating from peak 236
searching in Database Manager 86
fragmentation
drawing reactions 140
generating pathways 112–114
libraries, searching 152
prediction using library reactions 150
Fragmentation Library 137–154
Fragmentation Library window, illustrated 143
Fragments & Mechanisms 107–135
Fragments & Mechanisms window, illustrated 116
Fragments Comparator 155–157
Fragments Comparator window, illustrated 157
fragments, link with spectrum 126
fuzzy clustering (FC) 165
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Index: I
I
Kohonen networks 164
Fragmentation Library 137–154
Fragments & Mechanisms 107–135
Fragments Comparator 155–157
Isostope Pattern 18
Neural Networks 185–192
overview 12–25
Periodic Table 19
Report Creator 241–244
Spectra Classifier 169–175
Spectra Projector 177–184
Structure Editor 27–47
MS calculations
structures 45
n
MS experiments, simulating 128
L
N
libraries, fragmentation 137–154
libraries, spectral
backing up 104
creating libraries 96
importing 93
records, adding 99
SQL Server 92, 103
tools 102
uninstalling 98
limitations, Mass Frontier 9
names, searching in Database Manager 86
Neural Networks 185–192
Neural Networks window, illustrated 189
NIST library 93
nodes, spectral trees 52
noise elimination 206
importing spectra from Excel 233
installing Mass Frontier 4–7
Isotope Pattern module 18
J
Joint Component Detection (JCD) algorithm 213
K
O
Object Linking and Embedding (OLE) features, Microsoft
Office 229
M
maps (SOM), self-organizing 164, 185
mass settings
precision 228
resolution 226
mass spectra
bar code 133
classification 159–167
classifying 173
comparing 69
importing 233
searching in Database Manager 78
transformation 166
mass spectral data, exchanging between modules 89
mass spectral databases 93
mechanism extraction 146
Microsoft Office features 229
modules
Chromatogram Processor 193–220
Database Manager 61–90
exchanging data between 89
Formula Generator 235–239
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P
peaks
fragment assignment 90
fragments, proposed 130
generating formulas 236
unexplained 129
periodic table 19
precision, mass settings 228
principal component analysis (PCA) 162
R
Rapid Component Detection (RCD) algorithm 211
reaction formalism 111
reaction mechanisms overview 110
reaction restrictions 117
reaction symbols 148
Thermo Electron Corporation
Index: S
records
adding to libraries 99
Database Manager 65–67, 70
Fragmentation Library 142, 145
Report Creator 241–244
Report Creator window, illustrated 242
reports, creating 243
resolution, mass settings 226
S
searching
components 223
Database Manager 77–88
libraries, fragmentation 152
self-organizing maps (SOM) 164, 185
smoothing 207
Spectra Classifier 169–175
Spectra Classifier window, illustrated 171
Spectra Manager, molecular mass search 86
Spectra Projector 177–184
Spectra Projector window, illustrated 179
spectra, mass
bar code 133
classification 159–167
classifying 173
comparing 69
importing 233
searching in Database Manager 78
transformation 166
spectral libraries. see libraries, spectral
spectral trees
creating 54–59
description 49–53
Direct Infusion algorithm 217
illustrated 50
overview 49
searching in Database Manager 80
spreadsheets, Database Manager 75
SQL Server
installing 4
library utilities 92, 102, 103
starting Mass Frontier 8
Structure Editor 27–47
Structure Editor window, illustrated 28
structures
atom properties, changing 36, 37
atoms and bonds, selecting 34
bond properties, changing 38
bonds, unspecified location 46
charge sites 47
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checking 44
cleaning 43
copying 39
Database Manager 72–74
layout, changing 31, 31
moving and resizing 41
MS calculations 45
opening and saving 29
searching 30
searching in Database Manager 82–85
templates, using 33
text, adding 32
system requirements 3
T
thresholding 205
toolbar, Fragmentation Library 139
Total Extraction Component Detection (TECD) algorithm 216
X
n
Xcalibur MS data, processing 219
Xcalibur software, installing 4
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