LTQ Orbitrap Velos Getting Started

LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos™
Getting Started
Tune Plus 2.6
Revision B - 1250590
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Read This First
Welcome to the Thermo Scientific, LTQ Orbitrap Velos™ system! The
LTQ Orbitrap Velos is a member of the family of LTQ™ mass
spectrometer (MS) detectors.
About This Guide
This LTQ Orbitrap Velos Getting Started manual provides information
on how to set up, calibrate, and tune the LTQ Orbitrap Velos.
Procedures in Chapters 1–4 can be performed from the Xcalibur™ Tune
Plus window.
Who Uses This Guide
This LTQ Orbitrap Velos Getting Started manual is intended for all
personnel that need to operate the LTQ Orbitrap Velos, especially the
key operator. This manual should be kept near the instrument to be
available for quick reference.
Scope of This Guide
LTQ Orbitrap Velos Getting Started includes the following chapters:
Thermo Fisher Scientific
•
Chapter 1: “Introduction” provides general information about this
manual.
•
Chapter 2: “Tune Plus Window” provides information on the Tune
Plus window.
•
Chapter 3: “Calibrating the Instrument for FTMS Measurements”
provides procedures to calibrate the LTQ Orbitrap Velos for
FTMS measurements.
•
Chapter 4: “Performing Diagnostics/Checks” describes several
diagnostic procedures.
•
Chapter 5: “Instrument Setup” describes the FTMS relevant topics
of the data dependent settings in the Instrument Setup.
•
Chapter 6: “Instrument Configuration” gives instructions about
configuring the instrument.
LTQ Orbitrap Velos Getting Started
i
Read This First
About This Guide
ii
LTQ Orbitrap Velos Getting Started
•
Chapter 7: “LTQ Orbitrap Velos ETD Instruments”describes the
Orbitrap relevant differences in instrument settings and procedures
with respect to using a LTQ Orbitrap Velos ETD instrument.
•
Appendix A: “Miscellaneous Information” gives additional
information about various topics.
Thermo Fisher Scientific
Read This First
Related Documentation
Related Documentation
In addition to this guide, Thermo Fisher Scientific provides the
following documents for LTQ Orbitrap Velos and LTQ Orbitrap
Velos ETD:
•
LTQ Orbitrap Series Preinstallation Requirements Guide
•
LTQ Orbitrap Velos Hardware Manual
•
LTQ Velos manual set
You can access PDF files of the documents listed above from the data
system computer. The software also provides Help.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
iii
Read This First
Contacting Us
Contacting Us
There are several ways to contact Thermo Fisher Scientific.
Assistance
For technical support and ordering information, visit us on the Web:
www.thermo.com/advancedms
Customer Information Service
cis.thermo-bremen.com is the Customer Information Service site aimed
at providing instant access to
•
latest software updates
•
manuals, application reports, and brochures.
Note Thermo Fisher Scientific recommends that you register with the
site as early as possible. ▲
To register, visit register.thermo-bremen.com/form/cis and fill in the
registration form. Once your registration has been finalized, you will
receive confirmation by e-mail.
Changes to the Manual
❖
To suggest changes to this manual
•
Please send your comments (in German or English) to:
Editors, Technical Documentation
Thermo Fisher Scientific (Bremen) GmbH
Hanna-Kunath-Str. 11
28199 Bremen
Germany
•
Send an e-mail message to the Technical Editor at
documentation.bremen@thermofisher.com
You are encouraged to report errors or omissions in the text or index.
Thank you.
iv
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Read This First
Typographical Conventions
Typographical Conventions
This section describes typographical conventions that have been
established for Thermo Fisher Scientific manuals.
Data Input
Throughout this manual, the following conventions indicate data input
and output via the computer:
•
Messages displayed on the screen are represented by capitalizing the
initial letter of each word and by italicizing each word.
•
Input that you enter by keyboard is identified by quotation marks:
single quotes for single characters, double quotes for strings.
•
For brevity, expressions such as “choose File > Directories” are used
rather than “pull down the File menu and choose Directories.”
•
Any command enclosed in angle brackets < > represents a single
keystroke. For example, “press <F1>” means press the key labeled
F1.
•
Any command that requires pressing two or more keys
simultaneously is shown with a plus sign connecting the keys. For
example, “press <Shift> + <F1>” means press and hold the <Shift>
key and then press the <F1> key.
•
Any button that you click on the screen is represented in bold face
letters. For example, “click on Close”.
Topic Headings
The following headings are used to show the organization of topics
within a chapter:
Chapter 1
Chapter Name
Second Level Topics
Third Level Topics
Fourth Level Topics
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
v
Read This First
Safety and EMC Information
Safety and EMC Information
In accordance with our commitment to customer service and safety,
these instruments have satisfied the requirements for the European CE
Mark including the Low Voltage Directive.
Designed, processed, and tested in an ISO9001 registered facility, this
instrument has been shipped to you from our manufacturing facility in a
safe condition.
This instrument must be used as described in this manual. Any use of
this instrument in a manner other than described here may result in
instrument damage and/or operator injury.
Notice on Lifting and Handling of Thermo Scientific Instruments
For your safety, and in compliance with international regulations, the
physical handling of this Thermo Scientific instrument requires a team
effort for lifting and/or moving the instrument. This instrument is too
heavy and/or bulky for one person alone to handle safely.
Notice on the Proper Use of Thermo Scientific Instruments
In compliance with international regulations: If this instrument is used
in a manner not specified by Thermo Fisher Scientific, the protection
provided by the instrument could be impaired.
Notice on the Susceptibility to Electromagnetic Transmissions
Your instrument is designed to work in a controlled electromagnetic
environment. Do not use radio frequency transmitters, such as mobile
phones, in close proximity to the instrument.
Safety and Special Notices
Make sure you follow the precautionary statements presented in this
guide. The safety and other special notices appear different from the
main flow of text. Safety and special notices include the following:
Warning Warnings highlight hazards to human beings. Each Warning is
accompanied by a Warning symbol. ▲
Caution Cautions highlight information necessary to protect your
instrument from damage. ▲
Note Notes highlight information that can affect the quality of your
data. In addition, notes often contain information that you might need
if you are having trouble. ▲
vi
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
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Safety and EMC Information
Identifying Safety Information
The LTQ Orbitrap Velos Getting Started contains precautionary
statements that can prevent personal injury, instrument damage, and
loss of data if properly followed. Warning symbols alert the user to check
for hazardous conditions. These appear throughout the manual, where
applicable. The most common warning symbols are:
Warning This general symbol indicates that a hazard is present that
could result in injuries if it is not avoided.
The source of danger is described in the accompanying text. ▲
Warning High Voltages capable of causing personal injury are used in
the instrument. The instrument must be shut down and disconnected
from line power before service is performed. Do not operate the
instrument with the top cover off. Do not remove protective covers from
PCBs. ▲
Warning Treat heated zones with respect. Parts of the instrument might
be very hot and might cause severe burns if touched. Allow hot
components to cool before servicing them. ▲
Warning Wear gloves when handling toxic, carcinogenic, mutagenic, or
corrosive/irritant chemicals. Use approved containers and procedures for
disposal of waste solution. ▲
In addition to the above described, every instrument has specific
hazards. So, be sure to read and comply with the precautions described
in the subsequent chapters of this guide. They will help ensure the safe,
long-term use of your system.
General Safety Precautions
Observe the following safety precautions when you operate or perform
service on your instrument:
Thermo Fisher Scientific
•
Before plugging in any of the instrument modules or turning on the
power, always make sure that the voltage and fuses are set
appropriately for your local line voltage.
•
Only use fuses of the type and current rating specified. Do not use
repaired fuses and do not short-circuit the fuse holder.
•
The supplied power cord must be inserted into a power outlet with a
protective earth contact (ground). When using an extension cord,
make sure that the cord also has an earth contact.
LTQ Orbitrap Velos Getting Started
vii
Read This First
Safety and EMC Information
viii
LTQ Orbitrap Velos Getting Started
•
Do not change the external or internal grounding connections.
Tampering with or disconnecting these connections could endanger
you and/or damage the system.
•
The instrument is properly grounded in accordance with regulations
when shipped. You do not need to make any changes to the
electrical connections or to the instrument’s chassis to ensure safe
operation.
•
Never run the system without the housing on. Permanent damage
can occur.
•
Do not turn the instrument on if you suspect that it has incurred
any kind of electrical damage. Instead, disconnect the power cord
and contact a service representative for a product evaluation. Do not
attempt to use the instrument until it has been evaluated. (Electrical
damage may have occurred if the system shows visible signs of
damage, or has been transported under severe stress.)
•
Damage can also result if the instrument is stored for prolonged
periods under unfavorable conditions (e.g., subjected to heat, water,
etc.).
•
Always disconnect the power cord before attempting any type of
maintenance.
•
Capacitors inside the instrument may still be charged even if the
instrument is turned off.
•
Never try to repair or replace any component of the system that is
not described in this manual without the assistance of your service
representative.
•
Do not place any objects – especially not containers with liquids –
upon the instrument. Leaking liquids might get into contact with
electronic components and cause a short circuit.
Thermo Fisher Scientific
Read This First
Safety and EMC Information
Safety Advice for Possible Contamination
Hazardous Material Might Contaminate Certain Parts of Your
System During Analysis.
In order to protect our employees, we ask you to adhere to special
precautions when returning parts for exchange or repair.
If hazardous materials have contaminated mass spectrometer parts,
Thermo Fisher Scientific can only accept these parts for repair if they
have been properly decontaminated. Materials, which due to their
structure and the applied concentration might be toxic or which in
publications are reported to be toxic, are regarded as hazardous.
Materials that will generate synergetic hazardous effects in combination
with other present materials are also considered hazardous.
Your signature on the Repair-Covering letter confirms that the
returned parts have been decontaminated and are free of hazardous
materials.
The Repair-Covering letter can be ordered from your service engineer or
downloaded from the Customer Information Service (CIS) site. Please
register under http://register.thermo-bremen.com/form/cis.
Parts contaminated by radioisotopes are not subject to return to Thermo
Fisher Scientific – either under warranty or the exchange part program.
If parts of the system may be possibly contaminated by hazardous
material, please make sure the Field engineer is informed before the
engineer starts working on the system.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
ix
Contents
Thermo Fisher Scientific
Chapter 1
Introduction................................................................................1-1
Chapter 2
Tune Plus Window....................................................................2-1
Preliminary Remarks......................................................... 2-2
View Menu ....................................................................... 2-3
Spectrum View............................................................... 2-3
Graph View ................................................................... 2-5
Status View .................................................................... 2-5
Scan Mode Menu.............................................................. 2-8
Define Scan.................................................................... 2-8
Scan Ranges ................................................................. 2-14
Centroid/Profile........................................................... 2-14
Positive/Negative ......................................................... 2-14
Display Menu ................................................................. 2-15
Spectrum Averaging ..................................................... 2-15
Setup Menu .................................................................... 2-16
FT Transfer Optics ...................................................... 2-16
FT Injection Control ................................................... 2-17
FT Vacuum ................................................................. 2-19
FT Temperature Monitor ............................................ 2-20
FT Lock Masses ........................................................... 2-20
Tune Methods ................................................................ 2-21
Parameters with Differentiation between Ion Trap
and FT Scans ............................................................... 2-21
Parameters without Differentiation between Ion Trap
and FT Scans ............................................................... 2-22
Parameters not saved in a Tune Method ...................... 2-22
Chapter 3
Calibrating the Instrument for FTMS Measurements.........3-1
Preliminary Remarks......................................................... 3-2
Calibration Files and their Backups................................... 3-3
Backup Current Calibration........................................... 3-3
Restore Backup Calibration............................................ 3-3
Calibration Solutions ........................................................ 3-4
Chemicals for Preparing Calibration Solutions............... 3-4
Preparing Stock Solutions .............................................. 3-6
LTQ/FT-Hybrid Positive Ion Mode Calibration
Solution ......................................................................... 3-8
LTQ/FT-Hybrid Negative Ion Mode Calibration
Solution ......................................................................... 3-9
LTQ Orbitrap Velos Getting Started
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Contents
Applicable Calibration Solutions for FT Manual
Calibration................................................................... 3-10
Calibration and Tuning of the Ion Trap ......................... 3-11
Calibration of the Ion Trap.......................................... 3-11
Tuning the Ion Trap for Positive Ion Mode................. 3-11
Tuning the Ion Trap for Negative Ion Mode ............... 3-13
Automatic Calibration Page ............................................ 3-15
Semi-Automatic Calibration Page ................................... 3-17
HCD Calibration......................................................... 3-18
Check Calibration Page................................................... 3-19
FT Manual Calibration Page........................................... 3-22
Mass List Group Box ................................................... 3-23
Chapter 4
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LTQ Orbitrap Velos Getting Started
Performing Diagnostics/Checks............................................ 4-1
System Evaluation Procedures ........................................... 4-2
FT CLT-RF Pulser Evaluation ....................................... 4-2
FT Dynamic Range Test................................................ 4-3
FT Energy Dependence Evaluation................................ 4-3
FT ETD Fragmentation Efficiency ................................ 4-3
FT HCD Collision Cell Ejection Evaluation.................. 4-3
FT HCD Multipole Evaluation ..................................... 4-3
FT High Mass Range Target Compensation .................. 4-3
FT Isolation Test ........................................................... 4-4
FT Noise Test ................................................................ 4-4
FT Preamp Evaluation ................................................... 4-4
FT Pulser Evaluation...................................................... 4-4
FT Reagent Ion Source Drift Time Evaluation .............. 4-4
FT Reagent Ion Source Transfer Multipole
Evaluation...................................................................... 4-5
FT Sensitivity Test ......................................................... 4-5
FT Stability Test ............................................................ 4-5
FT Temperature Control Evaluation.............................. 4-6
FT Temperature Monitor .............................................. 4-6
Toggles ............................................................................. 4-8
FT Advanced Calibration............................................... 4-9
FT Analyzer Ion Gauge.................................................. 4-9
FT Analyzer Temperature Control................................. 4-9
FT Apodization.............................................................. 4-9
FT HCD Collision Gas.................................................. 4-9
FT Include Transients.................................................... 4-9
FT Manual Calibration for Single Range ..................... 4-10
FT Profile Mode .......................................................... 4-10
FT SIM and MSn Injection Waveforms....................... 4-10
FT Storage Evaluation Mode ....................................... 4-10
FT View Frequency...................................................... 4-11
FT Zero Offset............................................................. 4-11
Isolate Reagent Ion....................................................... 4-11
Reagent Ion AGC ........................................................ 4-11
Set Device ....................................................................... 4-12
Thermo Fisher Scientific
Contents
FT Lockmass Abundance ............................................. 4-13
FT Mass Check Test Duration..................................... 4-13
Setting new FT Transfer Optics Parameters ................. 4-13
Display Settings .............................................................. 4-15
Thermo Fisher Scientific
Chapter 5
Instrument Setup .......................................................................5-1
Using Locking in Automated Runs ................................... 5-2
Data Dependent Settings .................................................. 5-3
Using Masses instead of Mass-to-Charge Ratios ............. 5-3
Preview Mode ................................................................ 5-6
Monoisotopic Precursor Selection .................................. 5-7
Enabling Charge State Dependent ETD Time............... 5-8
Data Dependent FT SIM Scans ..................................... 5-9
Activation Type ........................................................... 5-13
FT HCD...................................................................... 5-14
FT ETD....................................................................... 5-15
MSn Settings for HCD Experiments............................ 5-16
MSn Settings for ETD Experiments............................. 5-16
Chapter 6
Instrument Configuration.........................................................6-1
Starting Instrument Configuration.................................... 6-2
FT Settings Page ............................................................... 6-3
FT Mass Lists Page............................................................ 6-4
Chapter 7
LTQ Orbitrap Velos ETD Instruments .....................................7-1
Tune Plus Window of the LTQ Orbitrap Velos ETD....... 7-2
Status View .................................................................... 7-2
Scan Mode Menu........................................................... 7-3
Setup Menu ................................................................... 7-4
Reagent Ion Source Dialog Box...................................... 7-5
Configuring the Reagent Ion Source ................................. 7-8
Powering On the ETD Module and Viewing Reagent
Ion Spectra...................................................................... 7-10
Powering On the ETD Module ................................... 7-10
Turning On the Reagent Ion Source and Viewing
Reagent Ion Spectra ..................................................... 7-10
Tuning the Reagent Ion Optics....................................... 7-12
Automatically Tuning the Reagent Ion Source............. 7-12
Manually Tuning the Reagent Ion Source.................... 7-14
Semi-Automatically Tuning the Reagent Ion Optics .... 7-17
Viewing the Current Reagent Ion Optics Settings........ 7-20
Saving Your ETD Tune Method.................................. 7-21
Tuning the Quadrupole Mass Filter ............................. 7-21
Performing an ETD Infusion Experiment ....................... 7-27
Viewing the Injection Reagent Settings ........................ 7-27
Troubleshooting an AGC Target Error ........................ 7-28
Obtaining an ETD Spectrum for Angiotensin I ........... 7-29
LTQ Orbitrap Velos Getting Started
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Contents
Optimizing the Reagent Ion Reaction Time................. 7-31
Creating an Xcalibur Instrument Method That Uses
ETD Activation .............................................................. 7-35
Angiotensin I Solutions................................................... 7-41
Preparing the Angiotensin I Stock Solution.................. 7-42
Preparing the Angiotensin I Test Solution.................... 7-42
Appendix A Miscellaneous Information ................................................... A-1
FT Analyzer Information in Scan Header..........................A-2
FT Analyzer Settings ......................................................A-2
FT Analyzer Messages ....................................................A-3
Data Size of FT Raw Files .................................................A-4
Glossary ................................................................................... G-1
Index ............................................................................................I-1
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Figures
Tune Plus window ................................................................................ 2-2
Spectrum View page .............................................................................. 2-3
Spectrum Display Options dialog box – FT page .................................. 2-4
Graph view page ................................................................................... 2-5
Status view – All page ............................................................................ 2-6
User Status Display Configuration dialog box ....................................... 2-7
Define Scan dialog box (Advanced Scan Features enabled) .................... 2-8
Scan Time Settings dialog box – FT page ............................................ 2-10
Lock Masses dialog box ....................................................................... 2-11
Define Scan dialog box with HCD selected as activation type ............. 2-14
Spectrum Averaging dialog box ........................................................... 2-15
FT Transfer Optics dialog box ............................................................ 2-16
Ion Trap page of the Injection Control dialog box .............................. 2-17
FT page of the Injection Control dialog box ....................................... 2-18
Ion Trap page of the Vacuum dialog box ............................................ 2-19
FT page of the Vacuum dialog box ..................................................... 2-19
FT Temperature Monitor dialog box .................................................. 2-20
Recommended settings in the Define Scan dialog box for an
automatic tune of the ion trap ............................................................. 3-12
Ion trap spectrum of positive ion mode calibration solution, scan
range m/z 130–2000, positive ion polarity mode ................................. 3-12
Ion trap spectrum of the positive ion mode calibration solution
(lower range), positive ion polarity mode ............................................ 3-13
Ion trap spectrum of the negative ion mode calibration solution,
scan range m/z 150–2000, negative ion polarity mode ........................ 3-14
Automatic page of the Calibrate dialog box ......................................... 3-15
Semi-Automatic page of the Calibrate dialog box ................................ 3-17
Check page of the Calibrate dialog box ............................................... 3-19
FT Manual page of the Calibrate dialog box ....................................... 3-22
System evaluation page of the Diagnostics dialog box
(LTQ Orbitrap Velos) ........................................................................... 4-2
Result of the FT stability test displayed in the Graph View ................... 4-6
Toggles page of the Diagnostics dialog box ........................................... 4-8
Set device page of the Diagnostics dialog box ...................................... 4-12
Display settings page of the Diagnostics dialog box ............................. 4-15
MS Detector Setup View – MS Detector Setup Page ............................ 5-2
Data Dependent Settings dialog box – Global page ............................... 5-3
Data Dependent Settings dialog box – Current Segment page .............. 5-6
Data Dependent Settings dialog box – Charge State page
(Advanced Features on) ......................................................................... 5-7
Enabling charge state dependent ETD time .......................................... 5-8
Data Dependent Settings dialog box – Current Scan Event page ........... 5-9
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
xv
Figures
Current Scan Event page – Repeat previous scan event with
HCD .................................................................................................. 5-10
Current Scan Event page – Repeat previous scan event with
ETD ................................................................................................... 5-11
Data Dependent Settings dialog box – Scan Widths page ................... 5-12
Data Dependent Settings dialog box – Activation page ....................... 5-13
Data Dependent Settings dialog box – FT HCD page ........................ 5-14
Data Dependent Settings dialog box – FT ETD page ......................... 5-15
MS Detector Setup Page – Scan event settings with
HCD experiment ................................................................................ 5-16
MS Detector Setup Page – Scan event settings with
ETD experiment ................................................................................. 5-17
Instrument Configuration dialog box .................................................... 6-2
LTQ Orbitrap Velos Configuration dialog box – FT Settings page ....... 6-3
LTQ Orbitrap Velos Configuration dialog box – FT Mass Lists
page ...................................................................................................... 6-4
Status View for LTQ Orbitrap Velos ETD ........................................... 7-2
Define Scan dialog box for LTQ Orbitrap Velos ETD .......................... 7-3
Injection Control dialog box – Reagent page ......................................... 7-4
Activating Reagent Ion Optics dialog box ............................................. 7-5
Reagent Ion Source dialog box .............................................................. 7-6
Reagent page of the Vacuum dialog box ................................................ 7-7
Enabling the reagent ion source ............................................................. 7-8
LTQ Orbitrap Series Configuration dialog box – Reagent Ion
Source page ........................................................................................... 7-9
Message box: Reagent Vial NOT At Temperature! .............................. 7-10
Tune Plus window showing the fluoranthene radical anion mass
spectrum ............................................................................................. 7-11
Activating Reagent Ion Source tuning ................................................. 7-13
Tune Plus window with Automatic page of Tune dialog box
displayed ............................................................................................. 7-14
Tune Plus window with Reagent Ion Optics dialog box ...................... 7-15
Reagent Ion Source Tune dialog box – Manual page ........................... 7-16
Tune Plus window showing the Display Graph view for manual
tuning of the Reagent Ion Source ........................................................ 7-17
Semi-Automatic page of the Tune dialog box ...................................... 7-18
Reagent Ion Optics dialog box ............................................................ 7-20
Activating Reagent Ion Isolation ......................................................... 7-21
Semi-automatic tune of the Back Multipole Offset dc voltage ............. 7-22
Calibrating the Reagent Ion Selection ................................................. 7-23
Dependence of the signal from the DC Offset .................................... 7-24
Chosen DC Offset (10 V) is too low for calibration ............................ 7-24
Point of optimum performance ........................................................... 7-25
Chosen DC Offset (32 V) is too high for calibration ........................... 7-25
“Noding” in quadrupoles .................................................................... 7-26
Injection Control dialog box – Reagent page ....................................... 7-27
Tune plus window showing a mass scan of infused
Angiotensin I ...................................................................................... 7-29
Define Scan window with the Activation Type ETD .......................... 7-30
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Figures
ETD MS/MS spectrum of Angiotensin I ............................................ 7-31
Tune and Define Scan windows open in Tune Plus ............................ 7-32
Tune window showing Product Ion Mass selected for Reagent Ion
Reaction Time Optimization .............................................................. 7-33
Xcalibur Roadmap Home Page ........................................................... 7-35
New Method view in Xcalibur Instrument Setup ................................ 7-36
Xcalibur MS Detector Setup view ....................................................... 7-37
Xcalibur Instrument Setup .................................................................. 7-38
Data Dependent Settings dialog box in MS Detector
Setup – Activation page ....................................................................... 7-39
Save As window in Xcalibur Instrument Setup, MS Detector
Setup view ........................................................................................... 7-40
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LTQ Orbitrap Velos Getting Started
xvii
Tables
Calibration compounds for LTQ Orbitrap Velos .................................. 3-4
Recommended solvents and reagents ..................................................... 3-5
Actual settings of manual toggles ...........................................................A-2
Typical data sizes (per scan) of an FT spectrum .....................................A-4
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
xix
Chapter 1
Introduction
This manual describes only the FTMS detector relevant settings and
procedures of the LTQ Orbitrap Velos software (Tune Plus 2.6). For ion
trap relevant settings and procedures, refer to the LTQ Series Getting
Started manual.
In addition to this manual, the LTQ Orbitrap Velos Tune Plus Online
Help gives information to specific topics. Nevertheless, it is
recommended to read this manual entirely.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
1-1
Chapter 2
Tune Plus Window
This chapter provides LTQ Orbitrap Velos specific information about
the Tune Plus window. It contains the following topics:
Thermo Fisher Scientific
•
“Preliminary Remarks” on page 2-2
•
“View Menu” on page 2-3
•
“Scan Mode Menu” on page 2-8
•
“Display Menu” on page 2-15
•
“Setup Menu” on page 2-16
•
“Tune Methods” on page 2-21
LTQ Orbitrap Velos Getting Started
2-1
Tune Plus Window
Preliminary Remarks
Preliminary Remarks
The Tune Plus window shows the schematic view of the LTQ Orbitrap
Velos and the instrument name. See Figure 2-1.
Figure 2-1.
Tune Plus window
To access the functions of the Tune Plus window, use the menu
commands, toolbar buttons, and display views. The FT relevant changes
or additions of the menu commands, toolbar buttons, and display views
are explained in the following chapters.
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
View Menu
View Menu
This section describes those elements of the View menu that are
different from the LTQ Velos version of the Tune Plus window.
Spectrum View
The Spectrum view displays real-time ion trap or FT mass spectra
depending on the analyzer type selected in the Define Scan dialog box.
See Figure 2-2.
Figure 2-2.
Spectrum View page
The Spectrum view page has a shortcut menu, which is displayed when
you right-click anywhere on the page. To open the Spectrum Display
Options dialog box, choose Display Options. The dialog box has two
pages: the Ion Trap page and the FT page.
Use the FT page to determine the number of decimals shown on peak
labels. See Figure 2-3 on page 2-4. To change the number of decimals,
click the arrows in the spin box to increment [up arrow] or decrement
[down arrow] the value. You can set the number of decimals to any value
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-3
Tune Plus Window
View Menu
from 0 to 5. Alternatively, you can enter a value in the spin box text
field. The LTQ Orbitrap Velos changes the number of decimals when
you click Apply or OK.
Figure 2-3.
Spectrum Display Options dialog box – FT page
A check box allows showing additional analyzer information for
FTMS scans. This information will be displayed above the spectrum
graph if the box is selected. See “FT Analyzer Messages” on page A-3 for
a list of items that may be displayed as analyzer information.
You can also decide whether or not to show the resolution and/or the
charge state of peaks in the FT spectrum by clearing or selecting the
corresponding check boxes.
If the FTMS analyzer is used, it is possible to display different diagnostic
views in the Spectrum view. See Chapter 4: “Performing
Diagnostics/Checks” for diagnostic features that involve the Spectrum
view.
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
View Menu
Graph View
The Graph view displays, in a variety of traces, real-time data generated
during calibration, tuning, and diagnostic tests. For example, the right
side of Figure 2-4 shows the progress of the transfer efficiency
evaluation.
Figure 2-4.
Graph view page
Status View
The Status view displays real-time status information for the
LTQ Orbitrap Velos. See Figure 2-5 on page 2-6. The Status view has
two pages: the All page and the User page. The All page displays the
real-time status information for about 80 parameters of the
LTQ Orbitrap Velos. You can scroll through the list to observe the status
of the parameters. The User page displays real-time status information
for LTQ Orbitrap Velos parameters that you have selected in the User
Status Display Configuration dialog box. (See page 2-6.)
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-5
Tune Plus Window
View Menu
Figure 2-5.
Status view – All page
User Status Display Configuration Dialog Box
Figure 2-6 on page 2-7 shows the User Status Display Configuration
dialog box.
❖
To configure the User page
1. Choose View > Display Status View.
2. Click the User tab. Right-click the User page to display the shortcut
menu.
3. Choose Configure. The User Status Display Configuration dialog
box is displayed. See Figure 2-6.
4. Select the check boxes that represent the status parameters you want
to have displayed on the User page.
5. Click OK to close the dialog box.
The User page displays real-time status information for the selected
parameters.
2-6
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
View Menu
Figure 2-6.
Thermo Fisher Scientific
User Status Display Configuration dialog box
LTQ Orbitrap Velos Getting Started
2-7
Tune Plus Window
Scan Mode Menu
Scan Mode Menu
This section describes the elements of the Scan Mode menu that are
different from the ion trap.
Define Scan
Choose Define Scan to display the Define Scan dialog box. The Define
Scan dialog box allows defining a scan in various ways depending on the
scan mode and scan type combination. Also, this dialog box allows
choosing the ion trap or the Orbitrap (FTMS) as analyzer. Figure 2-7
shows the Define Scan dialog box showing the Advanced Scan features.
The Advanced Scan features can be activated in the Scan Mode menu of
Tune plus.
Figure 2-7.
2-8
Define Scan dialog box (Advanced Scan Features enabled)
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
Scan Mode Menu
Scan Description
The Scan Description group box contains the following elements:
Analyzer
At the top, the Analyzer list box allows selecting
the analyzer type used during the currently
selected scan event. The following options are
available:
•
Mass Range
FTMS selects the Orbitrap analyzer.
• Ion Trap selects the ion trap analyzer.
The following mass ranges are available:
•
Low: m/z 15–200 for ion trap analyzer only
•
Normal: m/z 50–2000 for ion trap analyzer
and FTMS analyzer
High: m/z 100–4000 for ion trap analyzer
and FTMS analyzer
When you have selected the entry Ion Trap in
the Analyzer list box, this list box allows setting
the scan rate (Normal, Enhanced, Turbo, Zoom,
UltraZoom).
•
Scan Rate /
Resolution
Scan Type
When you have selected the entry FTMS in the
Analyzer list box, this list box allows setting the
resolution of the FT mass spectra.
Available resolution settings (FWHM at
m/z 400) are 7500, 15000, 30000, 60000, and
100000.
Usage of the scan types Full MS, SIM, SRM, or
CRM is analogous to the ion trap. However,
only one scan range is available for FTMS SIM,
FTMS SRM, and FTMS CRM scans.
Scan Time
The Scan Time group box contains the following elements:
Microscans
The number of microscans determines how
many spectra are averaged in one analytical scan.
If FTMS is chosen as analyzer, transients are
averaged for one analytical scan.
The number of microscans can be set
individually for FTMS, Ion Trap MS, FTMS
SIM, Ion Trap SIM, FT MSn, Ion Trap MSn, and
Ion Trap Zoom.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-9
Tune Plus Window
Scan Mode Menu
Max Inject Time
Scan Time Settings
Figure 2-8.
2-10
LTQ Orbitrap Velos Getting Started
The inject time is automatically controlled by the
Automatic Gain Control™ (AGC™). The entry in
this spin box limits the inject time to a maximum
value. To ensure the high mass accuracy of the
LTQ Orbitrap Velos, the maximum inject time
should not be reached. Otherwise, the number of
ions does not correspond to the AGC target
value.
The maximum inject time can be set individually
for FTMS, Ion Trap MS, FTMS SIM, Ion Trap
SIM, FT MSn, Ion Trap MSn, and Ion Trap
Zoom.
Note If the maximum inject time is reached, the
number of ions does not correspond to the
current AGC target value. This may affect the
mass accuracy of FTMS spectra. ▲
Click All to display the Scan Time Settings
dialog box. See Figure 2-8. It allows displaying
and setting all scan time settings for all scan types
at the same time for both the ion trap and the
FT analyzer.
Scan Time Settings dialog box – FT page
Thermo Fisher Scientific
Tune Plus Window
Scan Mode Menu
Locking
Locking allows using one or more peaks in the spectrum as internal
reference to improve mass accuracy. Locking is available for
FTMS scans.
Select the On check box in the Locking group box to enable locking.
Then, click Masses to display a dialog box for entering and editing lock
mass lists. See Figure 2-9.
Lock mass lists can consist of one or more lock masses. If the list
contains lock masses that are (temporarily) not found in the spectrum,
these lock masses are ignored (temporarily) and the instrument steps
back to the external calibration. Thus, even when lock masses are used,
the instrument should be external calibrated as well. For standard full
scan experiments, it is expected that the spectrum shows at least one
peak that corresponds to a lock mass.
Figure 2-9.
Lock Masses dialog box
There are two situations where the instrument makes use of a special
mode to artificially mix the lock mass into the spectrum:
•
If none of the given lock masses is found in the full spectrum, the
instrument tries to improve the abundance of the lock mass by
performing additional SIM injections of the specified lock mass.
•
If the given lock mass cannot be found in the spectrum because the
instrument runs in MSn or SIM scan type, the instrument adds the
lock mass by using SIM injections.
This way, lock masses can be used for all FTMS scan types and for
varying lock mass abundances. There is no need for user interaction
other than specifying a list of reference mass candidates.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-11
Tune Plus Window
Scan Mode Menu
Note Injection of lock masses is turned off completely, when you set the
target value of the lock mass abundance to 0% on the Set device page of
the Diagnostics dialog box. See page 4-13. ▲
See “FT Analyzer Messages” on page A-3 on how to view information
about the instruments locking state. See “Using Locking in Automated
Runs” on page 5-2 on how to set FTMS locking in Instrument Setup.
MSn Settings
The table in this group box allows specifying the parameters for each
segment of an MSn experiment.1
Act. Type
The Activation Type list box becomes available
when you enter a parent mass. It allows specifying
how the ions are activated for fragmentation and
has the following options:
•
CID (Collision-induced dissociation)
•
PQD (Pulsed-Q dissociation)
•
ETD (electron transfer dissociation)1
Use ETD to fragment peptides and proteins.
•
Normalized
Collision Energy
HCD (higher energy CID)
To use HCD you must select FTMS in the
Analyzer list box. Use HCD to obtain triple
quadrupole-like fragment ion spectra. HCD is
available only for the final step in an
MSn experiment – it is not possible to set up
an experiment where the first activation
method is HCD and the second method is
CID, for example. If you enter a new step
after an HCD experiment, Tune Plus will
change it to a CID experiment.
When ETD is selected in the Activation Type list
box, the Normalized Collision Energy spin box is
disabled. For ETD activation, no RF amplitude is
used to fragment ions.1
1
This feature is available only for the LTQ Orbitrap Velos ETD.
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
Scan Mode Menu
1Act.
Q
When HCD is selected in the Activation Type list
box, the Activation Q spin box is disabled. For
HCD activation, no Q value is used for
calculating voltages or amplitudes.
The Activation Q spin box is also disabled for
ETD activation.1
HCD Charge State The required absolute collision energy for the
fragmentation of precursor ions depends on their
charge states. A lower collision energy is required
for higher charge states. The algorithm for
calculating the absolute collision energy is based
on empirical data taken from measurements on
peptides. For example, the required absolute
energy to fragment [M+2H]2+ ions is about 75%
of that of the corresponding [M+H]+ ions. For
[M+3H]3+ ions, the value goes down to 60%.
To take advantage of this, enter the charge state of
the ions to be fragmented into the spin box. To
change the displayed value, click the arrows in the
spin box to increment [up arrow] or decrement
[down arrow] the value. Alternatively, you can
enter a value in the spin box text field. You can set
the HCD charge state to any value from 1 to 99.
The default value is 1.
The HCD Charge State spin box is available only
if HCD is selected as activation type, regardless of
the status of the Advanced Scan features. See
Figure 2-10.
1
This feature is available only for the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-13
Tune Plus Window
Scan Mode Menu
Figure 2-10. Define Scan dialog box with HCD selected as activation type
Scan Ranges
When HCD is selected as the activation method in the MSn Settings
area, the First Mass (m/z) is set to either 0.05×LastMass or 100,
whichever is higher.
Centroid/Profile
This pair of buttons allows toggling between the Centroid and the
Profile format. The Profile format for FTMS data is a compressed
Profile format. “FT Profile Mode” on page 4-10 describes how to switch
to full Profile format for FTMS data for diagnostic purposes.
For further information, see also “Data Size of FT Raw Files” on
page A-4.
Positive/Negative
This pair of buttons allows toggling between positive ion and negative
ion polarity. Different FT transfer, storage, and mass calibration
parameters are used for the different polarities.
2-14
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
Display Menu
Display Menu
This section describes the elements of the Display menu that are
different from the ion trap.
Spectrum Averaging
This toggle allows switching on or off spectrum averaging. If spectrum
averaging is enabled, the displayed spectrum is the moving average of
several spectra before and is shown in red. Averaging FTMS scans is an
averaging of transients. Use this functionality in analogy to ion trap
scans.
❖
To average FTMS scans
1. In the Tune Plus window, choose Display > Spectrum Averaging >
Settings… to display the Spectrum Averaging dialog box. See
Figure 2-11.
2. Enter the number of transients to average into the spin box.
3. Click OK to save your changes and close the dialog box.
Figure 2-11. Spectrum Averaging dialog box
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-15
Tune Plus Window
Setup Menu
Setup Menu
This section describes the elements of the Setup menu that are different
from the LTQ Velos.
FT Transfer Optics
The FT transfer parameters are only changed by an FT transmission
calibration, which is usually only necessary when the hardware of the
system has been modified. This dialog box displays the actual
FT readback values for the current scan mode. See Figure 2-12.
Figure 2-12. FT Transfer Optics dialog box
2-16
LTQ Orbitrap Velos Getting Started
❖
To open this dialog box
•
From the Tune Plus window, choose Setup > FT Ion Optics, or
•
click
in the Instrument Control toolbar.
Thermo Fisher Scientific
Tune Plus Window
Setup Menu
FT Injection Control
The Injection Control dialog box allows setting the automatic gain
control (AGC) target values. In addition, the Injection Control dialog
box allows enabling or disabling the injection waveforms.
❖
To open this dialog box
•
From the Tune Plus window, choose Setup > FT Injection Control,
or
•
click
in the Instrument Control toolbar.
The Injection Control dialog box has two pages to enable the
independent selection of target values for ion trap scans and FT scans.
Ion Trap Page
Recommended target values for the ion trap:
Full MS Target:
SIM Target:
MSn Target:
Zoom Target:
3e+04
1e+04
1e+04
3000
Figure 2-13. Ion Trap page of the Injection Control dialog box
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
2-17
Tune Plus Window
Setup Menu
FT Page
For FTMS measurements, only the Full MS target, the SIM target, and
the MSn target are used.
Recommended target values for the FT analyzer:
Full MS Target:
SIM Target:
MSn Target:
1e+06
5e+04
5e+04
Note Lower target values than those listed above may be used to obtain
shorter inject times. For MSn scans, lower target values may also
improve the isolation/fragmentation efficiency. Higher target values
than those listed above can be used to improve the dynamic range.
However, target values far above the recommended settings may affect
isolation/fragmentation efficiency and mass accuracy for the
FTMS analyzer. ▲
Figure 2-14. FT page of the Injection Control dialog box
Enable Full Scan Injection Waveforms
You can enable or disable the injection waveforms for FT scans. For ion
trap scans, the injection waveforms are always enabled.
If the injection waveforms are enabled, a filter is applied on the ions that
are injected into the ion trap. The ions above and below the selected ion
or ion range selected are rejected. This option is often useful if the ion
trap is being filled with ions of greater or lesser mass than the ion mass
or ion mass range of interest. For example, this option can be used to
remove high mass ions that are not of interest and to ensure that more
target ions can enter the trap before the trap is full.
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LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
Setup Menu
Note The FT injection waveforms option only applies to full scan
MS scans performed with the Orbitrap mass analyzer. In FT SIM and
FT MSn scans, the injection waveforms are automatically enabled. ▲
FT Vacuum
The Vacuum dialog box allows monitoring the vacuum system
parameters. The Vacuum dialog box has two pages to enable an
independent selection of displaying the vacuum data of the ion trap or
the FT part.
Figure 2-15. Ion Trap page of the Vacuum dialog box
Figure 2-16. FT page of the Vacuum dialog box
Thermo Fisher Scientific
❖
To open this dialog box
•
From the Tune Plus window, choose Setup > Vacuum…, or
•
click
in the Instrument Control toolbar.
LTQ Orbitrap Velos Getting Started
2-19
Tune Plus Window
Setup Menu
FT Temperature Monitor
The FT Temperature Monitor dialog box allows to view the status of the
FTMS analyzer temperature regulation. Deviations of the actual
temperature from the temperature setpoint can affect instrument
performance. It is not possible to operate the instrument when the
bakeout procedure is active.
Figure 2-17. FT Temperature Monitor dialog box
❖
To open this dialog box
•
From the Tune Plus window, choose Setup > FT Temperature
Monitor…, or
•
click
in the Instrument Control toolbar.
FT Lock Masses
Choose Setup > FT Lock Masses to display the Lock Masses dialog
box. See Figure 2-9 on page 2-11. This dialog box allows entering and
editing lock mass lists for positive and negative ion mode. See “Locking”
on page 2-11 for details.
2-20
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Tune Plus Window
Tune Methods
Tune Methods
Several parameters, like the ion source parameters, ion trap ion optics
parameters, or AGC target values are stored in tune methods. This topic
points out for which parameters a differentiation between ion trap and
FTMS is made. The Tune Plus title bar displays the name of the current
tune method. If you are not currently editing a preexisting tune method,
the title bar displays the word Untitled.
Parameters with Differentiation between Ion Trap and FT Scans
A differentiation between ion trap scans and FTMS scans is made for
the following tune parameters.
AGC Target Values
They can be set and saved independently for these experimental modes
(no differentiation between positive and negative ion polarity mode):
•
Ion Trap Full MS Target
•
Ion Trap SIM Target
•
Ion Trap MSn Target
•
Ion Trap Zoom Target
•
FT Full MS Target
•
FT SIM Target
•
FT MSn Target
Microscans and Maximum Inject Time
They can be set and saved independently for these experimental modes:
Thermo Fisher Scientific
•
Ion Trap Full MS, positive ion mode
•
Ion Trap SIM, positive ion mode
•
Ion Trap MSn, positive ion mode
•
Ion Trap Zoom, positive ion mode
•
FT Full MS, positive ion mode
•
FT SIM, positive ion mode
•
FT MSn, positive ion mode
•
Ion Trap Full MS, negative ion mode
LTQ Orbitrap Velos Getting Started
2-21
Tune Plus Window
Tune Methods
•
Ion Trap SIM, negative ion mode
•
Ion Trap MSn, negative ion mode
•
Ion Trap Zoom, negative ion mode
•
FT Full MS, negative ion mode
•
FT SIM, negative ion mode
•
FT MSn, negative ion mode
Inject Waveform Flags
The flag whether the inject waveform is enabled or disabled can be set
and saved independently for
•
Ion trap scans
•
FT full scans.
Parameters without Differentiation between Ion Trap and FT Scans
No differentiation between ion trap scans and FT scans is made for all
ESI parameters, and for all ion source and ion optics parameters.
Parameters not saved in a Tune Method
All parameters that can be set in an instrument method are not saved in
the tune method. Thus, the following parameters are not saved in a tune
method:
•
Analyzer (Ion Trap or FTMS)
•
Mass Range (Low, Normal, or High)
•
Scan Rate
•
Resolution
•
Scan Type (Full, SIM, SRM, CRM)
•
Scan Range
•
Polarity* (positive or negative)
•
Data type* (centroid or profile)
* Only the data format (centroid or profile) and the ion polarity are
saved in a tune file that are set after a new start of Tune Plus.
2-22
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for
FTMS Measurements
Chapter 3
This chapter provides procedures to calibrate the LTQ Orbitrap Velos
for FTMS measurements. It contains the following topics:
Thermo Fisher Scientific
•
“Preliminary Remarks” on page 3-2
•
“Calibration Files and their Backups” on page 3-3
•
“Calibration Solutions” on page 3-4
•
“Calibration and Tuning of the Ion Trap” on page 3-11
•
“Automatic Calibration Page” on page 3-15
•
“Semi-Automatic Calibration Page” on page 3-17
•
“Check Calibration Page” on page 3-19
•
“FT Manual Calibration Page” on page 3-22
LTQ Orbitrap Velos Getting Started
3-1
Calibrating the Instrument for FTMS Measurements
Preliminary Remarks
Preliminary Remarks
There are no specific tune procedures for the FTMS part. All FTMS ion
transfer and excitation parameters are treated as calibration parameters
and are determined in automatic calibration procedures.
In the automatic calibration, the FT transmission calibration and the
FT mass calibration are automatically performed for all calibration
ranges. In the semi-automatic calibration, it is possible to decide
whether the transmission and/or mass calibration are performed only for
the positive ion mode, only for the negative ion mode or for both
polarities. See “Automatic Calibration Page” on page 3-15 and
“Semi-Automatic Calibration Page” on page 3-17 for further details.
On the FT Manual page of the Calibrate dialog box, you can select your
own calibration masses for FT ion transmission, storage transmission,
and FT mass calibration. See “FT Manual Calibration Page” on
page 3-22 for further details.
Note Thermo Fisher Scientific recommends using the semi-automatic
calibration. ▲
3-2
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration Files and their Backups
Calibration Files and their Backups
After a successful or partly successful calibration, the ion trap and
FT calibration parameters are saved automatically. All ion trap and
FT calibration parameters are stored in the calibration file
master.LTQCal, which is located in the folder:
C:\Thermo\Instruments\LTQ\system\msx
Backup Current Calibration
It is possible to create a backup of the current calibration file manually
or by choosing File > Backup Current Calibration in the Tune Plus
window. The Backup Current Calibration and Restore Backup
Calibration items work by copying the master.LTQCal to user.LTQCal
and vice versa.
If a backup calibration user.LTQCal was already generated, the old
user.LTQCal will be backed-up to a file named userXYZ.LTQCal. If
you perform backup calibrations at regular intervals, then a history of
your calibration files is generated in the folder:
C:\Thermo\Instruments\LTQ\system\msx
Using the Backup Calibration command regularly allows to return to
previous calibrations in case a new calibration is suspected to have
worsened instrument performance.
Restore Backup Calibration
Upon Restore Backup Calibration, the calibration values saved in
user.LTQCal are automatically downloaded to the instrument.
Therefore, it is recommended to generate a current backup after a
successful calibration.
It is also recommended to use the Restore Backup Calibration
command instead of the Restore Factory Calibration command
because the backup calibration file is newer than the factory calibration
file.
Note Pressing the reset button of the instrument loads the
master.LTQCal file into the internal computer of the instrument. ▲
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-3
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
Calibration Solutions
This section provides information about preparing the calibration
solutions for the LTQ Orbitrap Velos.
The positive ion mode calibration solution allows calibrating Thermo
Scientific MS detectors with ESI source in positive ion mode.
Supported instruments are the LTQ Orbitrap Velos, other LTQ based
hybrid instruments (LTQ FT, LTQ FT Ultra, and LTQ Orbitrap series),
and the Exactive. The positive ion mode calibration solution covers a
mass range from m/z 74 to m/z 1822 and is therefore usable for
calibrations between m/z 50 and m/z 2000.
The negative ion mode calibration solution allows calibrating Thermo
Scientific MS detectors with ESI source in negative ion mode.
Supported instruments are the LTQ Orbitrap Velos, other LTQ based
hybrid instruments (LTQ FT, LTQ FT Ultra, and LTQ Orbitrap series),
and the Exactive. The negative ion mode calibration solution covers a
mass range from m/z 265 to m/z 1880 and is therefore usable for
calibrations between m/z 50 and m/z 2000.
Chemicals for Preparing Calibration Solutions
The n-butylamine, caffeine, MRFA, Ultramark 1621, sodium dodecyl
sulfate, and sodium taurocholate needed to make the calibration
solutions are supplied with your chemical accessory kit. When ordering
replacements, use the information listed in Table 3-1.
Table 3-1.
Calibration compounds for LTQ Orbitrap Velos
Description
Quantity
Supplier Product Number
Supplier: Sigma Chemical Company, see below.
n-Butylamine*
25 mL
471305-25ML
Caffeine Methanol Solution
1 mL
C6035-1ML
Met-Arg-Phe-Ala acetate salt
5 mg
M1170-5MG
Sodium Dodecyl Sulfate
10 g
L4509-10G
Sodium Taurocholate Hydrate
250 mg
T4009-250MG
Supplier: ABCR GmbH & Co. KG, see below.
Ultramark® 1621 Mass Spec. Standard
* If
3-4
LTQ Orbitrap Velos Getting Started
250 mg
AB172435
ordering elsewhere, use only mass spec grade quality.
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
To order more of these compounds, contact:
Sigma Chemical Company
P. O. Box 14508
St. Louis, Missouri, USA 63178-9916
Phone
(800) 325-3010 (in the U.S. & Canada)
(314) 771-3750 (U.K. & International)
Web site www.sigma-aldrich.com
or
ABCR GmbH & Co. KG
Im Schlehert 10
D-76187 Karlsruhe, Germany
Phone
+49 (0)721 950 61-0
Fax
+49 (0)721 950 61-80
Email
info@abcr.de
Web site www.abcr.de/english.htm
Solvents and Modifiers
You can also order specific chemicals from Thermo Fisher Scientific,
which are sold under its Fisher Chemical brand. As specified in
Table 3-2 use only LCMS grade chemicals for calibrating your
LTQ Orbitrap Series system.
Table 3-2. Recommended solvents and reagents
Solvent / Reagent
Specifications
Fisher Chemical P/N
Methanol
LCMS grade
A456-4
Acetonitrile
LCMS grade
A955-4
Water
LCMS grade
W6-4
Isopropyl alcohol
LCMS grade
A461-4
Acetic acid (modifier)
LCMS grade
A507-500 or A35-500
Formic acid (modifier)
99–100% (This acid must be
supplied in a glass bottle.)
A117-50
For a complete selection of LCMS-grade consumables from Fisher
Scientific, visit www.FisherLCMS.com.
Safety Advice
Potentially hazardous chemicals used in procedures throughout this
chapter include the following:
Thermo Fisher Scientific
•
Glacial acetic acid
•
Acetonitrile
LTQ Orbitrap Velos Getting Started
3-5
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
•
Methanol
•
Formic acid
Note Store and handle all chemicals in accordance with standard safety
procedures. The Material Safety Data Sheets (MSDS) describing the
chemicals being used are to be freely available to lab personnel for them
to examine at any time. Material Safety Data Sheets (MSDS) provide
summarized information on the hazard and toxicity of specific chemical
compounds. The MSDS also provides information on the proper
handling of compounds, first aid for accidental exposure, and
procedures for the remedy of spills or leaks. Producers and suppliers of
chemical compounds are required by law to provide their customers
with the most current health and safety information in the form of an
MSDS. Read the material safety data sheets for each chemical you use. ▲
Caution Please consider the following cautions when preparing
calibration solutions:
•
Do not filter solvents. Filtering solvents can introduce
contamination.
•
Do not use plastic pipettes to prepare your tuning and calibration
standards. Plastic products can release phthalates that can interfere
with your analyses. ▲
Preparing Stock Solutions
Use the chemicals described in the previous section to prepare the
calibration solutions from the following stock solutions:
•
”Caffeine Stock Solution”, next topic
•
“MRFA Stock Solution” on page 3-7
•
“Ultramark 1621 stock solution” on page 3-7
•
“N-Butylamine Stock Solution” on page 3-7
•
“Sodium Dodecyl Sulfate Stock Solution” on page 3-8
•
“Sodium Taurocholate Stock Solution” on page 3-8
Warning Avoid exposure to potentially harmful materials.
Always wear protective gloves and safety glasses when you handle
solvents or corrosives. Also contain waste streams and use proper
ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS)
for proper handling of a particular solvent. ▲
3-6
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
Caffeine Stock Solution
A 1 mg/mL stock solution of caffeine in 100% methanol is provided
with your LTQ Orbitrap Series MS detector. You can also order this
solution through Sigma. The Sigma product number for this solution is
C6035.
MRFA Stock Solution
❖
To prepare the MRFA stock solution
1. Obtain the vial of L-methionyl-arginyl-phenylalanyl-alanine
acetate × H2O (MRFA) in your accessory kit. In this form, the
MRFA sample has an average molecular weight of 523.7 u.
Carefully weigh 3.0 mg of the MRFA sample.
2. Dissolve the MRFA sample in a total volume of 1.0 mL of 50:50
methanol:water. Mix the solution (5.0 nmol/μL) thoroughly.
3. Transfer 50 μL of the 5 nmol/μL solution into a clean polypropylene
tube.
4. Add 1.45 mL of 50:50 methanol:water to the tube. Mix this
solution (166.7 pmol/μL) thoroughly.
5. Label the tube MRFA stock solution and store it in a freezer until it is
needed.
Ultramark 1621 stock solution
❖
To prepare the Ultramark 1621 stock solution
1. Obtain the vial of Ultramark 1621 in your accessory kit.
2. Using a syringe, measure out 10 μL of Ultramark 1621, and dissolve
it in 10 mL of acetonitrile.
3. Mix the solution thoroughly.
4. Label the vial Ultramark 1621 stock solution and store it in a freezer
until it is needed.
N-Butylamine Stock Solution
❖
To prepare the n-butylamine stock solution
1. Using a syringe, transfer 5 μL of n-butylamine to a 25 mL
(minimum) volumetric glass flask.
2. Add 9995 μL of 50:50 methanol/water to the flask.
3. Mix the solution thoroughly.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-7
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
4. Transfer the solution to a vial.
5. Label the vial N-butylamine stock solution (5/1000 dilution).
Sodium Dodecyl Sulfate Stock Solution
❖
To prepare the sodium dodecyl sulfate stock solution
1. Obtain the vial of sodium dodecyl sulfate. In this form, the sample
has an average molecular weight of 288.4 u.
2. Prepare the stock solution of sodium dodecyl sulfate by dissolving
2.88 mg in 10 mL of 50:50 methanol:water.
3. Mix the solution (1.0 nmol/μL) thoroughly.
4. Label the vial Sodium Dodecyl Sulfate stock solution (1 nmol/μL).
Sodium Taurocholate Stock Solution
❖
To prepare the sodium taurocholate stock solution
1. Obtain the vial of sodium taurocholate. In this form, the sample has
an average molecular weight of 537.7 u.
2. Prepare the stock solution of sodium taurocholate by dissolving
5.38 mg in 10 mL of 50:50 methanol:water.
3. Mix the solution (1.0 nmol/μL) thoroughly.
4. Label the vial Sodium Taurocholate stock solution (1 nmol/μL).
LTQ/FT-Hybrid Positive Ion Mode Calibration Solution
The LTQ/FT-hybrid positive ion mode calibration solution consists of
caffeine, MRFA, Ultramark 1621, and n-butylamine in an
acetonitrile:methanol:water solution containing 1% acetic acid.
❖
To prepare the positive ion mode calibration solution
1. Pipet 20 μL of the caffeine stock solution into a light-protected,
clean, dry 10 mL volumetric flask.
2. Pipet 100 μL of the MRFA stock solution into the flask.
3. Pipet 100 μL of the Ultramark 1621 stock solution into the flask.
4. Pipet 100 μL of the stock solution of n-butylamine into the flask.
3-8
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
Caution Use only glass pipets or stainless steel syringes when measuring
glacial acetic acid. Using plastic pipet tips causes contamination of acid
stock solutions that can introduce contaminants in the calibration
solution. ▲
5. Pipet 100 μL of glacial acetic acid into the flask.
6. Pipet 5 mL of acetonitrile into the flask.
7. Bring the volume of the solution up to the 10 mL-mark on the flask
with 50:50 methanol:water.
8. Mix the calibration solution thoroughly.
9. Transfer the solution to a light-protected, clean, dry vial.
10. Label the vial Positive Ion Mode Calibration Solution and store it in a
freezer until it is needed.
LTQ/FT-Hybrid Negative Ion Mode Calibration Solution
The LTQ/FT-hybrid negative ion mode calibration solution consists of
sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1621 in an
acetonitrile:methanol:water solution containing 1% acetic acid.
❖
To prepare the negative ion mode calibration solution
1. Pipet 100 μL of the sodium dodecyl sulfate stock solution into a
light-protected, clean, dry 10 mL volumetric flask.
2. Pipet 100 μL of the sodium taurocholate stock solution into the
flask.
3. Pipet 100 μL of the Ultramark 1621 stock solution into the flask.
Caution Use only glass pipets or stainless steel syringes when measuring
glacial acetic acid. Using plastic pipet tips causes contamination of acid
stock solutions that can introduce contaminants in the calibration
solution. ▲
4. Pipet 100 μL of glacial acetic acid into the flask.
5. Pipet 5 mL of acetonitrile into the flask.
6. Bring the volume of the solution up to the 10 mL-mark on the flask
with 50:50 methanol:water.
7. Mix the solution thoroughly.
8. Transfer the solution to a light-protected, clean, dry vial.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-9
Calibrating the Instrument for FTMS Measurements
Calibration Solutions
9. Label the vial Negative Ion Mode Calibration Solution and store it in
a freezer until it is needed.
Applicable Calibration Solutions for FT Manual Calibration
Because the FT Manual page of the Calibrate dialog box allows using
your own calibration masses, it is possible to use custom calibration
solution here. However, there are some requirements for the calibration
masses. The scan ranges of the instrument need to be covered properly
by the given masses.
3-10
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration and Tuning of the Ion Trap
Calibration and Tuning of the Ion Trap
This chapter describes the calibration and tuning of the ion trap for
FT measurements.
Calibration of the Ion Trap
To perform an FT calibration, the ion trap has to be successfully
calibrated before. It is very important that the electron multiplier gain is
correctly calibrated because the AGC prescan is performed in the ion
trap. Thus, the electron multiplier gain calibration should be checked
before an FT calibration is performed.
For the ion trap calibrations, the positive ion mode calibration solution
or the negative ion mode calibration solution can be used.
Tuning the Ion Trap for Positive Ion Mode
For the positive ion mode, it is recommended to perform an automatic
tune of m/z 524 at a Full MS Target of 1e4–3e4. Use the positive ion
mode calibration solution or the negative ion mode calibration solution
with the settings in the Define Scan dialog box that are shown on
Figure 3-1 on page 3-12.
The spectrum should look similar to the spectrum shown in Figure 3-2
on page 3-12 and Figure 3-3 on page 3-13. Make sure the peaks at
m/z 74, 138, 195, 524, and the highest Ultramark peaks are all present,
ideally above 30% of the base peak.
The inject time should be stable and less than 1 ms (if a Full MS target
of 1e4 is used). Do not forget to save the tune method after a successful
tuning.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-11
Calibrating the Instrument for FTMS Measurements
Calibration and Tuning of the Ion Trap
Figure 3-1.
Recommended settings in the Define Scan dialog box for an automatic tune of the ion trap
* Label for m/z 138.00 added manually
*
Figure 3-2.
3-12
Ion trap spectrum of positive ion mode calibration solution, scan range m/z 130–2000, positive ion
polarity mode
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Calibration and Tuning of the Ion Trap
Caffeine fragment peak
n-Butylamine peak
Figure 3-3.
Ion trap spectrum of the positive ion mode calibration solution (lower range), positive ion polarity mode
Tuning the Ion Trap for Negative Ion Mode
For the negative ion mode, it is recommended to perform an automatic
tune of m/z 514 at a Full MS Target of 1e4–3e4. Use the negative ion
mode calibration solution with the settings shown in Figure 3-1 on
page 3-12.
After the automatic tuning, a manual adjustment of the S-lens RF level
should be used to get an ion trap spectrum in the scan range
m/z 150–2000. At m/z 265 is the base peak (100%) and the highest
Ultramark adduct ion peaks are at about 80%, as shown in Figure 3-4
on page 3-14.
The S-lens RF level affects the mass spectrum as follows:
•
Thermo Fisher Scientific
Decreasing the S-lens RF level will decrease the amount of
fragmentation of fragile ions in the S-lens.
Decreasing the S-lens RF level will decrease the transmission of high
m/z ions through the S-lens and increase the transmission of the low
m/z ions.
LTQ Orbitrap Velos Getting Started
3-13
Calibrating the Instrument for FTMS Measurements
Calibration and Tuning of the Ion Trap
•
Increasing the S-lens RF level will increase the amount of
fragmentation of fragile ions in the S-lens.
Increasing the S-lens RF level will increase the transmission of high
m/z ions through the S-lens and decrease the transmission of the low
m/z ions.
If the S-lens RF level is set very low, in-source fragmentation of the
Ultramark adduct ions may occur. Thus, if you observe rather ions at
m/z 906, 1006, … than ions at m/z 1280, 1380, … the S-lens RF level
has to be increased. The inject time should be stable and less than 1 ms
(if a target of 1e4 is used). Do not forget to save the tune method after a
successful tuning.
# 3906 IT: 0.426 ST: 0.83 uS: 5 NL: 8.25E5
F: ITMS - p ESI Full ms [ 150.00-2000.00]
265.2
100
95
90
85
80
75
514.4
70
1579.6
65
60
Rel
ativ55
e
Inte
50
nsit
y
45
1679.5
1479.7
40
1779.5
1379.7
35
30
25
1106.1 1206.1
20
1006.1
15
1306.0
1785.5
1406.0
10
1179.8
906.1
5
1879.5
1279.8
210.9
379.9
0
200
400
470.1
596.4
600
678.3
806.1
800
994.1
1885.5
1505.8
1079.9
1000
1200
1400
1600
1800
2000
m/z
Figure 3-4.
3-14
Ion trap spectrum of the negative ion mode calibration solution, scan range m/z 150–2000, negative ion
polarity mode
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Automatic Calibration Page
Automatic Calibration Page
The Automatic page of the Calibrate dialog box allows performing an
automatic calibration of all the calibration parameters, including all ion
trap calibrations and all FT calibrations. See Figure 3-5.
Figure 3-5.
Automatic page of the Calibrate dialog box
In an automatic calibration, the FT calibration procedures are
performed automatically one after another, following the ion trap
calibration. To perform an automatic calibration, the negative ion mode
calibration solution has to be used.
The calibration masses and all experimental parameters (for example,
target values, scan ranges, or resolution settings) are set automatically
and cannot be influenced by the user.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-15
Calibrating the Instrument for FTMS Measurements
Automatic Calibration Page
Note Usually, you do not need to perform a complete ion trap
calibration or an FT ion transmission calibration unless the hardware is
modified in some way. However, it is necessary to repeat the electron
multiplier calibration and the FT mass calibration on a regular basis.
Thus, in the most cases it is not recommended to perform an automatic
calibration of the LTQ Orbitrap Velos. In this case, all calibrations are
performed, which takes about 1 hour. To run a multiplier gain
calibration or an FT mass calibration (which takes only some minutes),
it is recommended to use the semiautomatic calibration. ▲
3-16
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Semi-Automatic Calibration Page
Semi-Automatic Calibration Page
The Semi-Automatic page of the Calibrate dialog box allows selecting
specific calibration parameters to calibrate, for example only the ion trap
calibrations or only the FT calibrations. (See Figure 3-6.) For
FT calibrations, it is also possible to differentiate between positive and
negative ion mode.
Figure 3-6.
Semi-Automatic page of the Calibrate dialog box
To calibrate one or more selected parameters, clear the Select All check
box to make the individual calibration parameters available. Select the
parameter(s) you want to calibrate, then click Start.
For example:
•
Thermo Fisher Scientific
To run a complete automatic calibration (ion trap and FT), select
the Select All check box. Then, click Start. This is analogous to the
automatic calibration. As already described before, it is not
recommended to perform an automatic calibration of the
LTQ Orbitrap Velos if not necessary because all calibrations are
performed, which takes about 1 hour.
LTQ Orbitrap Velos Getting Started
3-17
Calibrating the Instrument for FTMS Measurements
Semi-Automatic Calibration Page
•
To run an automatic calibration of the ion trap, select the Select All
– Ion Trap check box. Then, click Start.
•
To run an automatic calibration of the FT part, select the Select All
– FT check box. Then, click Start.
•
To run an FT mass calibration, select the Mass Calibration check
box. Then, click Start.
In a semi-automatic calibration, the selected FT calibration procedure(s)
are performed automatically one after another.
All calibrations apart from the FT calibrations for the negative ion mode
can be performed with either the positive ion mode calibration solution
or the negative ion mode calibration solution. To run the FT ion
transmission and/or the mass calibration for the negative ion mode, the
negative ion mode calibration solution has to be used.
The calibration masses and all experimental parameters like target
values, scan ranges, resolution settings, inject waveforms etc. are set
automatically and cannot be influenced by the user.
HCD Calibration
The Semi-Automatic page of the Calibrate dialog box allows calibrating
the HCD collision energy and the HCD transmission for either ion
mode.
Note Calibrate the HCD transmission before you calibrate the
HCD collision energy. ▲
HCD Collision Energy
This check box allows specifying whether to perform a calibration of the
higher energy collision-induced dissociation (HCD) energy of the
Orbitrap mass analyzer in positive ion mode.
HCD Transmission
This check box allows specifying whether to perform a calibration of the
higher energy collision-induced dissociation (HCD) transmission
efficiency of the Orbitrap mass analyzer in positive ion mode.
3-18
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Check Calibration Page
Check Calibration Page
The Check page of the Calibrate dialog box allows automatically
checking several calibration settings. See Figure 3-7.
Figure 3-7.
Check page of the Calibrate dialog box
All calibration checks apart from those for the FT negative ion mode
can be performed with either the negative ion mode calibration solution
or the positive ion mode calibration solution. To check the
FT calibrations in the negative ion mode, the negative ion mode
calibration solution has to be used.
The calibration masses and all experimental parameters like target
values, scan ranges, resolution settings, etc. are set automatically.
At the conclusion of the check procedure, the LTQ Orbitrap Velos
displays a message that indicates whether the parameter(s) are calibrated
properly or not.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-19
Calibrating the Instrument for FTMS Measurements
Check Calibration Page
Using the Check page of the Calibrate dialog box, you can select the
following parameters:1
Select All
This check box allows specifying whether or
not to check all calibration parameters. To
check all calibration parameters, select the
Select All check box. In this case, all ion trap
calibration parameters and all FT calibration
parameters are checked. You can also check
each calibration parameter individually. To
make the individual calibration parameters
available, clear the Select All check box.
Select All Ion Trap
This check box allows specifying whether or
not to check the calibration of only the ion
trap parameters.
1
Reagent Ion Selection This check box allows specifying whether or
not to check the reagent ion source selection.
Select All-FT
This check box allows specifying whether or
not to check the calibration of only the FT ion
transfer optics and mass analyzer.
Reagent Ion Source
This check box allows specifying whether or
Transfer Multipole RF not to check the frequency of the RF voltage of
Frequency1
the transfer multipole in the reagent ion source
optics.
Transfer Multipole RF This check box allows specifying whether or
Frequency
not to check the frequency of the RF voltage of
the transfer multipole in the FT transfer ion
optics.
Storage Multipole RF This check box allows specifying whether or
Frequency
not to check the frequency of the RF voltage of
the storage multipole in the FT transfer ion
optics.
Positive Ion Mode
This check box allows specifying whether or
not to check the FT ion transmission
calibration and FT mass calibration for the
positive ion mode.
Negative Ion Mode
This check box allows specifying whether or
not to check the FT ion transmission
calibration and FT mass calibration for the
negative ion mode.
1
This feature is available only in the LTQ Orbitrap Velos ETD.
3-20
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
Check Calibration Page
Storage Transmission
This check box allows specifying whether or
not to check the ion storage transmission
calibration. The storage transmission is
checked by transferring ions form the ion trap
to the ion storage device and back, then
scanning in the ion trap. The FT storage
transmission calibration can be checked for the
positive and negative ion mode independently.
FT Transmission
This check box allows specifying whether or
not to check the FT ion transmission
calibration. The ion transmission from the ion
trap to the Orbitrap is checked by means of
the calibration masses in SIM experiments at
different AGC target values. The
FT transmission calibration can be checked for
the positive and negative ion mode
independently.
Mass Calibration
This check box allows specifying whether or
not to check the mass calibration of the
Orbitrap mass analyzer. By doing so, the
current mass calibration is checked, i.e it is a
check of the external mass calibration. The
FT mass calibration can be checked for the
positive and negative ion mode independently.
HCD Collision Energy This check box allows specifying whether or
not to check the HCD collision energy
calibration. To check the HCD collision
energy calibration, first make this check box
available by clearing the Select All check box.
Then, select the HCD Collision Energy check
box.
HCD Transmission
This check box allows specifying whether or
not to check the HCD transmission
calibration. To check the HCD transmission
calibration, first make this check box available
by clearing the Select All check box. Then,
select the HCD Transmission check box.
The Last Check Date readback column gives the date of the last
successful check for each item. If a check is performed that fails, the last
successful check date still appears in the Last Check Date readback
column. The last successful check continues to be in effect in the
instrument. However, the result column will show a red x mark
indicating that the current attempt check has failed or was aborted.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-21
Calibrating the Instrument for FTMS Measurements
FT Manual Calibration Page
FT Manual Calibration Page
The FT Manual page of the Calibrate dialog box allows performing or
checking an FT mass calibration with user-defined calibration masses.
See Figure 3-8.
Figure 3-8.
FT Manual page of the Calibrate dialog box
All experimental parameters like target values, scan ranges, resolution
settings, etc. must be set manually.
Note When starting from the FT Manual page, the calibration is
performed for the currently selected polarity only. ▲
3-22
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Calibrating the Instrument for FTMS Measurements
FT Manual Calibration Page
Mass List Group Box
The calibration masses for manual calibration can be defined in the
corresponding mass list on the FT Manual page of the Calibrate dialog
box. Mass lists can be imported and exported via the Instrument
Configuration page. See further details in Chapter 6: “Instrument
Configuration”.
Note Ensure that you have calculated the calibration masses with
sufficient accuracy (sub ppm). ▲
Name
This list box lists the names of the factory supplied and user
created mass lists.
Mass List
This table lists the mass-to-charge ratios of the ions that
you are using to calibrate the Orbitrap mass analyzer. You
can select an existing mass list in the Name list box, or you
can create or modify a mass list by clicking on it and editing
the entries in the Mass List table.
Save
Click Save to save the mass list with the name that is
selected in the Name list box.
Save As
Click Save As a to save the mass list with a new name.
Delete
Click Delete to delete the mass list that is selected in the
Name list box.
Factory-Supplied Mass Lists
There are also two factory supplied mass lists, calmix_positive (factory)
and calmix_negative (factory). They contain the exact masses of all main
ion peaks, which should appear if the negative ion mode calibration
solution is used in positive or negative ion mode, respectively.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
3-23
Chapter 4
Performing Diagnostics/Checks
This chapter describes several diagnostic procedures for the
LTQ Orbitrap Velos. It contains the following topics:
Thermo Fisher Scientific
•
“System Evaluation Procedures” on page 4-2
•
“Toggles” on page 4-8
•
“Set Device” on page 4-12
•
“Display Settings” on page 4-15
LTQ Orbitrap Velos Getting Started
4-1
Performing Diagnostics/Checks
System Evaluation Procedures
System Evaluation Procedures
The System evaluation page in the Diagnostics dialog box allows
evaluating the system performance. See Figure 4-1.
Figure 4-1.
System evaluation page of the Diagnostics dialog box (LTQ Orbitrap Velos)
❖
To display this page
From the Tune Plus window, choose Diagnostics > Diagnostics >
Tools > System evaluation.
Besides several ion trap relevant system evaluation procedures, you can
perform various FT system evaluation procedures, which are described
in the following topics.
FT CLT-RF Pulser Evaluation1
This procedure determines the pulser timing characteristics of the
CLT RF board. This procedure requires the infusion of the calibration
solution in positive ion polarity mode.
1
This feature is available only in the LTQ Orbitrap Velos ETD.
4-2
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
System Evaluation Procedures
FT Dynamic Range Test
This test is only applicable for an infusion experiment with a solution
containing MRFA, for example the positive ion mode calibration
solution or a MRFA alone solution (for example 5×10-6 M in
100% methanol/water, 1% acetic acid). This test determines the
signal-to-noise ratio of an isolated MRFA signal.
FT Energy Dependence Evaluation
This procedure determines the change in mass calibration when varying
the ion energy (HV Offset). This procedure can help evaluating the
FTMS analyzer components.
FT ETD Fragmentation Efficiency1
This procedure determines the ETD fragmentation efficiency, which is
the percentage of ions that fragment relative to the number of parent
ions. The test can run in both ion trap modes and FTMS modes. To run
the test, you must infuse a solution of Angiotensin I to generate a
spectrum that shows a triply-charged Angiotensin I ion at m/z 433 with
an intensity of greater than 5E+5.
FT HCD Collision Cell Ejection Evaluation
This procedure analyzes the dynamics of ions in the HCD collision cell.
This procedure can help evaluating the HCD collision cell components.
FT HCD Multipole Evaluation
This procedure evaluates the linearity and maximum amplitude of the
HCD multipole.
FT High Mass Range Target Compensation
This procedure determines an AGC target compensation factor, which
ensures that the FT mass calibration is still valid if the instrument is set
into the high mass range mode. The resulting compensation factor will
be saved in the calibration file. Usually, it is sufficient to run this
procedure once. It is not necessary to repeat this procedure on a regular
basis. It is recommended to use the positive ion mode calibration
solution for this test. However, you can also use any other solution that
gives reasonable ion signals at 1000 < m/z < 2000.
1
This feature is available only in the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-3
Performing Diagnostics/Checks
System Evaluation Procedures
FT Isolation Test
This test is only applicable for an infusion experiment with a solution
containing MRFA, for example the positive ion mode calibration
solution or a MRFA alone solution (for example 5×10-6 M in 100%
methanol/water, 1% acetic acid). This test is analogous to the “Check of
the ion isolation waveform” on the Check page of the Calibrate dialog
box. Here, the isolation of m/z 525.3 is performed at an AGC target of
2e+03 and analyzed by the ion trap. In contrast to this, the FT isolation
test is performed at higher targets and uses the FT analyzer. Thus this
test determines the maximum AGC target value that allows performing
a unit isolation of m/z 525.3 at the presence of m/z 524.3 and 526.3.
FT Noise Test
This test determines resistant noise peaks in the selected scan range. In
this test ions are “switched off ” automatically. At the conclusion of the
FT noise test, a list of resistant noise peaks is displayed in the Testing
text.
FT Preamp Evaluation
This evaluation allows checking the basic FTMS analyzer signal
detection path. The instrument needs to run in FTMS analyzer mode.
Thermo Fisher Scientific recommends switching to diagnostic transient
view. See “FT Include Transients” on page 4-9.
During the evaluation, the preamplifier input protection switches are
activated with a period of 100 ms. This switching can be observed as
periodic incidences in the transient if the electronic signal path is
operational.
FT Pulser Evaluation
This procedure tests characteristics of the high voltage pulser. This
procedure can help evaluating the FTMS analyzer electronics.
FT Reagent Ion Source Drift Time Evaluation1
This evaluation determines the drift time of reagent ions from the
reagent ion source to the linear trap mass analyzer. This procedure
requires the reagent ion source and filament emission to be on.
1
This feature is available only in the LTQ Orbitrap Velos ETD.
4-4
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
System Evaluation Procedures
FT Reagent Ion Source Transfer Multipole Evaluation1
This procedure evaluates the linearity and maximum amplitude of the
reagent ion transfer multipole.
FT Sensitivity Test
The FT sensitivity test is only applicable for an infusion experiment
with reserpine. The test assumes that a reserpine solution of 5×10-9 M
(100% methanol, 1% acetic acid) is used. The following test are
performed one after another:
1. SIM of m/z 609.3 using the ion trap as analyzer and an AGC target
of 2e+03.
2. SIM of m/z 609.3 using the Orbitrap detector as analyzer and an
AGC target of 5e+03.
3. SIM of m/z 609.3 using the Orbitrap detector as analyzer and an
AGC target of 5e+04.
4. MS/MS of m/z 609.3 using the Orbitrap detector and an
AGC target of 5e+04.
The test fails
•
if the inject time, which is necessary to reach the selected
AGC target value, is too high;
•
if the ratio of the reserpine signal to the overall signal inside the
SIM window is too low;
•
if the ion transmission from the ion trap to the Orbitrap detector is
too low, or
•
if the intensity of the product ions of reserpine is too low.
FT Stability Test
This test is applicable for an infusion experiment with any sample
solution. This test procedure checks the stability of the FT TIC (total
ion current) detected in the selected scan range by means of 600 scans.
In principle, the test can be performed at any experimental conditions.
It is recommended, however, to perform this test in Full scan mode
using one microscan, a resolution setting of 60000 and a FT Full MS
Target of 5e+05 or 1e+06. At the conclusion of the FT stability test, the
AGC stability and the corresponding signal variation is displayed. See
Figure 4-2.
1
This feature is available only in the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-5
Performing Diagnostics/Checks
System Evaluation Procedures
Figure 4-2.
Result of the FT stability test displayed in the Graph View
FT Temperature Control Evaluation
This evaluation procedure allows examining the temperature regulation
behavior of the instrument by intentionally driving temperatures to
extreme values.
Note The evaluation will usually take more than 12 hours where no
measurements can be done. After stopping the evaluation, the
instrument needs to stabilize temperatures for several hours before high
mass accuracy measurements can be started. ▲
FT Temperature Monitor
Mass accuracy of the Orbitrap detector with external mass calibration
depends on a stable temperature of the analyzer and the electronic
components. This evaluation plots a history of the temperature
regulation results to the Graph view. See “Graph View” on page 2-5.
4-6
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
System Evaluation Procedures
Reagent CI Gas Pressure Evaluation1
This evaluation procedure plots the CI gas pressure of the reagent ion
source against the anion intensity; the instrument then calculates the
optimum reagent gas pressure. Enter this value into the CI Gas Pressure
spin box of the Reagent Ion Source dialog box. See Figure 7-5 on
page 7-6.
Note Thermo Fisher Scientific recommends performing this procedure
after replacing the filament and/or the ion volume. ▲
1
This feature is available only in the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-7
Performing Diagnostics/Checks
Toggles
Toggles
The Toggles page in the Diagnostics dialog box allows toggling
(switching) a subsystem from one state to another state. See Figure 4-3.
Figure 4-3.
Toggles page of the Diagnostics dialog box
❖
To display this page
From the Tune Plus window, choose Diagnostics > Diagnostics >
Tools > Toggles.
Caution All toggles should only be used for diagnostic purposes. The
functionality of the LTQ Orbitrap Velos may be harmed if a toggle is
switched to a status that differs from its default value. ▲
If one of the FT toggles is (accidentally) different from its default value
during data acquisition, the FT Analyzer Settings of the Scan Header of
a raw file contains a reference to this. See Appendix A: “Miscellaneous
Information” for further details.
Note The status of a toggle is not saved in the tune method and is set
back to its default value after an instrument reset. The toggle state
shown by the radio buttons next to the list box does not necessarily
correspond to the actual settings. ▲
4-8
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
Toggles
FT Advanced Calibration
With this toggle, the Advanced Calibration Features are switched on.
The default setting is Off (disabled).
FT Analyzer Ion Gauge
With this toggle, the ion gauge for the FT analyzer vacuum can be
disabled manually for diagnostic purposes. The default setting is On
(enabled).
FT Analyzer Temperature Control
With this toggle, the FT analyzer temperature control regulation
electronics can be disabled for diagnostic purposes. The default setting is
On (enabled).
FT Apodization
With this toggle, the apodization can be switched on or off. The default
setting is On.
FT HCD Collision Gas
With this toggle, the HCD collision gas can be switched on or off. The
default setting is On. The default setting can be changed in the
Instrument Configuration application.
FT Include Transients
If this toggle is on, it is possible to display transients in the Spectrum
view by choosing Show FT Transient in the shortcut menu of the
Spectrum view. The menu is displayed when you right-click anywhere
on that page. See “Spectrum View” on page 2-3 for further details.
Note A transient view is only possible if profile (instead of centroid) is
chosen as data format. ▲
During transient display in Spectrum view, the x-coordinate is
misleadingly labeled with m/z instead of milliseconds. The default
setting is Off.
Note It is not possible to acquire transients into an Xcalibur raw file. ▲
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-9
Performing Diagnostics/Checks
Toggles
FT Manual Calibration for Single Range
This toggle can be used to influence the behavior of the FT manual
calibration procedures. See “FT Manual Calibration Page” on
page 3-22. In the default behavior, the FT manual calibration
procedures calibrate the whole scan mass range for the actual polarity.
To be able to use non-standard calibration substances that cover a
limited mass range only, advanced users may enable this toggle. With
this toggle enabled, there is no check for mass range coverage of
reference mass lists. Instead, the instrument stays in the chosen mass
range and calibrates this range only. With this toggle enabled, the user is
responsible to cover the whole mass range needed, possibly by
calibrating manually in several steps with different substances. If the
performed FT manual calibration is not suitable for the scan settings
used in an FTMS analyzer data acquisition, the scan header of a raw
data file contains a reference to this. See Chapter A: “Miscellaneous
Information” for further details.
FT Profile Mode
Use this toggle to select whether the FT profile mode corresponds to a
Full Profile format or to a Reduced Profile format. It is recommended to
use the Reduced Profile Mode for data acquisition because the data size
of the raw file is significantly decreased by using the Reduced Profile.
The default setting is Reduced. For further information, see also “Data
Size of FT Raw Files” on page A-4.
FT SIM and MSn Injection Waveforms
Usually, for FT SIM scans and FT MSn scans the injection waveforms
are automatically enabled. It is not possible to change this setting in the
Injection Control dialog box. With this toggle, it is possible to disable or
enable the injection waveforms manually for diagnostic purposes. The
default setting is On.
FT Storage Evaluation Mode
This toggle can be used to test the characteristics of ion transfer
components for the FTMS analyzer. The default setting is On
(enabled).
4-10
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
Toggles
FT View Frequency
If this toggle is switched on, the FT spectrum is shown as a frequency
spectrum. If the system is on and the FT is chosen as analyzer, the
frequency spectrum is displayed in the Spectrum view. The default
setting is Off.
Note The x-coordinate is misleadingly labeled with m/z instead of
kHz. ▲
This toggle is for diagnostic purposes only. Therefore, it is not possible
to acquire frequency spectra.
FT Zero Offset
If this toggle is switched on, an offset is added to the spectrum. This
enables to view the full noise band. The default setting is Off, if the
Reduced Profile format is used. The setting is On, if the Full Profile
format is used.
Isolate Reagent Ion
Enabling this toggle allows manually adjusting the Back Multipole
DC Offset voltage. See “Tuning the Quadrupole Mass Filter” on
page 7-21. The default setting is Off.1
Reagent Ion AGC
If this toggle is switched on, the instrument automatically performs an
AGC scan of the reagent ion source in regular intervals. The default
setting is Off.1
1
This feature is available only in the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-11
Performing Diagnostics/Checks
Set Device
Set Device
The Set device page of the Diagnostics dialog box allows selecting
devices or experimental parameters from the list and setting the value for
that device or parameter. See Figure 4-4.
❖
To set a device
1. Select the device or parameter you would like to set from the Device
list box.
2. Enter the device parameter's value in the text box below the Device
list box.
3. Click Set to apply the change to the device value or parameter.
Figure 4-4.
Set device page of the Diagnostics dialog box
Note The value in the text box below the Device list box, which is
displayed after the call of this page, does not necessarily correspond to
the actual value. ▲
The following topics describe several FT relevant parameters that may
be changed from this page.
4-12
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
Set Device
Note After an instrument reset, the manual settings are overwritten with
the corresponding calibration parameters. ▲
Caution Changing the instrument settings can harm the functionality of
the LTQ Orbitrap Velos, especially if followed by saving the calibration
parameters (manually or at conclusion of a calibration procedure).
Thus, this option should only be used by very advanced users. ▲
FT Lockmass Abundance
This device allows changing the target value of an injected lock mass
relative to the actual FT scan target value. The recommended default is
10 percent. Also see “Locking” on page 2-11.
Note Injection of lock masses is turned off completely, when you set the
target value to 0%. ▲
FT Mass Check Test Duration
This device allows changing the duration of FT Manual mass calibration
checks. See “FT Manual Calibration Page” on page 3-22. By changing
this value, a long-term mass stability evaluation can be run. The default
procedure of the FT manual mass calibration check is to perform
100 scans checking the mass accuracy. The test duration may be
extended to up to 72 hours. The default procedure can be restored by
setting the duration to zero. If the duration is set between two and
24 hours, the FT manual mass calibration check will specially control
the syringe pump to allow running long-term test with a single syringe
filling. For durations above 24 hours, it is assumed that an external
syringe pump is used.
Setting new FT Transfer Optics Parameters
Two set device items can be used to overwrite the FT optics values:
•
FT Optics Device Address
•
FT Optics Set Value
These values are originally determined during instrument calibration
and set automatically. Overwriting calibration values will influence
instrument performance and should only be done for diagnostic
purposes.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
4-13
Performing Diagnostics/Checks
Set Device
Note If you have changed FT transfer optics settings, Thermo Fisher
Scientific recommends performing a full FT instrument calibration
afterwards to assure good instrument performance. ▲
4-14
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Performing Diagnostics/Checks
Display Settings
Display Settings
Use the Display Settings page in the Diagnostics dialog box to select a
variety of instrument settings for display. See Figure 4-5. Select the
instrument settings you want to display and click Start. The
LTQ Orbitrap Velos displays the requested instrument settings in the
Testing text box.
Figure 4-5.
Display settings page of the Diagnostics dialog box
The following FT relevant instrument settings can be displayed:
Display FT calibration settings
Display FT diagnostics
Display FT instrument settings
Thermo Fisher Scientific
Displays all FT relevant calibration
parameters in the diagnostics text
box.
Displays current diagnostic
readback values of the FT electronic
boards.
Displays the current values of those
FT instrument settings that depend
on the scan range and ion polarity
mode and can be changed manually
on the Set Device page of the
Diagnostics dialog box.
LTQ Orbitrap Velos Getting Started
4-15
Performing Diagnostics/Checks
Display Settings
In the LTQ Orbitrap Velos ETD, an additional item is available:
Display reagent ion Displays the current values of the reagent ion
source settings
source parameters.
4-16
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Chapter 5
Instrument Setup
This chapter explains the “Locking” feature in automated runs and
describes the FT relevant topics of the data dependent settings in the
Instrument Setup. It contains the following topics:
Thermo Fisher Scientific
•
“Using Locking in Automated Runs” on page 5-2
•
“Data Dependent Settings” on page 5-3
LTQ Orbitrap Velos Getting Started
5-1
Instrument Setup
Using Locking in Automated Runs
Using Locking in Automated Runs
To use locking in an automated run, use the Instrument Setup program.
See Figure 5-1.
Note See “Locking” on page 2-11 for a basic description on using
locking with FTMS analyzer scans. ▲
Figure 5-1.
MS Detector Setup View – MS Detector Setup Page
The Lock Mass List button on the right side of the Segment settings
group box displays a lock mass list editor dialog. See Figure 2-9 on
page 2-11. Segment-related lock masses can be entered here. There are
separate lists for positive ion and negative ion mode. If the lock mass for
a segment is empty, no locking will be applied in the run and the
external mass calibration will be used.
5-2
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Data Dependent Settings
This section describes the FT relevant topics of the data dependent
settings in the Instrument Setup.
Using Masses instead of Mass-to-Charge Ratios
Figure 5-2 shows the Global page of the Data Dependent Settings dialog
box. It allows selecting dependent scan settings that apply to all
dependent scans.
Figure 5-2.
Data Dependent Settings dialog box – Global page
Instrument Setup allows using mass units in the input fields of dialog
boxes instead of mass-to-charge ratios. To enable this feature, select the
check box on the Global page and click OK. From now on, you can
enter just the uncharged masses of the most intensive peaks. It is not
necessary anymore to consider all possible charge states. LTQ Orbitrap
Velos determines the charge state from the full scan and converts these
masses to mass-to-charge ratios. Enabling this feature affects the
parameters in the data dependent settings as described below.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-3
Instrument Setup
Data Dependent Settings
Mass Units in Global Parameters
Global
Mass Range is interpreted as masses.
Therefore, a mass range of m/z 500–2000
will allow selecting a M2+ peak at m/z 251,
but will ignore a M2+ peak at m/z 1001.
Mass Widths
Exclusion mass widths are interpreted as
masses. Note that the isotope exclusion
functionality will be based on masses.
Therefore, if both a M2+ and M3+ peak
show up for the same peptide in a full scan,
only the more abundant charge state will
be selected.
Parent mass widths are interpreted as
masses.
Reject mass widths are interpreted as
masses.
Dynamic Exclusion
Dynamic exclusion mass widths are
interpreted as masses. When a peak is
dynamically excluded, the neutral mass is
put on the list. Therefore, other charge
states of the same mass will be excluded in
future cycles.
Mass Tags
Mass deltas are interpreted as masses. The
mass deltas are converted back to
mass-to-charge ratios based on the detected
charge state of the selected peak. The
partner must also have the same charge
state. Mass tolerances are based on
exclusion mass widths.
Isotopic Data Dependence Mass differences are interpreted as masses.
The mass differences are converted back to
mass-to-charge ratios based on the detected
charge state of the selected peak.
Neutral Loss
Neutral loss mass widths are interpreted as
masses.
Product
Product mass widths are interpreted as
masses.
5-4
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Mass Units in Segment Parameters
Parent Mass List
Reject Mass List
Charge State
Neutral Loss
Product Mass List
Add/Sub
Thermo Fisher Scientific
Input will be interpreted as neutral masses
– not [M+H]+ or [M-H]+. If “MS Charge
State” is specified in the mass list, this value
is used to convert the mass back to a
mass-to-charge ratio. If this is not specified,
masses are converted to mass-to-charge
ratios based on all charge states that are not
on the rejection list. “4 and up” is an
allowed charge state, but the search is only
performed for 4, and not 5, 6, etc. If more
than one charge state is identified, only the
most abundant is selected.
Input will be interpreted as neutral masses
– not [M+H]+ or [M-H]+.
Because masses must be converted back to
mass-to-charge ratios, the charge state must
be known for any peak that is analyzed.
Therefore, rejection of unassigned charge
states is automatically enforced.
Masses are interpreted as masses. The
charge state used to convert back to
mass-to-charge ratio is based on the peak
being analyzed. The charge state of the
potential partner must match the charge
state of the peak.
Input will be interpreted as neutral masses
– not [M+H]+ or [M-H]+.
Mass is interpreted as a mass and is
converted to mass-to-charge based on the
charge state of the selected peak.
LTQ Orbitrap Velos Getting Started
5-5
Instrument Setup
Data Dependent Settings
Preview Mode
Figure 5-3 shows the Current Segment page of the Data Dependent
Settings dialog box.
Figure 5-3.
Data Dependent Settings dialog box – Current Segment page
If the preview mode for FTMS master scans is enabled on the Current
Segment page, the data dependent decision is made on the basis of the
FT master scan with lower resolution to increase the duty cycle. The
resolution of the FTMS scan itself is not changed. Because the high
resolution is usually not required to make the data dependent decision,
it is recommended to enable the preview mode.
To prevent making data dependent decisions on basis of
lower-resolution preview spectra, disable this option. For example, if
there are ions with high charge states to be examined, and the data
dependent settings require charge state recognition of precursor ions,
this might be a reason to turn off this option. Otherwise, the high
charge state clusters may not be resolved and charge states will not be
recognized in preview mode.
5-6
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Monoisotopic Precursor Selection
Figure 5-4 shows Charge State page of the Data Dependent Settings
dialog box.
Figure 5-4.
Data Dependent Settings dialog box – Charge State page (Advanced Features on)
If the monoisotopic precursor selection is enabled on the Charge State
page, the data dependent scan is only performed for one molecular ion
of the corresponding overall isotopic distribution if Dynamic Exclusion
is enabled.
This check box is only available on the Charge State page if the
Advanced Features are turned on in the LTQ Orbitrap Velos menu of
the Instrument Setup.
Note The algorithm for isotopic cluster recognition will handle correctly
only such isotope distributions where the third peak (A + 2) is lower in
intensity than the second peak (A + 1). For more complex isotope
patterns (for example, ion containing Sn, Br, or multiple Cl atoms), it is
recommended to clear the Enable monoisotopic precursor selection
check box. ▲
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-7
Instrument Setup
Data Dependent Settings
Use Non-Peptide Monoisotopic Recognition
This check box is only available if monoisotopic precursor selection is
active. If monoisotopic precursor selection is active and this box is not
checked, precursor ions in FT master scans must match peptide-type
isotopic distribution to identify the monoisotopic peak. If this box is
selected, monoisotopic peaks will also be identified for small molecules
and precursor ions with non-peptide-type isotopic distributions.
Enabling Charge State Dependent ETD Time1
For data-dependent scans that use ETD activation, you can allow the
instrument to adjust the reaction time according to the charge state of
the ions. Select this check box to have the instrument reduce the
reaction time for highly charged ions and increase it for lowly charged
ions.
Figure 5-5.
Enabling charge state dependent ETD time
1
This feature is available only for the LTQ Orbitrap Velos ETD.
5-8
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Data Dependent FT SIM Scans
A data dependent FT SIM scan is performed around the center mass
determined in a previous reference scan event if the check box “Same
MS order as referenced scan event” is selected on the Current Scan
Event page as shown in Figure 5-6.
Figure 5-6.
Data Dependent Settings dialog box – Current Scan Event page
Repeat previous Scan Event with HCD
The Repeat previous scan event with HCD check box will only be
available when all of the following requirements are fulfilled:
Thermo Fisher Scientific
•
Select an experiment type other than data dependent triple play,
data dependent neutral loss MS3, or data dependent product MS3.
•
Select a scan event greater than or equal to 3.
•
Specify one or more parent ions in a previous scan event.
•
FTMS is selected as the mass analyzer for the current scan event.
•
A neutral loss or product ion list is specified for the referenced scan
event.
•
Either option From neutral loss list or From product list is selected.
LTQ Orbitrap Velos Getting Started
5-9
Instrument Setup
Data Dependent Settings
•
HCD is selected as activation type on the Activation page. See
page 5-13.
Select the check box to repeat the previous scan event with
HCD activation type. A typical experiment to use this feature would be
to trigger an HCD fragmentation experiment with a neutral loss
observed in an ion trap MS/MS experiment. See Figure 5-7.
Figure 5-7.
Current Scan Event page – Repeat previous scan event with HCD
Repeat previous Scan Event with ETD1
The Repeat previous scan event with ETD check box will only be
available when all of the following requirements are fulfilled:
•
A neutral loss or product ion list is specified for the referenced scan
event.
•
Either option From neutral loss list or From product list is selected.
•
ETD is selected as activation type on the Activation page. See
page 5-13.
1
This feature is available only for the LTQ Orbitrap Velos ETD.
5-10
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Select the check box to repeat the previous scan event with
ETD activation type. A typical experiment to use this feature would be
to trigger an ETD fragmentation experiment with a neutral loss
observed in an ion trap MS/MS experiment. See Figure 5-8.
Figure 5-8.
Current Scan Event page – Repeat previous scan event with ETD
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-11
Instrument Setup
Data Dependent Settings
Scan Width
The scan width of the data dependent FT SIM scan can be selected on
the Scan widths page of the Data Dependent Settings dialog box. See
Figure 5-9.
Figure 5-9.
5-12
Data Dependent Settings dialog box – Scan Widths page
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
Activation Type
On the Activation page of the Data Dependent Settings dialog box, the
Activation type list box allows specifying how the ions are activated for
fragmentation during a data dependent experiment. See Figure 5-10. It
has the following options:
•
CID (Collision-induced dissociation)
•
PQD (Pulsed-Q dissociation)
•
ETD (electron transfer dissociation)1
Use ETD to fragment peptides and proteins. After selecting ETD,
Normalized collision energy and Activation Q are not available and
Supplemental Activation becomes available. See Figure 7-36 on
page 7-39.
•
HCD (higher energy CID)
Use HCD to obtain triple quadrupole-like fragment ion spectra. If
you select HCD, the Activation Q spin box becomes unavailable.
Figure 5-10. Data Dependent Settings dialog box – Activation page
1
This feature is available only for the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-13
Instrument Setup
Data Dependent Settings
FT HCD
The FT HCD page of the Data Dependent Settings dialog box will only
be available when FTMS is selected as the mass analyzer for the current
scan event. See Figure 5-11.
Figure 5-11. Data Dependent Settings dialog box – FT HCD page
The FT HCD page offers two modes for choosing the first mass of the
dependent scan:
•
a mass with a fixed m/z value
To change the m/z value, click the arrows in the spin box to
increment [up arrow] or decrement [down arrow] the value. You can
set m/z to any value from 50 to 4000; default is 100. Alternatively,
enter a value in the spin box text field.
•
a mass with an m/z value that is relative to the precursor mass.
To change the percentage, click the arrows in the spin box to
increment [up arrow] or decrement [down arrow] the value. You can
set the percentage to any value from 0 to 4; default is 0.25.
Alternatively, enter a value in the spin box text field.
Note The highest m/z that the LTQ Orbitrap Velos can analyze is equal
to approximately 20 times the m/z of the first mass. ▲
5-14
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
FT ETD1
The FT ETD page of the Data Dependent Settings dialog box will only
be available when FTMS is selected as the mass analyzer for the current
scan event. See Figure 5-12.
Figure 5-12. Data Dependent Settings dialog box – FT ETD page
Note The highest m/z that the LTQ Orbitrap Velos can analyze is equal
to approximately 20 times the m/z of the first mass. ▲
The FT ETD page offers two modes for choosing the first mass of the
dependent scan:
•
a mass with a fixed m/z value
To change the m/z value, click the arrows in the spin box to
increment [up arrow] or decrement [down arrow] the value. You can
set m/z to any value from 50 to 4000; default is 100. Alternatively,
enter a value in the spin box text field.
1
This feature is available only for the LTQ Orbitrap Velos ETD.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-15
Instrument Setup
Data Dependent Settings
•
a mass with an m/z value that is relative to the precursor mass.
To change the percentage, click the arrows in the spin box to
increment [up arrow] or decrement [down arrow] the value. You can
set the percentage to any value from 0 to 4; default is 0.25.
Alternatively, enter a value in the spin box text field.
MSn Settings for HCD Experiments
Similar to the Define Scan dialog box, the Instrument Setup allows
selecting the Activation type (CID/PQD/HCD). See “MSn Settings” on
page 2-12. Use HCD to obtain triple quadrupole-like fragment ion
spectra. If you select HCD as activation type, the HCD charge state
spin box becomes available and the Activation Q spin box is disabled.
See Figure 5-13.
Figure 5-13. MS Detector Setup Page – Scan event settings with HCD experiment
MSn Settings for ETD Experiments
In the LTQ Orbitrap Velos ETD, the Instrument Setup allows selecting
ETD as the Activation type. See Figure 5-14. Use ETD to fragment
peptides and proteins at the amide-N to alpha-C bond to produce cand z-type fragment ions. Selecting ETD as activation type has the
following consequences:
•
5-16
LTQ Orbitrap Velos Getting Started
The Supplemental Activation Check box becomes available and
allows entering the corresponding charge state in the spin box.
Thermo Fisher Scientific
Instrument Setup
Data Dependent Settings
•
The Normalized Collision Energy spin box and the Activation Q
spin box are disabled.
Figure 5-14. MS Detector Setup Page – Scan event settings with ETD experiment
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
5-17
Chapter 6
Instrument Configuration
This chapter gives instructions on how to configure your instrument. It
contains the following topics:
Thermo Fisher Scientific
•
“Starting Instrument Configuration” on page 6-2
•
“FT Settings Page” on page 6-3
•
“FT Mass Lists Page” on page 6-4
LTQ Orbitrap Velos Getting Started
6-1
Instrument Configuration
Starting Instrument Configuration
Starting Instrument Configuration
From the Instrument Configuration dialog box, click the LTQ Orbitrap
Velos MS button in the Configured Devices group box. See Figure 6-1.
Then, click Configure to open the LTQ Orbitrap Velos Configuration
dialog box.
Figure 6-1.
Instrument Configuration dialog box
The LTQ Orbitrap Velos Configuration dialog box allows entering
LTQ Orbitrap Velos configuration information by using several pages,
including the FT Settings page and the FT Manual Calibration page.
The elements of the pages are described in the following topics.
See “Configuring the Reagent Ion Source” on page 7-8 for information
about the Reagent Ion Source page of the LTQ Orbitrap Velos
Configuration dialog box, which is available for the LTQ Orbitrap
Velos ETD.
6-2
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Instrument Configuration
FT Settings Page
FT Settings Page
The FT Settings page of the LTQ Orbitrap Velos Configuration dialog
box allows editing the FTMS analyzer temperature setpoint and
enabling the HCD collision gas. See Figure 6-2.
Figure 6-2.
LTQ Orbitrap Velos Configuration dialog box – FT Settings page
Using the FT Settings page of the LTQ Orbitrap Velos Configuration
dialog box, you can select the following parameters:
Enable HCD collision gas Select the check box to enable the
HCD collision gas. Otherwise, using
HCD fragmentation is not possible. It is
recommended to clear this check box only
for diagnostic purposes.
FTMS analyzer
Enter the desired temperature for the
temperature setpoint (°C) Orbitrap analyzer chamber. The default
value is 26.
Note Configuration changes will become effective when you reboot
your instrument. ▲
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
6-3
Instrument Configuration
FT Mass Lists Page
FT Mass Lists Page
The FT Mass Lists page of the LTQ Orbitrap Velos Configuration
dialog box allows editing the calibration mass lists, which are displayed
in the FT Manual page of the Calibrate dialog box. See Figure 6-3. It is
also possible to import or export a mass list as a text file.
Figure 6-3.
LTQ Orbitrap Velos Configuration dialog box – FT Mass Lists page
The FT Mass Lists page of the LTQ Orbitrap Velos Configuration
dialog box has the following parameters:
6-4
LTQ Orbitrap Velos Getting Started
Name
This list box lists the names of the factory supplied
and user created mass lists.
Mass List
This table lists the mass-to-charge ratios of the ions
that you are using to calibrate the Orbitrap mass
analyzer. You can select an existing mass list in the
Name list box, or you can create or modify a mass
list by clicking on and editing the entries in the
Mass List table.
Save
Click Save to save the mass list with the name that
is selected in the Name list box.
Save As
Click Save As a to save the mass list with a new
name.
Delete
Click Delete to delete the mass list that is selected
in the Name list box.
Import
Click Import to import a mass list that is a text file.
Thermo Fisher Scientific
Instrument Configuration
FT Mass Lists Page
Export
Click Export to export a mass list to a text file.
Show Factory Lists Select this check box to show the factory calibration
mass lists in Tune Plus.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
6-5
Chapter 7
LTQ Orbitrap Velos ETD Instruments
This chapter describes only the Orbitrap relevant differences in
instrument settings and procedures with respect to using an
LTQ Orbitrap Velos ETD instrument. See also the previous chapters of
this manual and the LTQ Orbitrap Velos Hardware Manual.
This chapter contains the following topics:
•
“Tune Plus Window of the LTQ Orbitrap Velos ETD” on page 7-2
•
“Configuring the Reagent Ion Source” on page 7-8
•
“Powering On the ETD Module and Viewing Reagent Ion Spectra”
on page 7-10
•
“Tuning the Reagent Ion Optics” on page 7-12
•
“Performing an ETD Infusion Experiment” on page 7-27
•
“Creating an Xcalibur Instrument Method That Uses ETD
Activation” on page 7-35
•
“Angiotensin I Solutions” on page 7-41
For additional information concerning ETD, refer to the ETD Module
Hardware Manual and the ETD Module Getting Started Guide.
Note If your instrument is equipped with a reagent ion source, you need
to configure the reagent ion source in Instrument Configuration to get
access to all reagent ion source relevant settings of Tune Plus and
Instrument Setup. See “Configuring the Reagent Ion Source” on
page 7-8. ▲
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-1
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
Tune Plus Window of the LTQ Orbitrap Velos ETD
This section describes the differences in the Tune Plus window of an
LTQ Orbitrap Velos ETD instrument. Spectrum view and Graph view
show no differences to the standard instrument. They are described in
Chapter 2: “Tune Plus Window”.
Status View
The Tune Plus window shows a schematic view of the LTQ Orbitrap
Velos ETD instrument. See Figure 7-1. The instrument control icon in
the toolbar allows accessing parameters for the reagent ion source and
the reagent ion optics.
Reagent Ion Optics icon
Figure 7-1.
7-2
Reagent Ion Source instrument control i
Status View for LTQ Orbitrap Velos ETD
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
The color of the Reagent Ion Source control icon indicates the status of
the reagent ion source:
Reagent ion source off
Reagent ion source on
The status of the filament is shown in the Status view – All
page.
The Status view – All page additionally displays information about
reagent ion source, reagent ion optics, reagent vacuum, reagent
turbopump, and reagent power supplies.
Scan Mode Menu
This section describes the elements of the Scan Mode menu that are
different from the standard instrument.
Define Scan Dialog Box
As described in Chapter 2: “Tune Plus Window”, you can choose the
analyzer type (Ion Trap or FTMS), mass range, resolution, and scan
type. See Figure 7-2. You can as well define the scan range (for example,
First Mass–Last Mass window).
Figure 7-2.
Define Scan dialog box for LTQ Orbitrap Velos ETD
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-3
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
When you select ETD as Activation Type in the MSn Settings, the
Supplemental Activation check box becomes available. Select this check
box to enable supplemental activation of ETD MS/MS and MSn.
Use the SA Energy spin box to enter the percentage of the energy that
should be used for supplemental activation. The available range is 0 to
20%.
Use the SA Charge State spin box to enter the charge state of the parent
ion for supplemental activation. Available charge states are 2 to 10.
Setup Menu
This section describes the elements of the Setup menu that are different
from the standard instrument.
Injection Control Dialog Box – Reagent Page
The number of reagent ions admitted into the linear trap is regulated by
the parameters in the Reagent page in the Injection Control dialog box.
See Figure 7-3. Open the Reagent page in the Injection Control window
by clicking the Injection Control instrument control graphic or click
Setup > Injection Control > Reagent.
Figure 7-3.
Injection Control dialog box – Reagent page
See “Viewing the Injection Reagent Settings” on page 7-27 for
information about the parameters available on this dialog box.
7-4
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
Reagent Ion Optics Dialog Box
The Reagent Ion Optics dialog box allows specifying voltages for the
reagent ion source lenses, the back lens, and the transfer multipole.
Usually, the reagent ion optics parameters are optimized by the
automatic tune procedure to maximize the transmission of reagent ions
from the reagent ion source to the linear ion trap.
You may obtain small improvement in the signal by manual tuning and
changing the settings. You can change the settings in this dialog box
only after selecting the View Reagent Ion Spectra check box in the
Reagent Ion Source dialog box. See Figure 7-4. See “Tuning the Reagent
Ion Optics” on page 7-12 for more information.
Figure 7-4.
Activating Reagent Ion Optics dialog box
Note The reagent ion optics lenses have broad optimums – except the
back lens. Increasing or decreasing the back lens offset by 3 or 4 V can
significantly reduce the signal. ▲
Reagent Ion Source Dialog Box
The Reagent Ion Source dialog box allows setting selected ETD reagent
ion source parameters. See Figure 7-5 on page 7-6. The actual parameter
values are shown on the right side of the dialog box.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-5
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
Use the Reagent Ion Source On check box to turn on or off the reagent
ion source, including the vial heaters. If the reagent vials are not at their
target temperatures, a “Please Wait” message window appears. Typically,
heating takes 5 to 10 minutes. Turn off the reagent ion source if you do
not plan to use it for an extended period, overnight for example.
Use the Filament On check box to turn on or off the filament, which
produces electrons. When the reagent vials reach their target
temperatures, the filament automatically turns on and the Filament On
check box automatically shows a check mark. (You can force the
filament to turn on before the vials reach their target temperatures by
selecting the check box.) To prolong the lifetime of the filament, turn it
off if the reagent ion source will not be in use for an hour or more.
Figure 7-5.
Reagent Ion Source dialog box
Click Open Probe Interlock to evacuate the inlet valve block to a target
pressure of less than 0.1 mTorr. When the target pressure is achieved, a
message states that you can open the ball valve. Refer to the
LTQ Orbitrap Velos Hardware Manual for maintenance instructions for
the reagent ion source.
When you select the View Reagent Spectra check box, the following
actions become possible:
•
7-6
LTQ Orbitrap Velos Getting Started
The injection of analyte ions, but not reagent ions, into the mass
analyzer for mass analysis is stopped. This allows measuring reagent
ion intensity and also checking for reagent contamination by
observing the mass spectrum.
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tune Plus Window of the LTQ Orbitrap Velos ETD
•
You can change the parameter values in the Reagent Ion Optics
dialog box. See page 7-5.
•
You can autotune the reagent ion source. See page 7-12.
Vacuum Dialog Box – Reagent Page
The Reagent page of the Vacuum dialog box allows switching on the ion
gauge in the ETD Module. It also displays the pressure readings of the
ion gauge and the Convectron gauge in the ETD Module. See
Figure 7-6.
Figure 7-6.
Thermo Fisher Scientific
Reagent page of the Vacuum dialog box
LTQ Orbitrap Velos Getting Started
7-7
LTQ Orbitrap Velos ETD Instruments
Configuring the Reagent Ion Source
Configuring the Reagent Ion Source
After you have installed the reagent vials for the ETD Module as
described in the LTQ Orbitrap Velos Hardware Manual, you have to
configure the reagent ion source of the LTQ Orbitrap Velos ETD.
❖
To configure the reagent ion source
1. From the Instrument Configuration window, click LTQ Orbitrap
Velos MS in the Configured Devices group box.
2. Click Configure to open the LTQ Orbitrap Velos Configuration
dialog box.
3. Click Reagent Ion Source in the left hand section of the
LTQ Orbitrap Velos Configuration window. The Reagent Ion
Source page of the Configuration dialog box allows configuring the
reagent ion source and activating the cooling gas. See Figure 7-7.
4. Select the Reagent Ion Source Configured check box as shown in
Figure 7-7.
Figure 7-7.
Enabling the reagent ion source
5. In the Vial 1 row, click the Reagent Name list box and select
Fluoranthene. The Fluoranthene Vial 1 Reagent Mass, Activation
Type, and default Vial Temperature will appear in the table.
7-8
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Configuring the Reagent Ion Source
Caution The empty vial in the Vial 2 heater is an integral part of the
carrier/CI gas system. It is necessary to keep the carrier/CI gas system
closed to the laboratory. If no vial is placed in the Vial 2 heater:
•
The carrier/CI gas containing the reagent may escape to the
laboratory causing a safety problem.
•
The ETD Module will not operate correctly and the filament will
burn out. ▲
6. Select the Use Cooling Gas check box as shown in Figure 7-8.
Figure 7-8.
LTQ Orbitrap Series Configuration dialog box – Reagent Ion Source page
7. Click OK in the LTQ Orbitrap Velos Configuration dialog box.
A message box informs you that for the configuration changes to
take effect, you will need to reboot the data system and then the
MS detector.
8. Click Done in the Instrument Configuration window.
9. Reboot the data system.
10. Reboot the LTQ Orbitrap Velos ETD.
The system software is now configured for using the reagent ion source
of the LTQ Orbitrap Velos ETD system.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-9
LTQ Orbitrap Velos ETD Instruments
Powering On the ETD Module and Viewing Reagent Ion Spectra
Powering On the ETD Module and Viewing Reagent Ion Spectra
After the reagent vials have been installed as described in the
LTQ Orbitrap Velos Hardware Manual, power on the ETD Module by
placing the LTQ Orbitrap Velos ETD in On mode. Turn on the reagent
ion source to view the reagent ion spectra.
Powering On the ETD Module
❖
To power on the ETD Module
1. Toggle the ETD Module service switch to the Operating Mode
(ON) position if it is not already in this position.
2. Toggle the LTQ Orbitrap Velos MS FT Electronics switch to the On
position. This turns the ETD Module on if the ETD Module
service switch is already in the Operating Mode (ON) position.
For detailed instructions about starting up and shutting down the
instrument, refer to the LTQ Orbitrap Velos Hardware Manual.
Turning On the Reagent Ion Source and Viewing Reagent Ion Spectra
Even when the ETD module is turned on, the reagent ion source within
it is not turned on until you turn it on.
❖
To turn on the reagent ion source
1. Click the Reagent Ion Source instrument control icon in the
toolbar of the Tune Plus window. (See icon in margin and
Figure 7-1 on page 7-2.) The Reagent Ion Source dialog box
appears. (See Figure 7-5 on page 7-6.)
2. In the Reagent Ion Source dialog box, click the Reagent Ion Source
On check box. (See Figure 7-5.) If the reagent vials are not at their
target temperature, a message box appears. See Figure 7-9. When
the reagent vials reach their target temperature, voltage is applied to
the ETD Module ion optics. The filament automatically turns on;
the Filament On check box automatically shows a check mark and
its actual condition switches from Off to On.
Figure 7-9.
7-10
LTQ Orbitrap Velos Getting Started
Message box: Reagent Vial NOT At Temperature!
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Powering On the ETD Module and Viewing Reagent Ion Spectra
❖
To view the reagent ion spectra
1. Select the View Reagent Ion Spectra check box in the Reagent Ion
Source dialog box. See Figure 7-10.
2. Reagent ion peaks appear in the Spectrum view. See Figure 7-10.
View reagent ion
spectra
Figure 7-10. Tune Plus window showing the fluoranthene radical anion mass spectrum
If the spectrum is satisfactory, proceed to the next step. If the
spectrum is not satisfactory, tune the reagent ion optics as described
in “Tuning the Reagent Ion Optics” on page 7-12.
3. Clear the View Reagent Ion Spectra check box.
The Tune Plus Spectrum view from the ETD module is cleared.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-11
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Tuning the Reagent Ion Optics
This section describes how to tune the ETD Reagent Ion Optics settings
to obtain optimized reagent ion transmission.
There are three ways to tune the ion optics within the reagent ion
source: automatically, semi-automatically, and manually.
• “Automatically Tuning the Reagent Ion Source“, next section
• “Manually Tuning the Reagent Ion Source” on page 7-14
• “Semi-Automatically Tuning the Reagent Ion Optics” on page 7-17
Automatically tuning the reagent ion source is the best method for most
situations. In some cases it may be appropriate to perform manual
tuning. Choose manual tuning to manually optimize reagent ion optics
parameters and reagent ion source parameters that are not automatically
tuned such as Emission Current, Electron Energy, and CI gas pressure.
Manual tuning is done by observing the effects of adjusting these
parameters on the reagent ion signal intensity.
Note Tune Plus provides an evaluation procedure for CI gas pressure
under Diagnostics > Diagnostics > Tools > System evaluation >
Reagent CI gas pressure evaluation. Thermo Fisher Scientific
recommends performing this procedure after replacing the filament
and/or the ion volume. ▲
Use semi-automatic tuning to optimize each lens setting individually
within an optimization range and according to the step size you select.
Automatically Tuning the Reagent Ion Source
Automatically tuning the reagent ion source assures the best ion optics
settings for optimum transmission of reagent ions (fluoranthene).
❖
To automatically tune the reagent ion source
1. On the Windows taskbar, choose Start > All Programs > Xcalibur
> LTQ Tune.
The Tune Plus window opens. (See Figure 7-1 on page 7-2.)
2. Click the On/Off/Standby button to take the LTQ Orbitrap
Velos ETD out of Standby mode and turn it on.
On
Off
Standby
3. Click the Display Graph View button.
4. If the reagent ion source is not on, turn it on as described in
“Turning On the Reagent Ion Source and Viewing Reagent Ion
Spectra” on page 7-10.
7-12
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
5. In the Reagent Ion Source dialog box, select the View Reagent Ion
Spectra check box. (See Figure 7-11.)
Figure 7-11. Activating Reagent Ion Source tuning
6. Click the Tune button. (See icon in margin.) The Tune dialog box
appears.
7. The left side of the underlying view shows the reagent ion spectra
(fluoranthene). See Figure 7-12 on page 7-14. The center view
shows one of the parameters that are automatically tuned (back lens
potential tuned to maximize the signal intensity at m/z 202).
8. Click the Automatic tab in the Tune dialog box if it is not already
the active tab. The Automatic page of this window appears.
9. Click Start in the Automatic page of the Tune dialog box. The
system will begin automatically tuning the ion optics of the reagent
ion source. The Status box of the dialog box indicates that
automatic tuning is completed by displaying the message,
“Optimization Complete”. This message will also indicate the
percentage change in the reagent ion signal intensity at m/z 202
relative to the prior value. A typical reagent signal intensity is about
1–2E7 in centroid mode when the system has been cleaned and the
ion volume is new.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-13
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Automatic tab
Figure 7-12. Tune Plus window with Automatic page of Tune dialog box displayed
10. Rerun Automatic Tune if the percentage change is greater than
20%. This is an iterative process. At some point there will be no
more improvements in signal intensity.
Manually Tuning the Reagent Ion Source
❖
To manually tune the reagent ion source
1. Click the Display Graph View icon in Tune Plus.
2. If the reagent ion source is not on, open the Reagent Ion Source
dialog box and turn on the reagent ion source as described in
“Turning On the Reagent Ion Source and Viewing Reagent Ion
Spectra” on page 7-10.
3. In the Reagent Ion Source dialog box, select the View Reagent Ion
Spectra check box. See Figure 7-10 on page 7-11.
7-14
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
4. Click the Reagent Ion Optics icon at the top of the Tune Plus
window. (See icon in margin and Figure 7-13.) The Reagent Ion
Optics dialog box appears.
Reagent Ion Optics icon
Figure 7-13. Tune Plus window with Reagent Ion Optics dialog box
5. Click the Tune button in Tune Plus. The Tune dialog box appears.
You can also tune the reagent ion source. When the Reagent Ion
Source dialog box is open (See Figure 7-5 on page 7-6.), click the
Tune button in Tune Plus.
6. Click the Manual tab in the Tune dialog box if the Manual page is
not already visible. (See Figure 7-14 on page 7-16.)
7. Select the Reagent Ion from Vial 1 check box.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-15
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Figure 7-14. Reagent Ion Source Tune dialog box – Manual page
8. Click Start.
The Graph view displays a plot of the reagent ion intensity. See
Figure 7-15 on page 7-17. You can observe the response of the
reagent ion intensity to changes in the lens parameters (Reagent Ion
Optics dialog box) and Emission Current, CI gas pressure, and
Electron Energy (Reagent Ion Source dialog box). Adjust these
parameters to achieve the maximum reagent ion signal intensity.
Note Increasing the emission current might shorten the filament life
time. Therefore, Thermo Fisher Scientific recommends readjusting the
emission current only in exceptional circumstances. ▲
7-16
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Figure 7-15. Tune Plus window showing the Display Graph view for manual tuning of the Reagent Ion Source
Semi-Automatically Tuning the Reagent Ion Optics
Use the semi-automatic tuning method to fine-tune the lens parameters
to a range of settings and in step increments.
❖
To semi-automatically tune the reagent ion optics
1. On the Windows taskbar, choose Start > Programs > All Programs
> Xcalibur > LTQ Tune.
The Tune Plus window opens. (See Figure 7-1 on page 7-2.)
2. Click the Display Graph View button.
3. If the Reagent Ion Source is not on, turn it on:
a. In the Tune Plus window, choose Setup > Reagent Ion Source.
The Reagent Ion Source dialog box opens. (See Figure 7-5 on
page 7-6.)
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-17
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
b. Select the Reagent Ion Source On check box.
If the reagent vials are not at their target temperature, a message
appears:
Reagent Vial NOT At Temperature! Please wait . . .
When the reagent vials reach their target temperature, voltage is
applied to the LTQ Orbitrap Velos ETD system’s ion optics.
The filament automatically turns on (the Filament On check
box automatically shows a check mark and its actual condition
switches from Off to On).
4. In the Reagent Ion Source dialog box, select the View Reagent Ion
Spectra check box.
5. In the Control/Scan Mode toolbar, click the Tune button.
The Tune dialog box opens with the Automatic page shown by
default (Figure 7-11 on page 7-13).
Figure 7-16. Semi-Automatic page of the Tune dialog box
7-18
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
6. Click the Semi-Automatic tab. The Semi-Automatic page opens. See
Figure 7-16 on page 7-18.
7. From the What to Optimize list, select the item you want to tune:
•
Reagent ion lens 1 (V)
•
Reagent ion gate lens (V)
•
Reagent ion lens 2 (V)
•
Reagent ion lens 3 (V)
•
Back Multipole Offset (V); see also “Tuning the Quadrupole
Mass Filter” on page 7-21
•
Back lens (V)
8. Adjust the settings in the Optimization Range area:
•
Start
•
End
•
Step
9. Click Start.
The results of your settings are shown in the Results area.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-19
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Viewing the Current Reagent Ion Optics Settings
❖
To view the current Reagent Ion Optics settings
1. Click the Reagent Ion Optics instrument control icon in the Tune
Plus window to open the Reagent Ion Optics dialog box. The
parameters in the Reagent Ion Optics dialog box have been
optimized by the Auto Tune process. See Figure 7-17.
Note When the View Reagent Ion Spectra check box in the Reagent Ion
Source dialog box is not selected, the parameters in the Reagent Ion
Optics dialog box cannot be changed. ▲
2. Click OK to close the Reagent Ion Optics dialog box.
Figure 7-17. Reagent Ion Optics dialog box
7-20
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Saving Your ETD Tune Method
After tuning is complete, save the ETD Tune parameters in a tune
method.
❖
To save the tune method
1. On the File/Display toolbar, click the Save button.
The Save As dialog box opens.
2. Browse to choose a location and specify a file name.
3. Click Save.
Tuning the Quadrupole Mass Filter
Stable calibration of the quadrupole mass filter between the linear ion
trap and the C-Trap is achieved by manually adjusting the Back
Multipole DC Offset voltage.
❖
To adjust the Back Multipole DC Offset voltage
1. Open the Toggles page in the Diagnostics dialog box.
Figure 7-18. Activating Reagent Ion Isolation
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-21
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
2. Set FT HCD collision gas to On to switch on the collision gas in
the HCD cell.
3. Set Isolate reagent ion to On. See Figure 7-18 on page 7-21.
4. In the Reagent Ion Source dialog box, select the View Reagent Ion
Spectra check box.
5. Open the Tune dialog box and perform a semi-automatic tune of
the Back Multipole Offset with the settings shown in Figure 7-19 on
page 7-22 (Start 0, End 40, Step 0.35).
Figure 7-19. Semi-automatic tune of the Back Multipole Offset dc voltage
6. Search for a local maximum just before the oscillations on the signal
start. (In the example shown in Figure 7-21, that would be at about
17 V).
7. Open the Reagent Ion Optics dialog box. Enter the new value for
the Back Multipole Offset voltage into the respective spin box and
click Apply.
7-22
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
8. Open the Calibrate dialog box and perform a semi-automatic
calibration of the Reagent Ion Selection. See Figure 7-20 on
page 7-23 and Figure 7-23 on page 7-25.
Figure 7-20. Calibrating the Reagent Ion Selection
Choosing the Operating Point for the Quadrupole Mass Filter
The performance of the quadrupole mass filter strongly depends on the
kinetic energy (that is, DC Offset) of the ions. If the kinetic energy is
too low it causes a poor transmission; if the kinetic energy is too high it
leads to oscillations (noding) of the signal.
In the example shown in Figure 7-21, noding starts at above 20 V. In
this region, the signal is fragile. Depending on the DC Offset (for
comparison: 10 V, 17 V, and 32 V; red points) the calibration curves
look differently.
The best operating point for the filter is the immediate region before the
oscillations start. In the example, that would be the range between 15 V
and 20 V.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-23
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
Figure 7-21. Dependence of the signal from the DC Offset
If the DC Offset is chosen too low, intensity losses in the filter are too
large. During the calibration, the signal still increases after the stable
region is reached. See Figure 7-22. As a consequence, the transmission at
the operating point of the filter is poor.
DC Offset = 10V
1.20E+07
1.00E+07
Intensität
8.00E+06
q=0.35
q=0.45
6.00E+06
4.00E+06
2.00E+06
0.00E+00
350.00
400.00
450.00
500.00
550.00
600.00
650.00
700.00
Amplitude /V
Figure 7-22. Chosen DC Offset (10 V) is too low for calibration
With offset voltages between 15 V and 20 V, no noding is visible. See
Figure 7-23. The transmission reaches a maximum shortly after the start
of the stable region, resulting in a high transmission at optimum filter
efficiency. The calibration results in a broad plateau with constant
intensity.
7-24
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
DC Offset = 17V
1.20E+07
1.00E+07
Intensität
8.00E+06
q=0.35
q=0.45
6.00E+06
4.00E+06
2.00E+06
0.00E+00
350.00
400.00
450.00
500.00
550.00
600.00
650.00
700.00
Amplitude /V
Figure 7-23. Point of optimum performance
If the DC Offset is chosen too low, strong oscillation on the signal
occur. See Figure 7-24. This creates problems when calibrating the filter.
Because the maxima are very narrow, the anion signal is susceptible to
changes of the ion optics (thermal drift of voltages, deterioration of the
reagent source, etc.) Therefore, this region is not suitable for stable
operation of the ETD system even though it may include the maximum
of the anion intensity.
DC offset = 32V
1.20E+07
1.00E+07
Intensität
8.00E+06
q=0.35
q=0.45
6.00E+06
4.00E+06
2.00E+06
0.00E+00
350.00
400.00
450.00
500.00
550.00
600.00
650.00
700.00
Amplitude /V
Figure 7-24. Chosen DC Offset (32 V) is too high for calibration
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-25
LTQ Orbitrap Velos ETD Instruments
Tuning the Reagent Ion Optics
„Noding“ in Quadrupoles
In quadrupole filters, the intensity of the nodes increases with the
kinetic energy of the ions. See Figure 7-25.
Low kinetic energy: weakly developed nodes
High kinetic energy: strongly developed nodes
Figure 7-25. “Noding” in quadrupoles
Depending on the location of the nodes, the ions leave the quadrupole
as an either convergent beam (good transmission) or divergent beam
(bad transmission). Tuning the voltages moves the place of the nodes,
leading to oscillations of the signal.
7-26
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
Performing an ETD Infusion Experiment
Procedures for performing an ETD infusion experiment are described in
the following sections:
•
Viewing the Injection Reagent Settings
•
Troubleshooting an AGC Target Error
•
Obtaining an ETD Spectrum for Angiotensin I
•
Optimizing the Reagent Ion Reaction Time
Viewing the Injection Reagent Settings
The number of reagent ions admitted into the linear ion trap is
regulated by the parameters in the Reagent page of the Injection Control
dialog box.
❖
To view the injection reagent ion settings
1. On the Windows taskbar, choose Start > All Programs > Xcalibur
> LTQ Tune.
The Tune Plus window opens. (See Figure 7-1 on page 7-2.)
2. Click the Injection Control instrument control graphic or click
Setup > Injection Control to display the Injection control dialog
box.
3. Click the Reagent tab.
The Reagent page opens. See Figure 7-26.
Figure 7-26. Injection Control dialog box – Reagent page
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-27
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
The ETD Reagent Injection control consists of two parameters:
•
The AGC Target parameter sets the target number of reagent anions
to be injected into the linear ion trap to perform ETD. The default
value for this parameter is 3E5.
•
The Max. Inject Time (ms) parameter specifies the maximum
amount of time that the system allows for anions to be injected into
the trap. The default value for this time is 50 ms.
The reagent ion source injects reagent anions into the linear ion trap
until the ETD AGC target is reached. The time allowed to reach the
ETD AGC target cannot exceed the Maximum Injection time (the
Maximum Injection time takes precedence over the AGC target).
Troubleshooting an AGC Target Error
If the AGC target is not reached due to the Maximum Injection time
limit, the system displays an error message advising you that the
AGC target has not been reached within the specified time limit
(Maximum Injection time limit exceeded). This implies that the
sensitivity of the reagent ion source is too low. To deal with this error,
try the following procedures:
1. To increase the sensitivity of the source, run automatic tuning of the
Reagent Ion Source. (See “Automatically Tuning the Reagent Ion
Source” on page 7-12.)
2. The sensitivity decrease might be due to a dirty ion volume. A
sufficiently contaminated ion volume causes the Maximum
Injection time limit to be exceeded. Clean or change the ion
volume. Refer to the LTQ Orbitrap Velos Hardware Manual for the
procedure to do this.
3. The decrease of the signal might be due to a deformed filament.
Change the filament. Refer to the LTQ Orbitrap Velos Hardware
Manual for the procedure to do this.
4. The sensitivity decrease might be due to a dirty reagent ion source
and its optics. A sufficiently contaminated reagent ion source and its
optics causes the Maximum Injection time limit to be exceeded.
Clean or change the reagent ion source and its optics. Refer to the
LTQ Orbitrap Velos Hardware Manual for the procedure to do this.
5. Increase the emission current. However, doing this might shorten
the filament life time.
6. Increase the Maximum Injection time limit. This is a temporary way
to eliminate the error message. The Maximum Injection time limit
can be increased up to the limits imposed by the overall scan cycle
time.
7-28
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
Maximum Injection Time limit and AGC Target influence the scan
duration when obtaining an ETD spectrum.
Obtaining an ETD Spectrum for Angiotensin I
This section assumes that you are infusing Angiotensin I into the
LTQ Orbitrap Velos ETD according to the procedures described in the
LTQ Series Getting Started manual. The recipe for this solution is given
in “Angiotensin I Solutions” on page 7-41.
❖
To obtain an ETD spectrum of Angiotensin I
1. Open the Tune Plus application. The Tune Plus window appears.
2. Click On/Off/Standby to On. The mass spectrometer scans the
infused analyte and produces a mass spectrum. See Figure 7-27.
On
Off
Standby
Figure 7-27. Tune plus window showing a mass scan of infused Angiotensin I
3. Turn on the reagent ion source as explained in “Turning On the
Reagent Ion Source and Viewing Reagent Ion Spectra” on
page 7-10.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-29
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
4. Click Define Scan to display the Define Scan dialog box. See
Figure 7-28 on page 7-30.
5. Enter the parent ion m/z of the 3+ charge state of Angiotensin I in
the n=2 line of the Define Scan dialog box.
Note The molecular weight of Angiotensin I (acetate hydrate) is 1296 u
and the (M + 3H)3+ parent is giving rise of a signal at m/z 433.0. ▲
Activation Type=ETD
Figure 7-28. Define Scan window with the Activation Type ETD
6. Select ETD from the Activation Type list box in the Define Scan
dialog box.
7. Click Apply in the Define Scan dialog box.
8. Click OK in the Define Scan dialog box. The Define Scan dialog
box closes and the ETD MS/MS spectrum of Angiotensin I appears
in the Tune Plus window. See Figure 7-29 on page 7-31.
7-30
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
Figure 7-29. ETD MS/MS spectrum of Angiotensin I
Optimizing the Reagent Ion Reaction Time
Typically, the system default Reagent Ion Reaction Time of 100 ms is
appropriate for doubly charged ions. In some cases it is helpful to obtain
an optimized Reagent Ion Reaction Time for your specific analyte,
especially for ions with higher charge states. The procedures presented
in this topic assume that your system is generating the reagent ions as
described in “Turning On the Reagent Ion Source and Viewing Reagent
Ion Spectra” on page 7-10.
❖
To obtain an optimized reagent ion reaction time
1. Turn on ETD activation for the analyte of interest (Angiotensin I in
this case).
2. On the Control/Scan Mode toolbar, click the Define Scan button.
to display the Define Scan dialog box.
3. Enter the parent ion mass for the analyte of interest.
4. From the Act. Type list, select ETD. (See Figure 7-28 on page 7-30.)
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-31
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
5. Click Tune. The Tune dialog box opens with two additional tabs
displayed for tuning ETD. See Figure 7-30 on page 7-32.
Tune
Reagent Ion Reaction Time tab
Total Ion Current (TIC)
Figure 7-30. Tune and Define Scan windows open in Tune Plus
6. Click the Reagent Ion Reaction Time tab in the Tune dialog box.
See Figure 7-30.
7. Optimize on either the total ion current of the product ions (TIC)
or the m/z of particular product ions.
•
Optimize on the total ion current (TIC) of the product ions:
i. Click TIC in the What To Optimize On section of the Tune
dialog box. (See Figure 7-30.)
ii. Click Start. The software generates a graph of the product
ion TIC versus reaction time. The Status box of the Tune
dialog box shows the optimized reagent ion reaction time
after the Tune process is completed.
iii. A pop up dialog asks if you want to accept the optimized
value. If you accept the optimized value, the reagent ion
reaction time is set to this optimized value in the Define
7-32
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
Scan dialog box. Otherwise, it is restored to its previous
value.
The reagent ion reaction time is now optimized based on the total
ion current.
Product Ion Mass
Figure 7-31. Tune window showing Product Ion Mass selected for Reagent Ion Reaction Time Optimization
•
Optimize on the m/z of product ions:
i. Click Product Ion Mass in the What To Optimize On
section of the Tune dialog box. See Figure 7-31. The spin
box adjacent to Product Ion Mass becomes active.
ii. Enter the m/z of the fragment of interest into the spin box.
iii. Click Start. The software generates a graph of intensity of
the m/z of interest versus reaction time. The Status box of
the Tune dialog box shows a reagent ion reaction time after
the Tune process is completed.
iv. A pop up dialog asks if you want to accept the optimized
value. If you accept the optimized value, the reagent ion
reaction time is set to this optimized value in the Define
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-33
LTQ Orbitrap Velos ETD Instruments
Performing an ETD Infusion Experiment
Scan dialog box. Otherwise, it is restored to its previous
value.
The reagent ion reaction time is now optimized based on a
particular m/z of product ions.
7-34
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Creating an Xcalibur Instrument Method That Uses ETD Activation
❖
To create an Xcalibur instrument method that uses ETD activation
1. Open the Xcalibur application (for example, from the Microsoft
Windows desktop). The Roadmap Home Page appears. See
Figure 7-32.
2. Click the Instrument Setup icon in the Roadmap Home Page.
Figure 7-32. Xcalibur Roadmap Home Page
3. Click General MS or MSn in the Select Experiment Type section of
the New Method view. See Figure 7-33 on page 7-36.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-35
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Figure 7-33. New Method view in Xcalibur Instrument Setup
4. Click the MS Detector Setup tab if this is not already the selected
tab in the Untitled-Instrument Setup window. See Figure 7-34.
5. Load the appropriate Tune Method (for example, a method saved as
described in “Saving Your ETD Tune Method” on page 7-21).
7-36
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Figure 7-34. Xcalibur MS Detector Setup view
6. Choose Scan Events to be 2 or more in the Segment 1 Settings
portion of the MS Detector Setup view. See Figure 7-35 on
page 7-38.
7. Select the Scan Event 2 bar (or the bar for a Scan Event >2). See
Figure 7-35.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-37
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Scan Events
Dependent Scan
Scan Event bar
Settings (active when Dependent Scan is selected)
Figure 7-35. Xcalibur Instrument Setup
8. Select the Dependent Scan check box at the lower left corner of the
MS Detector Setup view. (See Figure 7-35.) The adjacent Settings
button becomes active.
9. Click Settings. A Data Dependent Settings dialog box appears. See
Figure 7-36 on page 7-39.
7-38
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Figure 7-36. Data Dependent Settings dialog box in MS Detector Setup – Activation page
10. In the Data Dependent Settings dialog box, do the following:
a. Choose Scan Event > Activation in the menu on the left side of
the window.
b. Select an Activation Type. Choose ETD, CID or PQD. The
choice of Activation Type may be different for each Scan Event.
c. For Default Charge State use values of 2 or more.
d. For Isolation Width use values between 2 and 4.
e. The Activation Time is either left at its default value or chosen
as discussed in “Optimizing the Reagent Ion Reaction Time” on
page 7-31.
f. Click OK to close the Data Dependent Settings dialog box.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-39
LTQ Orbitrap Velos ETD Instruments
Creating an Xcalibur Instrument Method That Uses ETD Activation
Figure 7-37. Save As window in Xcalibur Instrument Setup, MS Detector Setup view
11. Click File > Save As to save your Xcalibur method under the file
name of your choice. (See Figure 7-37.) This method can be chosen
and run when Sequence Setup is chosen in the Xcalibur Roadmap
Home Page. (See Figure 7-32 on page 7-35.) Refer to your Xcalibur
software online Help and the previous chapters of this manual for
information about the other Data Dependent settings.
7-40
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
LTQ Orbitrap Velos ETD Instruments
Angiotensin I Solutions
Angiotensin I Solutions
This section provides instructions for the preparation of solutions
containing Angiotensin I (acetate hydrate). A stock solution is diluted to
make a test solution. The test solution is used to demonstrate the
application of the LTQ Orbitrap Velos ETD and to optimize the
reagent ion reaction time.
Handle Angiotensin I in accordance with its Material Safety Data Sheet
(MSDS).
Warning Avoid exposure to potentially harmful materials.
Always wear protective gloves and safety glasses when you handle
solvents or corrosives. Also contain waste streams and use proper
ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS)
for proper handling of a particular solvent. ▲
Note Store and handle all chemicals in accordance with standard safety
procedures. The Material Safety Data Sheet (MSDS) describing the
chemicals being used should be freely available to laboratory personnel
for them to examine at any time. Material Safety Data Sheets provide
summarized information on the hazard and toxicity of specific chemical
compounds. MSDSs also provide information on the proper handling
of compounds, first aid for accidental exposure, and procedures for
cleaning spills or dealing with leaks. ▲
Producers and suppliers of chemical compounds are required by law to
provide their customers with the most current health and safety
information in the form of an MSDS. Read the MSDS for each
chemical you use. Dispose of all laboratory reagents in the appropriate
way. (Refer to the MSDS.)
The Angiotensin I in your ETD Reagent Kit (Thermo Reagent Kit
P/N 98000-62008, Thermo Angiotensin P/N 00301-15517) is
Sigma/Aldrich #A9650.
The Angiotensin I MSDS is obtained by clicking the MSDS link at:
www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A9650
Other potentially hazardous chemicals used in the procedures in this
section include:
•
Glacial acetic acid
•
Methanol
Handle these chemicals in accord with their MSDS documents.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
7-41
LTQ Orbitrap Velos ETD Instruments
Angiotensin I Solutions
Preparing the Angiotensin I Stock Solution
❖
To prepare an Angiotensin I stock solution
1. Obtain the 1 mg vial of Angiotensin I in your accessory kit.
2. Add 382 μL of water, 382 μL of methanol, and 8 μL of glacial acetic
acid to the 1 mg of Angiotensin I.
3. Ensure that the Angiotensin I is thoroughly dissolved.
4. Label the vial Angiotensin I stock solution and store it in a freezer until
it is needed.
Preparing the Angiotensin I Test Solution
❖
To prepare an Angiotensin I sample solution
1. Pipet 100 μL of the stock solution (1nmol/μL) of Angiotensin I into
a clean polypropylene microcentrifuge tube.
2. Add 900 μL of 50:50 methanol/water (0.1% acetic acid) to the tube.
3. Mix this solution (100 pmol/μL) thoroughly.
4. Pipet 19.8 mL of 0.1% acetic acid – 50:50 methanol/water into a
clean 20 mL glass scintillation vial.
5. Add 200 μL of the 100 pmol/μL solution into the scintillation vial
to bring the final volume to 20 mL.
6. Mix this 1 pmol/μL solution thoroughly.
7. Label the vial Angiotensin I test solution and store it in a freezer until
it is needed.
7-42
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Appendix A
Miscellaneous Information
This appendix contains supplemental information for the previous
chapters. It contains the following topics:
Thermo Fisher Scientific
•
“FT Analyzer Information in Scan Header” on page A-2
•
“Data Size of FT Raw Files” on page A-4
LTQ Orbitrap Velos Getting Started
A-1
Miscellaneous Information
FT Analyzer Information in Scan Header
FT Analyzer Information in Scan Header
The Qual Browser window allows you to open a raw file and to display
scan header information for a selected scan in any of the cells. Choose
View > Scan Header to display the Scan Header of the current scan in
the active cell.
FT Analyzer Settings
The scan header information of an FTMS scan includes information
about the FT Analyzer Settings that is not available in the usual Reports
(Tune method, Instrument method, Status log, or Error log):
T=1e5
PsIT=0.65
Tog=(…)
AGC Target for this scan (here: 1e+05)
Prescan Inject Time (here: 0.65 ms)
Manual diagnostic toggles are set different from
their default values. See Table A-1 below for
detailed information.
iWf
Inject waveform on for this scan.
PvR=2e4
Preview analysis active for this scan
DiagManualSettings Calibration parameters were manually changed
under Diagnostics.
Table A-1. Actual settings of manual toggles
A-2
LTQ Orbitrap Velos Getting Started
Tog = (…)
Relevant Toggle
Current setting
Default setting
ApoOff
FT apodization
Off
On
TrExp
FT include transient
On
Off
FullP
FT profile mode
Full
Reduced
IWFoff
FT SIM and MSn injection
waveforms
Off
On
Freq
FT view frequency
On
Off
Offset
FT zero offset
On
Off
Thermo Fisher Scientific
Miscellaneous Information
FT Analyzer Information in Scan Header
FT Analyzer Messages
The scan header of an FT scan includes also so-called FT Analyzer
Messages:
RF=1535V
HCD=148eV
Ufill=0.45
RF amplitude value (here: 1535 V)
HCD collision energy in eV (here:
148 eV)
Maximum ion time reached. Here: the
real number of ions is only ~45% of the
target value.
MCal=4d
Last mass calibration for this scan range
is several days old (here: 4 days)
Est=0x24
Machine-readable result message for
post-processing tools
DAC=0.98
FT transient measurement near
saturation, this might result in spectral
harmonics (typically target value too
high)
TCal=[195..]
This is a hint that the current scan range
settings for the FT analyzer are outside
the calibrated storage/transfer mass
range. Transfer parameters are
extrapolated.
Lock=(inj524.3,1/1,+3ppm) Information about lock mass settings,
extra SIM injection of lock mass ions,
number of identified lock masses in the
spectrum, and deviation of corrected
(locked) masses compared to the external
mass calibration.
Stable=15min
Shows the elapsed stabilization time of
the FTMS analyzer high voltage
electronics after last off state or polarity
switch. For best external mass accuracies,
it is required to let the FTMS analyzer
high voltage electronics stabilize before
performing an acquisition or mass
calibration.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
A-3
Miscellaneous Information
Data Size of FT Raw Files
TempDiff=1
PkOvf
There is a temperature difference in the
FTMS analyzer temperature between
mass calibration time and current state.
This may be caused by setting a different
analyzer temperature setpoint in
instrument configuration, by rapid
significant changes in the ambient
temperature, or by not waiting for
temperature stabilization after
instrument (temperature regulation) was
off.
Internal Peak detection overflow in the
FT spectrum analysis
Note The actual FT Analyzer Messages can also be displayed in the Tune
Spectrum view, see “Spectrum View” on page 2-3. ▲
Data Size of FT Raw Files
The data size of a raw file with FT data depends on many parameters:
for example, on the number of scans, the resolution setting, and the data
format.
Table A-2 below displays typical data sizes (per scan) of an FT spectrum
(negative mode calibration solution, scan range m/z 120–1200,
AGC target 5E5, 1 microscan, resolution setting 60000) at different
FT data formats.
Table A-2. Typical data sizes (per scan) of an FT spectrum
A-4
LTQ Orbitrap Velos Getting Started
FT Data Format
Typical data size / scan
Centroid
ca. 10 kB
Reduced Profile
ca. 20 kB
Full Profile
ca. 2800 kB
Thermo Fisher Scientific
Glossary
This section lists and defines terms used in this manual. It also includes acronyms, metric prefixes, symbols, and
abbreviations.
A
B
C
D
E
F
G
H
I
J
K
L
A
A ampere
ac alternating current
ADC analog-to-digital converter
adduct ion An ion formed by the joining together of
two species, usually an ion and a molecule, and often
within the ion source, to form an ion containing all
the constituent atoms of both species.
AGC™ See Automatic Gain Control™ (AGC).
APCI See atmospheric pressure chemical ionization
(APCI).
M N
O
P
Q
R
S
T
U
V W X
Y
APCI needle, corona discharge A needle to which a
sufficiently high voltage (typically ±3 to ±5 kV) is
applied to produce a chemical ionization plasma by
the corona discharge mechanism.
See also chemical ionization (CI), chemical ionization
(CI) plasma, atmospheric pressure chemical ionization
(APCI), and corona discharge.
APCI nozzle The nozzle in the APCI probe that sprays
the sample solution into a fine mist.
See also atmospheric pressure chemical ionization
(APCI).
APCI sample tube A fused silica tube that delivers
sample solution to the APCI nozzle. The APCI
sample tube extends from the sample inlet to the
APCI nozzle.
APCI corona discharge current The ion current
carried by the charged particles in the APCI source.
The voltage on the APCI corona discharge needle
supplies the potential required to ionize the particles.
The APCI corona discharge current is set; the APCI
corona discharge voltage varies, as required, to
maintain the set discharge current.
APCI source Contains the APCI probe assembly,
APCI manifold, and API stack.
See also corona discharge and APCI corona discharge
voltage.
See also atmospheric pressure chemical ionization
(APCI), APCI manifold, and API stack.
APCI corona discharge voltage The high voltage that
is applied to the corona discharge needle in the APCI
source to produce the APCI corona discharge. The
corona discharge voltage varies, as required, to
maintain the set APCI spray current.
APCI spray current The ion current carried by the
charged particles in the APCI source. The APCI
corona discharge voltage varies, as required, to
maintain the set spray current.
See also APCI spray current.
APCI manifold The manifold that houses the APCI
sample tube and nozzle, and contains the plumbing
for the sheath and auxiliary gas.
Thermo Fisher Scientific
Z
See also atmospheric pressure chemical ionization
(APCI), and API stack.
APCI vaporizer A heated tube that vaporizes the
sample solution as the solution exits the sample tube
and enters the atmospheric pressure region of the
APCI source.
See also atmospheric pressure chemical ionization
(APCI).
LTQ Orbitrap Velos Getting Started
G-1
Glossary: API
API See atmospheric pressure ionization (API).
API atmospheric pressure region The first of two
chambers in the API source. Also referred to as the
spray chamber.
API capillary-skimmer region The area between the
capillary and the skimmer, which is surrounded by the
tube lens. It is also the area of first-stage evacuation in
the API source.
API heated capillary A tube assembly that assists in
desolvating ions that are produced by the ESI or APCI
probe.
See also API heated capillary voltage.
API heated capillary voltage The dc voltage applied to
the heated capillary. The voltage is positive for positive
ions and negative for negative ions.
See also API source and API heated capillary.
API ion transfer capillary A tube assembly that assists
in desolvating ions that are produced by the ESI, NSI,
or APCI probe.
See also API ion transfer capillary offset voltage and
API ion transfer capillary temperature.
API ion transfer capillary offset voltage A dc voltage
applied to the ion transfer capillary. The voltage is
positive for positive ions and negative for negative
ions.
See also API source and API ion transfer capillary.
API ion transfer capillary temperature The
temperature of the ion transfer capillary, which should
be adjusted for different flow rates.
See also API source and API ion transfer capillary.
API source The sample interface between the LC and
the mass spectrometer. It consists of the API probe
(ESI or APCI) and API stack.
See also atmospheric pressure ionization (API), ESI
source, APCI source, ESI probe, and API stack.
API spray chamber The first of two chambers in the
API source. In this chamber the sample liquid exits
the probe and is sprayed into a fine mist (ESI or NSI)
or is vaporized (APCI) as it is transported to the
entrance end of the ion transfer capillary.
G-2
LTQ Orbitrap Velos Getting Started
API spray shield A stainless steel, cylindrical vessel
that, in combination with the ESI or APCI flange,
forms the atmospheric pressure region of the API
source.
See also atmospheric pressure ionization (API).
API stack Consists of the components of the API
source that are held under vacuum and includes the
API spray shield, API ion transfer capillary, API tube
lens, skimmer, the ion transfer capillary mount, and
the tube lens and skimmer mount.
See also atmospheric pressure ionization (API) and
API source.
API tube lens A lens in the API source that separates
ions from neutral particles as they leave the ion
transfer capillary. A potential applied to the tube lens
focuses the ions toward the opening of the skimmer
and helps to dissociate adduct ions.
See also API tube lens offset voltage, API source, API
ion transfer capillary, and adduct ion.
API tube lens and skimmer mount A mount that
attaches to the heated capillary mount. The tube lens
and skimmer attach to the tube lens and skimmer
mount.
API tube lens offset voltage A DC voltage applied to
the tube lens. The value is normally tuned for a
specific compound.
See also API tube lens, adduct ion, and source CID.
APPI See Atmospheric Pressure Photoionization
(APPI).
ASCII American Standard Code for Information
Interchange
atmospheric pressure chemical ionization (APCI) A
soft ionization technique done in an ion source
operating at atmospheric pressure. Electrons from a
corona discharge initiate the process by ionizing the
mobile phase vapor molecules. A reagent gas forms,
which efficiently produces positive and negative ions
of the analyte through a complex series of chemical
reactions.
See also electrospray ionization (ESI).
Thermo Fisher Scientific
Glossary: atmospheric pressure ionization (API)
atmospheric pressure ionization (API) Ionization
performed at atmospheric pressure by using
atmospheric pressure chemical ionization (APCI),
electrospray ionization (ESI), or nanospray ionization
(NSI).
chemical ionization (CI) The formation of new
ionized species when gaseous molecules interact with
ions. The process can involve transfer of an electron,
proton, or other charged species between the
reactants.
Atmospheric Pressure Photoionization (APPI) A soft
ionization technique in which an ion is generated
from a molecule when it interacts with a photon from
a light source.
chemical ionization (CI) plasma The collection of
ions, electrons, and neutral species formed in the ion
source during chemical ionization.
Automatic Gain Control™ (AGC) Sets the ion
injection time to maintain the optimum quantity of
ions for each scan. With AGC on, the scan function
consists of a prescan and an analytical scan.
auxiliary gas The outer-coaxial gas (nitrogen) that
assists the sheath (inner-coaxial) gas in dispersing
and/or evaporating sample solution as the sample
solution exits the APCI, ESI, or H-ESI nozzle.
See also chemical ionization (CI).
CI See chemical ionization (CI).
CID See collision-induced dissociation (CID).
CLT curved linear trap
cm centimeter
cm3 cubic centimeter
auxiliary gas flow rate The relative rate of flow of
auxiliary gas (nitrogen) into the API source reported
in arbitrary units.
collision gas A neutral gas used to undergo collisions
with ions.
auxiliary gas inlet An inlet in the API probe where
auxiliary gas is introduced into the probe.
collision-induced dissociation (CID) An ion/neutral
process in which an ion is dissociated as a result of
interaction with a neutral target species.
See also auxiliary gas and atmospheric pressure
ionization (API).
auxiliary gas plumbing The gas plumbing that delivers
outer coaxial nitrogen gas to the ESI or APCI nozzle.
auxiliary gas valve A valve that controls the flow of
auxiliary gas into the API source.
B
b bit
B byte (8 b)
baud rate data transmission speed in events per second
BTU British thermal unit, a unit of energy
consecutive reaction monitoring (CRM) scan type A
scan type with three or more stages of mass analysis
and in which a particular multi-step reaction path is
monitored.
Convectron™ gauge A thermocouple bridge gauge that
is sensitive to the pressure as well as the thermal
conductivity of the gas used to measure pressures
between X and Y.
corona discharge In the APCI source, an electrical
discharge in the region around the corona discharge
needle that ionizes gas molecules to form a chemical
ionization (CI) plasma, which contains CI reagent
ions.
See also chemical ionization (CI) plasma and
atmospheric pressure chemical ionization (APCI).
C
CPU central processing unit (of a computer)
°C degrees Celsius
CRM See consecutive reaction monitoring (CRM) scan
type.
CE central electrode (of the Orbitrap)
C-Trap curved linear trap
cfm cubic feet per minute
<Ctrl> control key on the terminal keyboard
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
G-3
Glossary: d
D
d depth
Da dalton
DAC digital-to-analog converter
damping gas Helium gas introduced into the ion trap
mass analyzer that slows the motion of ions entering
the mass analyzer so that the ions can be trapped by
the RF voltage fields in the mass analyzer.
data-dependent scan A scan mode that uses specified
criteria to select one or more ions of interest on which
to perform subsequent scans, such as MS/MS or
ZoomScan.
to a rich ladder of sequence ions derived from cleavage
at the amide groups along the peptide backbone.
Amino acid side chains and important modifications
such as phosphorylation are left intact.
See also fluoranthene.
electrospray ionization (ESI) A type of atmospheric
pressure ionization that is currently the softest
ionization technique available to transform ions in
solution into ions in the gas phase.
EMBL European Molecular Biology Laboratory
<Enter> Enter key on the terminal keyboard
ESD electrostatic discharge
dc direct current
ESI See electrospray ionization (ESI).
divert/inject valve A valve on the mass spectrometer
that can be plumbed as a divert valve or as a loop
injector.
ESI flange A flange that holds the ESI probe in
position next to the entrance of the heated capillary,
which is part of the API stack. The ESI flange also
seals the atmospheric pressure region of the API
source and, when it is in the engaged position against
the spray shield, compresses the high-voltage
safety-interlock switch.
DS data system
DSP digital signal processor
E
ECD See electron capture dissociation (ECD).
EI electron ionization
electron capture dissociation (ECD) A method of
fragmenting gas phase ions for tandem mass
spectrometric analysis. ECD involves the direct
introduction of low energy electrons to trapped gas
phase ions.
ESI probe A probe that produces charged aerosol
droplets that contain sample ions. The ESI probe is
typically operated at liquid flows of 1 μL/min to
1 mL/min without splitting. The ESI probe includes
the ESI manifold, sample tube, nozzle, and needle.
ESI source Contains the ESI probe and the API stack.
See also electrospray ionization (ESI), ESI probe, and
API stack.
See also electron transfer dissociation (ETD) and
infrared multiphoton dissociation (IRMPD).
ESI spray current The flow of charged particles in the
ESI source. The voltage on the ESI spray needle
supplies the potential required to ionize the particles.
electron multiplier A device used for current
amplification through the secondary emission of
electrons. Electron multipliers can have a discrete
dynode or a continuous dynode.
ESI spray voltage The high voltage that is applied to
the spray needle in the ESI source to produce the ESI
spray current. In ESI, the voltage is applied to the
spray liquid as it emerges from the nozzle.
electron transfer dissociation (ETD) A method of
fragmenting peptides and proteins. In electron
transfer dissociation (ETD), singly charged reagent
anions transfer an electron to multiply protonated
peptides within the ion trap mass analyzer. This leads
G-4
LTQ Orbitrap Velos Getting Started
See also ESI spray current.
ETD See electron transfer dissociation (ETD).
eV electron volt
Thermo Fisher Scientific
Glossary: external lock mass
external lock mass A lock that is analyzed in a separate
MS experiment from your sample. If you need to run
a large number of samples, or if accurate mass samples
will be intermingled with standard samples, you might
want to use external lock masses. These allow more
rapid data acquisition by eliminating the need to scan
lock masses during each scan.
See also internal lock mass.
F
f femto (10-15)
°F degrees Fahrenheit
.fasta file extension of a SEQUEST® search database
file
ft foot
fragmentation The dissociation of a molecule or ion
to form fragments, either ionic or neutral. When a
molecule or ion interacts with a particle (electron, ion,
or neutral species) the molecule or ion absorbs energy
and can subsequently fall apart into a series of charged
or neutral fragments. The mass spectrum of the
fragment ions is unique for the molecule or ion.
FT Fourier Transformation
FT-ICR MS See Fourier Transform - Ion Cyclotron
Resonance Mass Spectrometry (FT-ICR MS).
FTMS Fourier Transformation Mass Spectroscopy
full-scan type Provides a full mass spectrum of each
analyte or parent ion. With the full-scan type, the
mass analyzer is scanned from the first mass to the last
mass without interruption. Also known as single-stage
full-scan type.
Fast Fourier Transform (FFT) An algorithm that
performs a Fourier transformation on data. A Fourier
transform is the set of mathematical formulae by
which a time function is converted into a
frequency-domain function and the converse.
FWHM Full Width at Half Maximum
FFT See Fast Fourier Transform (FFT).
G Gauss; giga (109)
fluoranthene A reagent anion that is used in an
electron transfer dissociation (ETD) experiment.
GC gas chromatograph; gas chromatography
firmware Software routines stored in read-only
memory. Startup routines and low-level input/output
instructions are stored in firmware.
forepump The pump that evacuates the foreline. A
rotary-vane pump is a type of forepump.
Fourier Transform - Ion Cyclotron Resonance Mass
Spectrometry (FT-ICR MS) A technique that
determines the mass-to-charge ratio of an ion by
measuring its cyclotron frequency in a strong
magnetic field.
fragment ion A charged dissociation product of an
ionic fragmentation. Such an ion can dissociate
further to form other charged molecular or atomic
species of successively lower formula weights.
Thermo Fisher Scientific
G
g gram
GC/MS gas chromatography / mass spectrometer
GUI graphical user interface
H
h hour
h height
handshake A signal that acknowledges that
communication can take place.
HCD Higher Energy Collision Induced Dissociation
header information Data stored in each data file that
summarizes the information contained in the file.
H-ESI source Heated-electrospray ionization (H-ESI)
converts ions in solution into ions in the gas phase by
using electrospray ionization (ESI) in combination
with heated auxiliary gas.
LTQ Orbitrap Velos Getting Started
G-5
Glossary: high performance liquid chromatography (HPLC)
high performance liquid chromatography (HPLC)
Liquid chromatography in which the liquid is driven
through the column at high pressure. Also known as
high pressure liquid chromatography.
ion gauge Measures the pressure in the mass analyzer
region (high vacuum region) of the vacuum manifold.
ion optics Focuses and transmits ions from the API
source to the mass analyzer.
HPLC See high performance liquid chromatography
(HPLC).
ion source A device that converts samples to gas-phase
ions.
HV high voltage
IRMPD See infrared multiphoton dissociation
(IRMPD).
Hz hertz (cycles per second)
K
I
ICR ion cyclotron resonance
ID inside diameter
IEC International Electrotechnical Commission
IEEE Institute of Electrical and Electronics Engineers
in inch
infrared multiphoton dissociation (IRMPD) In
infrared multiphoton dissociation (IRMPD), multiply
charged ions consecutively absorb photons emitted by
a infrared laser until the vibrational excitation is
sufficient for their fragmentation. The fragments
continue to pick up energy from the laser pulse and
fall apart further to ions of lower mass.
k kilo (103, 1000)
K kilo (210, 1024)
KEGG Kyoto Encyclopedia of Genes and Genomes
kg kilogram
L
l length
L liter
LAN local area network
lb pound
LC See liquid chromatography (LC).
See also electron capture dissociation (ECD).
instrument method A set of experiment parameters
that define Xcalibur operating settings for the
autosampler, liquid chromatograph (LC), mass
spectrometer, divert valve, syringe pump, and so on.
Instrument methods are saved as file type .meth.
internal lock mass A lock that is analyzed during the
same MS experiment as your sample and is contained
within the sample solution or infused into the
LC flow during the experiment. Internal lock masses
provide the most accurate corrections to the data.
See also external lock mass.
I/O input/output
LC/MS See liquid chromatography / mass spectrometry
(LC/MS).
LED light-emitting diode
LHe liquid helium
liquid chromatography (LC) A form of elution
chromatography in which a sample partitions between
a stationary phase of large surface area and a liquid
mobile phase that percolates over the stationary phase.
liquid chromatography / mass spectrometry
(LC/MS) An analytical technique in which a
high-performance liquid chromatograph (LC) and a
mass spectrometer (MS) are combined.
LN2 liquid nitrogen
G-6
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Glossary: lock mass
lock mass A known reference mass in the sample that is
used to correct the mass spectral data in an accurate
mass experiment and used to perform a real-time
secondary mass calibration that corrects the masses of
other peaks in a scan. Lock masses with well-defined,
symmetrical peaks work best. You can choose to use
internal lock mass or external lock mass.
MS/MS Mass spectrometry/mass spectrometry, or
tandem mass spectrometry is an analytical technique
that involves two stages of mass analysis. In the first
stage, ions formed in the ion source are analyzed by an
initial analyzer. In the second stage, the mass-selected
ions are fragmented and the resultant ionic fragments
are mass analyzed.
log file A text file, with a .log file extension, that is used
to store lists of information.
MSn scan mode The scan power equal to 1 to 10,
where the scan power is the power n in the expression
MSn. MSn is the most general expression for the scan
mode, which can include the following:
M
μ micro (10-6)
m meter; milli (10-3)
M mega (106)
M+ molecular ion
MALDI See matrix-assisted laser desorption/ionization
(MALDI).
matrix-assisted laser desorption/ionization
(MALDI) Ionization by effect of illumination with a
beam of laser generated light onto a matrix containing
a small proportion of analyte. A mass spectrometric
technique that is used for the analysis of large
biomolecules.
MB Megabyte (1048576 bytes)
MH+ protonated molecular ion
min minute
mL milliliter
mm millimeter
MRFA A peptide with the amino acid sequence
methionine–arginine–phenylalanine–alanine.
MS mass spectrometer; mass spectrometry
MS MSn power: where n = 1
MS scan modes Scan modes in which only one stage of
mass analysis is performed. The scan types used with
the MS scan modes are full-scan type and selected ion
monitoring (SIM) scan type.
MSDS Material Safety Data Sheet
Thermo Fisher Scientific
• The scan mode corresponding to the one stage of
mass analysis in a single-stage full-scan experiment
or a selected ion monitoring (SIM) experiment
• The scan mode corresponding to the two stages of
mass analysis in a two-stage full-scan experiment or
a selected reaction monitoring (SRM) experiment
• The scan mode corresponding to the three to ten
stages of mass analysis (n = 3 to n = 10) in a
multi-stage full-scan experiment or a consecutive
reaction monitoring (CRM) experiment.
See also MS scan modes and MS/MS.
multipole A symmetrical, parallel array of (usually)
four, six, or eight cylindrical rods that acts as an ion
transmission device. An RF voltage and dc offset
voltage are applied to the rods to create an electrostatic
field that efficiently transmits ions along the axis of
the multipole rods.
m/z Mass-to-charge ratio. An abbreviation used to
denote the quantity formed by dividing the mass of an
ion (in u) by the number of charges carried by the ion.
For example, for the ion C7H72+, m/z=45.5.
N
n nano (10-9)
nanospray ionization (NSI) A type of electrospray
ionization (ESI) that accommodates very low flow
rates of sample and solvent on the order of 1 to
20 nL/min (for static nanospray) or 100
to 1000 nL/min (for dynamic nanospray).
NCBI National Center for Biotechnology Information
(USA)
LTQ Orbitrap Velos Getting Started
G-7
Glossary: NIST
NIST National Institute of Standards and Technology
(USA)
NMR Normal Mass Range
NSI See nanospray ionization (NSI).
octapole An octagonal array of cylindrical rods that acts
as an ion transmission device. An RF voltage and dc
offset voltage applied to the rods create an electrostatic
field that transmits the ions along the axis of the
octapole rods.
O
Q
quadrupole A symmetrical, parallel array of four
hyperbolic rods that acts as a mass analyzer or an ion
transmission device. As a mass analyzer, one pair of
opposing rods has an oscillating radio frequency (RF)
voltage superimposed on a positive direct current (dc)
voltage. The other pair has a negative dc voltage and
an RF voltage that is 180 degrees out of phase with the
first pair of rods. This creates an electrical field (the
quadrupole field) that efficiently transmits ions of
selected mass-to-charge ratios along the axis of the
quadrupole rods.
OD outside diameter
R
OT Orbitrap
RAM random access memory
OVC outer vacuum case
raw data Uncorrected liquid chromatograph and mass
spectrometer data obtained during an acquisition.
Xcalibur and Xcalibur-based software store this data in
a file that has a .raw file extension.
Ω ohm
P
p pico (10-12)
Pa pascal
PCB printed circuit board
PDA detector Photodiode Array detector is a linear
array of discrete photodiodes on an integrated circuit
chip. It is placed at the image plane of a spectrometer
to allow a range of wavelengths to be detected
simultaneously.
resolution The ability to distinguish between two
points on the wavelength or mass axis.
retention time (RT) The time after injection at which
a compound elutes. The total time that the compound
is retained on the chromatograph column.
RF radio frequency
RF lens A multipole rod assembly that is operated with
only radio frequency (RF) voltage on the rods. In this
type of device, virtually all ions have stable trajectories
and pass through the assembly.
PE protective earth
PID proportional / integral / differential
P/N part number
p-p peak-to-peak voltage
ppm parts per million
PQD pulsed-Q dissociation
psig pounds per square inch, gauge
PTM posttranslational modification
G-8
LTQ Orbitrap Velos Getting Started
RF voltage An ac voltage of constant frequency and
variable amplitude that is applied to the ring electrode
or endcaps of the mass analyzer or to the rods of a
multipole. Because the frequency of this ac voltage is
in the radio frequency (RF) range, it is referred to as
RF voltage.
RMS root mean square
ROM read-only memory
rotary-vane pump A mechanical vacuum pump that
establishes the vacuum necessary for the proper
operation of the turbomolecular pump. (Also called a
roughing pump or forepump.)
Thermo Fisher Scientific
Glossary: RS-232
RS-232 An accepted industry standard for serial
communication connections. This Recommended
Standard (RS) defines the specific lines and signal
characteristics used by serial communications
controllers to standardize the transmission of serial
data between devices.
RT An abbreviated form of the phrase retention time
(RT). This shortened form is used to save space when
the retention time (in minutes) is displayed in a
header, for example, RT: 0.00-3.75.
S
s second
selected ion monitoring (SIM) scan type A scan type
in which the mass spectrometer acquires and records
ion current at only one or a few selected
mass-to-charge ratio values.
See also selected reaction monitoring (SRM) scan
type.
selected reaction monitoring (SRM) scan type A scan
type with two stages of mass analysis and in which a
particular reaction or set of reactions, such as the
fragmentation of an ion or the loss of a neutral moiety,
is monitored. In SRM a limited number of product
ions is monitored.
SEM secondary electron multiplier
sheath gas plumbing The gas plumbing that delivers
sheath gas to the ESI or APCI nozzle.
sheath gas pressure The rate of flow of sheath gas
(nitrogen) into the API source. A measurement of the
relative flow rate (in arbitrary units) that needs to be
provided at the sheath gas inlet to provide the required
flow of inner coaxial nitrogen gas to the ESI or APCI
nozzle. A software-controlled proportional valve
regulates the flow rate.
See also sheath gas.
sheath gas valve A valve that controls the flow of
sheath gas into the API source. The sheath gas valve is
controlled by the data system.
signal-to-noise ratio (S/N) The ratio of the signal
height (S) to the noise height (N). The signal height is
the baseline corrected peak height. The noise height is
the peak-to-peak height of the baseline noise.
SIM See selected ion monitoring (SIM) scan type.
skimmer A vacuum baffle between the higher pressure
capillary-skimmer region and the lower pressure
region. The aperture of the skimmer is offset with
respect to the bore of the ion transfer capillary.
source CID A technique for fragmenting ions in an
atmospheric pressure ionization (API) source.
Collisions occur between the ion and the background
gas, which increase the internal energy of the ion and
stimulate its dissociation.
Serial Peripheral Interface (SPI) hardware and
firmware communications protocol
SPI See Serial Peripheral Interface (SPI).
serial port An input/output location (channel) for
serial data transmission.
SRM See selected reaction monitoring (SRM) scan
type.
sheath gas The inner coaxial gas (nitrogen), which is
used in the API source to help nebulize the sample
solution into a fine mist as the sample solution exits
the ESI or APCI nozzle.
sweep gas Nitrogen gas that flows out from behind the
sweep cone in the API source. Sweep gas aids in
solvent declustering and adduct reduction.
sheath gas flow rate The rate of flow of sheath gas into
the API source. A measurement of the relative flow
rate (in arbitrary units) that needs to be provided at
the sheath gas inlet to provide the required flow of
sheath gas to the ESI or APCI nozzle.
sheath gas inlet An inlet in the API probe where sheath
gas is introduced into the probe.
See also sweep gas flow rate.
sweep gas flow rate The rate of flow of sweep gas into
the API source. A measurement of the relative flow
rate (in arbitrary units) to provide the required flow of
nitrogen gas to the sweep cone of the API source.
See also sweep gas.
syringe pump A device that delivers a solution from a
syringe at a specified rate.
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started
G-9
Glossary: T
T
U
T Tesla
u atomic mass unit
target compound A compound that you want to
identify or quantitate or that a specific protocol (for
example, an EPA method) requires that you look for.
Target compounds are also called analytes, or target
analytes.
UHV ultra high vacuum
TIC See total ion current (TIC).
Ultramark 1621 A mixture of
perfluoroalkoxycyclotriphosphazenes used for ion trap
calibration and tuning. It provides ESI singly charged
peaks at m/z 1022.0, 1122.0, 1222.0, 1322.0, 1422.0,
1522.0, 1622.0, 1722.0, 1822.0, and 1921.9.
TMP See turbomolecular pump.
UMR Universal Mass Range
Torr A unit of pressure, equal to 1 mm of mercury and
133.32 Pa.
V
total ion current (TIC) The sum of the ion current
intensities across the scan range in a mass spectrum.
tube lens offset The voltage offset from ground that is
applied to the tube lens to focus ions toward the
opening of the skimmer.
See also source CID.
Tune Method A defined set of mass spectrometer tune
parameters for the ion source and mass analyzer. Tune
methods are defined by using the Exactive Tune, Tune
Plus (LCQ Series, LXQ, and LTQ), or Tune Master
(TSQ Quantum) window and saved as the file type
.mstune, .LCQTune, .LTQTune, or .TSQTune,
respectively.
A tune method stores tune parameters only.
(Calibration parameters are stored separately, not with
the tune method.)
tune parameters Instrument parameters whose values
vary with the type of experiment.
turbomolecular pump A vacuum pump that provides
a high vacuum for the mass spectrometer and detector
system.
V volt
V ac volts alternating current
V dc volts direct current
vacuum manifold A thick-walled, aluminum chamber
with machined flanges on the front and sides and
various electrical feedthroughs and gas inlets that
encloses the API stack, ion optics, mass analyzer, and
ion detection system.
vacuum system Components associated with lowering
the pressure within the mass spectrometer. A vacuum
system includes the vacuum manifold, pumps,
pressure gauges, and associated electronics.
vent valve A valve that allows the vacuum manifold to
be vented to air or other gases. A solenoid-operated
valve.
vol volume
W
w width
W watt
TWA time weighted average
G-10
LTQ Orbitrap Velos Getting Started
Thermo Fisher Scientific
Index
A
C
absolute collision energy 2-13
abundance, of lock masses 2-11
Activation page 5-13
Activation Q 2-13, 5-13, 5-16–5-17
activation type 2-12–2-13, 5-16
Advanced Calibration Features 4-9
Advanced Features, in instrument setup 5-7
Advanced Scan features 2-8
AGC
prescan 3-11
stability 4-5
target compensation factor 4-3
target values 2-10, 2-17, 2-21, 7-28
All page, of the Status view 2-5
analyzer
information 2-4
selecting 2-8
temperature control 4-9
temperature setpoint A-4
type 2-9
vacuum 4-9
Angiotensin I
ETD MS/MS spectrum 7-31
ETD spectrum 7-29
mass scan 7-29
molecular weight 7-30
stock solution 7-42
test solution 7-42
API accessory kit 3-9
apodization 4-9
automated run 5-2
automatic calibration 3-2, 3-15
automatic gain control (AGC) 2-10, 2-17
Automatic page
Calibrate dialog box 3-15
Tune dialog box 7-13
automatic tune, recommended settings 3-12
autotuning, the reagent ion source 7-7, 7-12
caffeine methanol solution 3-4
Calibrate dialog box
Automatic page 3-15
Check page 3-19
FT Manual page 3-22
Semi-Automatic page 3-17–3-18
calibration
automatic 3-15
automatic check settings 3-19
FT manual calibration 3-22
ion trap 3-11
negative ion mode 3-20
parameters 3-2, 4-15
positive ion mode 3-20
procedures 3-2
quadrupole mass filter 7-21
reagent ion selection 7-23
semi-automatic calibration 3-17
values 3-3
calibration checks 3-19
readback 3-21
calibration compounds 3-4
calibration files
backups 3-3
content 4-3
history 3-3
calibration masses 3-10, 3-23
calibration solutions
FT manual calibration 3-10
negative mode 3-9, 3-12, 3-23
positive mode 3-8
preparing 3-4
calmix 3-23
centroid format 2-14
changes, in the ambient temperature A-4
changing, instrument settings 4-13
Charge State page, of Data Dependent Settings dialog box 5-7
charge states
determination 5-3
HCD 2-13
peaks 2-4
recognition 5-6
rejection 5-5
Check page, of the Calibrate dialog box 3-19
checking, for reagent contamination 7-6
checks
calibration 3-19
mass duration 4-13
chemicals, ordering 3-5
B
back lens
offset 7-5
potential 7-13
Back Multipole DC Offset voltage 4-11, 7-21
bakeout procedure 2-20
Br, in molecules 5-7
burnout, of filament 7-9
Thermo Fisher Scientific
LTQ Orbitrap Velos Getting Started I-1
Index: D
CI gas pressure 4-7, 7-12, 7-16
CID 2-12
Cl, in molecules 5-7
CLT RF board 4-2
collision energy 3-21
collision gas, for HCD 4-9
compensation factor 4-3
complex isotopic pattern 5-7
compressed profile format 2-14
configuration changes 6-3
converting, masses to mass-to charge ratios 5-3
cooling gas, for ETD reagent vials 7-8–7-9
Current Scan Event page 5-9–5-11
Current Segment page 5-6
D
data dependent FT SIM scan 5-9
data dependent settings
charge state 5-7
current segment 5-3, 5-6
Data Dependent Settings dialog box, Activation page 7-39
data formats
storage 2-22
switching formats 2-14
data sizes, of FT spectrum A-4
DC Offset 7-23
define scan
analyzer type 2-9
inject time 2-10
locking feature 2-11
mass range 2-9
resolution 2-9
scan rate 2-9
scan time 2-9
scan type 2-9
settings for automatic ion trap tuning 3-12
Define Scan dialog box 2-8
diagnostic views 2-4
Diagnostics dialog box, Toggles page 4-8
Display menu 2-15
Display page, of Configuration dialog box 6-2, 7-8
Display Settings page 4-15
displaying, instrument settings 4-15
drift time, of reagent ions 4-4
dynamic exclusion 5-4, 5-7
dynamic range 2-18
dynamics of ions, in HCD collision cell 4-3
E
editing, lock mass lists 2-11, 2-20
electron energy 7-12, 7-16
I-2
LTQ Orbitrap Velos Getting Started
electron multiplier
calibration 3-16
gain calibration 3-11
emission current 7-12, 7-16, 7-28
enabling
HCD collision gas 6-3
injection waveforms 2-18
error message 7-28
ESI parameters 2-22
ETD
AGC target 7-28
fragmentation efficiency 4-3
ETD Module
powering on 7-10
service switch 7-10
exclusion mass widths 5-4
external calibration 2-11, 5-2
external mass accuracy 4-6
F
filament
activation 7-6
life time 7-6, 7-16
status 7-3
fluoranthene
enabling, usage of 7-8
mass spectrum, of radical anion 7-11
frequency spectrum 4-11
FT analyzer
automatic calibration 3-18
information in scan header A-2
ion gauge 4-9
messages A-3
settings A-2
target values 2-18
temperature control 4-9
FT apodization 4-9
FT calibration
displaying settings 4-15
modes 3-17
procedures 3-15
FT diagnostics, displaying 4-15
FT dynamic range test 4-3
FT Electronics switch 7-10
FT HCD page 5-14–5-15
FT include transients 4-9
FT injection waveforms 2-19
FT instrument calibration 4-14
FT instrument settings 4-15
FT isolation test 4-4
FT lockmass abundance 4-13
FT manual mass calibration 3-22, 4-13
FT Manual page 3-2, 3-10, 3-22, 6-4
FT mass calibration 3-2, 4-3
Thermo Fisher Scientific
Index: G
automatic calibration 3-18
check 3-21
frequency 3-16
FT Mass Lists page 6-4
FT mass spectrum, resolution 2-9
FT MSn scans 4-10
FT noise test 4-4
FT optics values 4-13
FT preamplifier evaluation 4-4
FT profile mode 4-10
FT sensitivity test 4-5
FT Settings page 6-3
FT SIM scans 5-9
injection waveforms 4-10
FT spectrum, label of x-coordinate 4-11
FT stability test 4-5
FT storage transmission 3-2, 3-21
FT target values 2-18
FT temperature control evaluation 4-6
FT Temperature Monitor
dialog box 2-20
evaluation 4-6
icon 2-20
FT TIC stability 4-5
FT transfer optics 2-16
FT transfer parameters 2-16
FT transmission calibration 2-16, 3-2, 3-21
FT vacuum 2-19
FT view frequency 4-11
FT zero offset 4-11
FTMS analyzer
signal detection path 4-4
temperature 2-20, A-4
Full MS target 2-18
full noise band 4-11
Full Profile format 4-10–4-11
G
Global page 5-3
Graph view 2-5
H
hazardous chemicals 3-5
HCD
activation type 2-12, 5-13
charge state 2-13, 5-16
collision energy 3-18, 3-21, A-3
collision gas 4-9, 6-3
dynamics of ions 4-3
enabling, collision gas 6-3
multipole 4-3
scan ranges 2-14
Thermo Fisher Scientific
transmission 3-18, 3-21
transmission calibration 3-21
transmission efficiency 3-18
high charge states 5-6
high mass accuracy measurements 4-6
high mass range mode 4-3
high voltage
electronics A-3
pulser 4-4
I
infusion experiment 4-3–4-5
inject time 2-10, 3-14
injection control
FT target values 2-18
ion trap target values 2-17
Injection Control dialog box 2-17, 4-10
Reagent page 7-27
injection waveforms
enabling/disabling 2-17–2-19, 4-10
flags 2-22
inlet valve block 7-6
in-source fragmentation 3-14
Instrument Configuration dialog box 6-2, 7-8
instrument method 2-22
instrument reset 4-13
instrument settings, displaying 4-15
Instrument Setup 5-1–5-3
intensity, of peaks 5-7
internal peak detection overflow A-4
internal reference 2-11
ion energy 4-3
ion mode 2-14
ion polarity
changing 2-14
mode 2-21
ion trap
automatic calibration 3-18
calibration 3-11
target values 2-17
tuning, recommended settings 3-12
isolation/fragmentation efficiency 2-18
isotope exclusion 5-4
isotopic cluster recognition 5-7
L
last successful check 3-21
lens parameters 7-16
lifetime, of filament 7-6
lock mass abundance 2-11, 4-13
Lock Mass List button 5-2
lock mass lists 2-11, 2-20
LTQ Orbitrap Velos Getting Started
I-3
Index: M
lock mass settings A-3
lock masses 2-11, 5-2
Lock Masses dialog box 2-11, 2-20, 5-2
locking
availability 2-11
in automated runs 5-2
LTQ Orbitrap Velos Configuration dialog box
description 6-2
FT Mass Lists page 6-4
FT Settings page 6-3–6-4, 7-9
M
manual toggles A-2
mass accuracy 2-18, 4-6
mass calibration 4-3
mass deltas 5-4
mass differences 5-4
mass lists 6-4
by factory 3-23, 6-5
mass range 2-9, 5-4
coverage 4-10
mass-to-charge ratio, as mass 5-3
mass-to-charge ratios 3-23
Material Safety Data Sheet (MSDS) 3-6, 7-41
maximum AGC target value 4-4
maximum inject time 2-21, 7-28
setting 2-10
microscans 2-9, 2-21
monoisotopic peaks 5-8
monoisotopic precursor selection 5-7–5-8
MRFA
alone solution 4-3–4-4
signal 4-3
stock solution 3-7
supplier 3-4
MS Charge State 5-5
MS Detector Setup page 5-2
MSn scans, injection waveforms 4-10
MSn settings 2-12
MSn target 2-18
multiplier gain calibration 3-16
O
Orbitrap
chamber temperature 6-3
checking, the calibration 3-20
selecting, as analyzer 2-9
ordering, chemicals 3-5
overriding, calibration values 4-13
P
parameters, of vacuum system 2-19
parent mass list 5-5
parent mass widths 5-4
peaks
charge state 2-4
intensity 5-7
labels 2-3
performing, diagnostics/checks 4-1
positive ion mode
calibration solution 3-8
checking calibration 3-20
FT calibration 3-17
spectrum, of calibration solution 3-12
PQD 2-12
preamplifier
evaluation 4-4
input protection switches 4-4
preview mode 5-6
product
mass list 5-5
mass widths 5-4
product ion mass 7-33
product ions 7-32
profile format 2-14
Q
Q value 2-13
quadrupole mass filter 7-23, 7-26
N
n-butylamine
quality 3-4
stock solution 3-7
negative ion mode 3-18
calibration solution 3-9, 3-12, 3-23
checking calibration 3-20
preparing calibration solution 3-9
spectrum, of calibration solution 3-14
tuning 3-13
I-4
neutral loss mass widths 5-4
non-peptide monoisotopic peak recognition 5-8
number, of reagent ions 7-4
LTQ Orbitrap Velos Getting Started
R
readback values 4-15
reagent ion
intensity 7-6, 7-16
isolation 7-21
peaks 7-11
reaction time 7-31
Thermo Fisher Scientific
Index: S
reagent ion optics
parameters 7-5
settings 7-20
Reagent Ion Optics dialog box 7-5, 7-20
Reagent Ion Optics icon 7-2, 7-15
reagent ion source
parameters 4-16
status 7-3
turning on 7-10
Reagent Ion Source dialog box 4-7, 7-5, 7-10
Reagent Ion Source instrument control icon
color 7-3
location 7-2
Reagent Ion Source Tune dialog box, Manual page 7-16
reagent ion spectra 7-10–7-11
reagent ion transfer multipole 4-5
Reagent page
Injection Control dialog box 7-4, 7-27
Vacuum dialog box 7-7
reagent signal intensity 7-13
reagent turbopump 7-3
reagent vacuum 7-3
reagent vials 7-6, 7-8
Reduced Profile format 4-10–4-11
rejecting
mass lists 5-5
mass widths 5-4
reserpine 4-5
resistant noise peaks 4-4
resolution
of FT mass spectrum 2-9
of FTMS scan 5-6
RF amplitude value A-3
Roadmap Home Page 7-35
S
safety problem, in the laboratory 7-9
saving, ETD Tune parameters 7-21
scan cycle time 7-28
scan description 2-9
scan events 7-37
scan header 4-8, 4-10, A-2
Scan Mode menu 2-8
scan range settings A-3
scan ranges, in HCD experiment 2-14
scan rate 2-9
scan time 2-9
Scan Time Settings dialog box 2-10
scan type 2-9
scan width 5-12
selecting
analyzer type 2-8
calibration masses 3-2
Thermo Fisher Scientific
devices 4-12
experimental parameters 4-12
semi-automatic calibration 3-2, 3-17
Semi-Automatic page, of Calibrate dialog box 3-17–3-18
sensitivity, of reagent ion source 7-28
Set device page 2-12, 4-12, 4-15
settings, of manual toggles A-2
Setup menu 2-16
shortcut menus
Spectrum view 2-3, 4-9
User page 2-6
signal-to-noise ratio 4-3
SIM
injections 2-11
target 2-18
S-lens, manual adjustment 3-13
Sn, in molecules 5-7
sodium dodecyl sulfate
stock solution 3-9
supplier 3-4
sodium taurocholate
stock solution 3-9
supplier 3-4
spectrum averaging 2-15
Spectrum Display Options dialog box 2-4
Spectrum view
display options 2-3
shortcut menu 2-3, 4-9
stabilization time A-3
starting, instrument configuration 6-2
Status box, of Tune dialog box 7-33
Status view 2-5
stock solutions
Angiotensin I 7-42
caffeine 3-7
MRFA 3-7–3-8
n-butylamine 3-7
sodium dodecyl sulfate 3-8
sodium taurocholate 3-8
Ultramark 1621 3-7
storage multipole 3-20
storage transmission calibration 3-21
supplemental activation 7-4
switching, data formats 2-14
syringe pump 4-13
System Evaluation page 4-2
system performance 4-2
T
target values
FT analyzer 2-18
ion trap 2-17
lock mass 4-13
temperature
LTQ Orbitrap Velos Getting Started
I-5
Index: U
monitor 4-6
of Orbitrap chamber 6-3
of reagent vials 7-10
setpoint 2-20
temperature regulation A-4
behavior 4-6
evaluation 4-6
FTMS analyzer 2-20
results 4-6
testing, HV pulser 4-4
tin, in molecules 5-7
Toggles page 4-8
total ion current (TIC) 4-5, 7-32
transfer multipole 3-20
transients
averaging 2-9, 2-15
data format 4-9
displaying 4-9
saving 4-9
tune methods 2-21
Tune Plus window
Display menu 2-15
LTQ Orbitrap Velos 2-2
LTQ Orbitrap Velos ETD 7-2
Scan Mode menu 2-8
Setup menu 2-16
version 1-1
View menu 2-3
tuning
ETD Reagent Ion Optics settings 7-12
ion trap for negative ion mode 3-13
ion trap for positive ion mode 3-11
I-6
LTQ Orbitrap Velos Getting Started
reagent ion source 7-14
U
Ultramark 1621
peaks 3-11
stock solution 3-7
supplier 3-4
User page, of Status view 2-5–2-6
User Status Display Configuration dialog box 2-5–2-7
V
vacuum
icon 2-19
system parameters 2-19
Vacuum dialog box 2-19
vial heaters 7-6
View menu 2-3
views
Graph view 2-5
Spectrum view 2-3
Status view 2-5
X
Xcalibur
instrument method 7-35
raw file 4-9
Thermo Fisher Scientific
Thermo Fisher Scientific Inc.
81 Wyman Street
P.O. Box 9046
Waltham, Massachussetts 02454-9046
United States
www.thermo.com
Part of Thermo Fisher Scientific
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