ETD Module Getting Started Guide Revision D

ETD Module Getting Started Guide Revision D
ETD Module
For LTQ XL and Velos Pro Mass Spectrometers
(compatible with LTQ Orbitrap XL, LTQ Orbitrap Velos,
Orbitrap Velos Pro, and Orbitrap Elite)
Getting Started Guide
98000-97003 Revision D
June 2012
© 2012 Thermo Fisher Scientific Inc. All rights reserved.
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The contents of this document are subject to change without notice. All technical information in this
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Release history: Revision A, March 2008; Revision B, Jan 2009; Revision C, Dec 2010; Revision D, June 2012
Software version: Thermo LTQ Tune Plus 2.7.0 and later; Microsoft Windows 7 Professional (32 bit) SP1—
Thermo Foundation 2.0 and later, and Thermo Xcalibur 2.2 and later; Windows XP Workstation SP3—
Foundation 1.0.2 SP2 or earlier, and Xcalibur 2.1 SP1 or earlier
For Research Use Only. Not for use in diagnostic procedures.
Regulatory Compliance
Thermo Fisher Scientific performs complete testing and evaluation of its products to ensure full compliance with
applicable domestic and international regulations. When the system is delivered to you, it meets all pertinent
electromagnetic compatibility (EMC) and safety standards as described in the next section or sections by product name.
Changes that you make to your system may void compliance with one or more of these EMC and safety standards.
Changes to your system include replacing a part or adding components, options, or peripherals not specifically
authorized and qualified by Thermo Fisher Scientific. To ensure continued compliance with EMC and safety standards,
replacement parts and additional components, options, and peripherals must be ordered from Thermo Fisher Scientific
or one of its authorized representatives.
Regulatory compliance results for the following Thermo Scientific products:
• LTQ XL/ETD System (November 2008)
• LTQ Velos/ETD System (November 2008)
• Velos Pro/ETD System (April 2011)
LTQ XL/ETD System (November 2008)
EMC Directive 89/336/EEC
EMC compliance has been evaluated by TÜV Rheinland of North America, Inc.
EN 61000-3-2: 2006
EN 61000-4-4: 2004
EN 61000-3-3: 1995, A1: 2001, A2: 2005
EN 61000-4-5: 2005
EN 61326-1: 2006
EN 61000-4-6: 2007
EN 61000-4-2: 1995, A1: 1999, A2: 2001
EN 61000-4-8: 1993, A1: 2001
EN 61000-4-3: 2006
EN 61000-4-11: 2004
FCC Class A, CFR 47 Part 15: 2007
CISPR 11: 1999, A1: 1999, A2: 2002
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
LTQ Velos/ETD System (November 2008)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TÜV Rheinland of North America, Inc.
EN 61326-1: 2006
EN 61000-4-4: 2004
EN 55011: 2007
EN 61000-4-5: 2005
EN 61000-3-2: 2006
EN 61000-4-6: 2007
EN 61000-3-3: 2005
EN 61000-4-11: 2004
EN 61000-4-2: 2001
FCC Part 15: 2007
EN 61000-4-3: 2006
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EEC and harmonized standard EN 61010-1:2001.
Velos Pro/ETD System (April 2011)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TÜV Rheinland of North America, Inc.
EN 61326-1: 2006
EN 61000-4-3: 2006
EN 55011: 2007, A2: 2007
EN 61000-4-4: 2004
CFR 47, FCC Part 15, Subpart B, Class A: 2009
EN 61000-4-5: 2005
EN 61000-3-2: 2006
EN 61000-4-6: 2007
EN 61000-3-3: 1995, A1: 2001, A2: 2005
EN 61000-4-8: 1993, A1: 2001
EN 61000-4-2: 1995, A1: 1999, A2: 2001
EN 61000-4-11: 2004
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EEC and harmonized standard EN 61010-1:2001.
FCC Compliance Statement
THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO
THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL
INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED,
INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION.
CAUTION Read and understand the various precautionary notes, signs, and symbols contained inside
this manual pertaining to the safe use and operation of this product before using the device.
Notice on Lifting and Handling of
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Notice on the Proper Use of
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In compliance with international regulations: Use of this instrument in a manner not specified by Thermo Fisher
Scientific could impair any protection provided by the instrument.
Notice on the Susceptibility
to Electromagnetic Transmissions
Your instrument is designed to work in a controlled electromagnetic environment. Do not use radio frequency
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For manufacturing location, see the label on the instrument.
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Thermo Fisher Scientific has contracted with one or more recycling or disposal companies in each European Union
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rohsweee for further information on Thermo Fisher Scientific’s compliance with these Directives and the recyclers in
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C
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Thermo Scientific
Chapter 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
MS/ETD System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Electron Transfer Dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Types of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
LTQ XL and LTQ Velos Experiment Types . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ETD Experiment Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Tuning and Calibration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Chapter 2
Setting Up Thermo Foundation Instrument Configuration . . . . . . . . . . . . . . . . . . . .7
Adding the Mass Spectrometer to the Instrument Configuration. . . . . . . . . . . . . 7
Specifying the Reagent Ion Source for the Mass Spectrometer. . . . . . . . . . . . . . . 9
Chapter 3
Tuning the ETD Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Opening the Tune Plus Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Tuning the Reagent Ion Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Automatic Tuning of the Reagent Ion Optics . . . . . . . . . . . . . . . . . . . . . . . . 14
Manually Tuning the Reagent Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Semi-Automatically Tuning the Reagent Ion Optics . . . . . . . . . . . . . . . . . . . 23
Viewing the Reagent Ion Optics Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Saving the ETD Tune Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Chapter 4
Performing an ETD Infusion Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Setting the Reagent Injection Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Troubleshooting an AGC Target Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Obtaining an ETD Spectrum for Angiotensin I. . . . . . . . . . . . . . . . . . . . . . . . . 30
Optimizing the Reagent Ion Reaction Time . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
ETD Module Getting Started Guide
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Contents
Chapter 5
Running ETD Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Nth Order Double Play (ETD) Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Data Dependent NL MS3 (ETD) Experiment. . . . . . . . . . . . . . . . . . . . . . . . . . 44
Data Dependent Decision Tree Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Chapter 6
Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
Appendix A Fluoranthene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
Appendix B Angiotensin I Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
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F
Figures
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Thermo Fisher Scientific
Xcalibur Instrument Setup window showing the New Method page
(Velos Pro/ETD system) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Thermo Foundation Instrument Configuration window with configured
devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Velos Pro Configuration dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Tune Plus window for the Velos Pro/ETD system . . . . . . . . . . . . . . . . . . . . . . . 12
Reagent Ion Source dialog box (default view) . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Reagent ion spectrum for the Velos Pro/ETD system (centroid mode) . . . . . . . 17
Tune dialog box showing the Automatic page. . . . . . . . . . . . . . . . . . . . . . . . . . 18
Reagent Ion Optics dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Trap Reagent Ion Transfer Optics dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Tune dialog box showing the Manual page . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Graph view of the reagent ion source tuning in Tune Plus
(Velos Pro/ETD system) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Tune dialog box showing the Semi-Automatic page . . . . . . . . . . . . . . . . . . . . . 23
Reagent Ion Optics dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Trap Reagent Ion Transfer Optics dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Injection Control dialog box showing the Reagent page . . . . . . . . . . . . . . . . . . 27
Reagent Ion Source dialog box (default view) . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Define Scan dialog box configured for ETD on angiotensin I . . . . . . . . . . . . . . 32
ETD MS/MS spectrum of angiotensin I (Velos Pro/ETD system) . . . . . . . . . . . 33
Tune dialog box showing the Reagent Ion Reaction Time page . . . . . . . . . . . . 35
ETD MS/MS spectrum of m/z 433.3 and the optimization graph displayed
in Tune Plus (Velos Pro/ETD system) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Tune dialog box showing the Reagent Ion Reaction Time page
(m/z 388.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
ETD MS/MS spectrum of m/z 433.3 and the optimization graph displayed
in Tune Plus (Velos Pro/ETD system) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Xcalibur Roadmap view of the Home Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
New Method page for the Velos Pro/ETD system . . . . . . . . . . . . . . . . . . . . . . . 41
Nth Order Double Play dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Nth Order Double Play with ETD template. . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Data Dependent Settings dialog box showing the Activation parameters . . . . . 43
Data Dependent NL MS3 dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Example data in the Neutral Loss Masses table . . . . . . . . . . . . . . . . . . . . . . . . . 45
Data Dependent NL MS3 template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Nth Order Double Play with ETD template (Scan Event 2 selected) . . . . . . . . . 49
Current Scan Event page in the Data Dependent Settings dialog box . . . . . . . . 50
ETD Module Getting Started Guide
xi
Figures
Figure 33.
Figure 34.
xii
ETD Module Getting Started Guide
Procedures dialog box for the Data Dependent Decision Tree
(Velos Pro/ETD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ETD reagent (fluoranthene radical anion) generation from fluoranthene . . . . . . 57
Thermo Fisher Scientific
P
Preface
The ETD Module Getting Started Guide describes how to set up, calibrate, and tune the
LTQ™ XL™/ETD, LTQ Velos™/ETD, or Velos Pro™/ETD system and how to acquire electron
transfer dissociation (ETD) data.
Note For the LTQ Velos/ETD system, follow the Velos Pro/ETD system information,
unless otherwise noted.
Contents
• Related Documentation
• Safety and Special Notices
• Contacting Us
 To suggest changes to documentation or to Help
Complete a brief survey about this document by clicking the button below.
Thank you in advance for your help.
Thermo Scientific
ETD Module Getting Started Guide
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Preface
Related Documentation
In addition to this guide, Thermo Fisher Scientific provides the following documentation
listed in Table 1 for the LTQ XL/ETD and Velos Pro/ETD systems. These PDF files are
accessible from the data system computer.
Table 1. LTQ XL/ETD and Velos Pro/ETD system documentation
Model
Related documents
LTQ XL and Velos Pro
LTQ Series Preinstallation Requirements Guide
LTQ Series Getting Connected Guide
LTQ Series Getting Started Guide
LTQ Series Hardware Manual
ETD module
ETD Module Hardware Manual
To access the manuals for the mass spectrometer (MS), from the Microsoft™ Windows™
taskbar, choose Start > All Programs > Thermo Instruments > Manuals > model, where
model is your specific LTQ Series model, and then click the PDF you want to view.
Note For Thermo Xcalibur™ data system version 2.0.7 or earlier (Microsoft Windows XP
operating system), choose Start > All Programs > Xcalibur > Manuals > LTQ > model.
The software also provides Help. To access the Help, choose Help from the menu bar.
Safety and Special Notices
Make sure that you follow the precautionary statements presented in this guide. The safety
and other special notices appear in boxes. Safety and special notices include the following.
CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION
notice is accompanied by an appropriate CAUTION symbol.
IMPORTANT Highlights information necessary to prevent damage to software, loss of
data, or invalid test results; or might contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.
Table 2 lists the additional caution-specific symbols that appear in the ETD Module Getting
Started Guide.
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ETD Module Getting Started Guide
Thermo Scientific
Preface
Table 2. Caution-specific symbols and their meanings
Symbol
Meaning
Chemical: Hazardous chemicals might be present in the instrument.
Wear gloves when handling carcinogenic, corrosive, irritant,
mutagenic, or toxic chemicals. Use only approved containers and
procedures for disposing of waste oil.
Eye Hazard: Eye damage could occur from splattered chemicals or
airborne particles. Wear safety glasses when handling chemicals or
servicing the instrument.
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the information you need.
 To contact Technical Support
Phone
800-532-4752
Fax
561-688-8736
E-mail
[email protected]
Knowledge base
www.thermokb.com
Find software updates and utilities to download at mssupport.thermo.com.
 To contact Customer Service for ordering information
Phone
800-532-4752
Fax
561-688-8731
E-mail
[email protected]
Web site
www.thermo.com/ms
 To get local contact information for sales or service
Go to www.thermoscientific.com/wps/portal/ts/contactus.
 To copy manuals from the Internet
Go to mssupport.thermo.com, agree to the Terms and Conditions, and then click
Customer Manuals in the left margin of the window.
Thermo Scientific
ETD Module Getting Started Guide
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Preface
 To suggest changes to documentation or to Help
• Fill out a reader survey online at www.surveymonkey.com/s/PQM6P62.
• Send an e-mail message to the Technical Publications Editor at
[email protected]
xvi
ETD Module Getting Started Guide
Thermo Scientific
1
Introduction
This chapter provides general information about the MS/ETD systems. For additional
information, such as procedures for daily operation, maintenance, and system startup and
shutdown, refer to the ETD Module Hardware Manual.
Note For the LTQ Velos/ETD system, follow the Velos Pro/ETD system information,
unless otherwise noted.
Contents
• MS/ETD System
• Electron Transfer Dissociation
• Types of Experiments
• Tuning and Calibration
• Diagnostics
MS/ETD System
The LTQ XL/ETD and Velos Pro/ETD systems are members of the Thermo Scientific family
of mass spectrometer (MS) detectors, and consist of an electron transfer dissociation (ETD)
module installed at the back of an LTQ XL or Velos Pro mass spectrometer. With the
MS/ETD system you can perform ETD mass spectroscopy on analytes.
The LTQ XL and Velos Pro mass spectrometers are advanced analytical instruments that
include a syringe pump, a divert/inject valve, an atmospheric pressure ionization (API) source,
a 2D linear ion trap, and the Xcalibur data system.
The MS/ETD system provides a source of fluoranthene reagent ions that react with analyte
molecules in the linear ion trap of the LTQ XL or Velos Pro mass spectrometer. The ETD
module contains two reagent vials, chemical ionization (CI)/carrier gas (nitrogen) handling
hardware and flow restrictors, ion volume and filament, ion optics, and heaters for these
components.
Thermo Scientific
ETD Module Getting Started Guide
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1
Introduction
Electron Transfer Dissociation
The nitrogen gas serves two functions in the ETD process:
• As a carrier gas, the nitrogen sweeps the reagent (fluoranthene) from the vial to the ion
source where the reagent radical anions form.
• As a CI vehicle, the nitrogen undergoes collisions with 70 eV electrons from the filament
in the ion volume. These 70 eV electrons knock electrons off of the nitrogen molecules
(creating nitrogen ions). The secondary electrons resulting from these collisions have near
thermal-kinetic energies. The fluoranthene molecules capture these thermal electrons to
form the reagent radical anions that react with the analyte.
Electron Transfer Dissociation
ETD provides peptide dissociation by transferring electrons to positively charged peptides,
leading to a rich ladder of sequence ions derived from cleavage at the amide groups along the
peptide backbone. Important posttranslational modifications, such as phosphorylation and
glycosylation, are left intact.
Types of Experiments
The New Method page of the Thermo Xcalibur Instrument Setup window for the LTQ XL or
Velos Pro (Figure 1) mass spectrometer contains links (buttons) to templates for various types
of experiments. To save time entering the parameters for the instrument method, open the
template designed for the experiment type that you want to perform, enter the parameters
specific to the experiment, and then save the entries as part of an Xcalibur instrument method
(.meth file).
The experiments are organized by the following categories:
• “LTQ XL and LTQ Velos Experiment Types” on page 4
• “ETD Experiment Types” on page 4
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ETD Module Getting Started Guide
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1 Introduction
Types of Experiments
Figure 1.
Xcalibur Instrument Setup window showing the New Method page (Velos Pro/ETD system)
Thermo Scientific
ETD Module Getting Started Guide
3
1
Introduction
Types of Experiments
LTQ XL and LTQ Velos Experiment Types
As described in the LTQ Series Getting Started Guide, the experiment templates available for
the LTQ XL and Velos Pro mass spectrometers are as follows:
• General MS or MSn experiment
• Data Dependent™ experiments:
–
Data Dependent MS/MS
–
Data Dependent Triple Play
–
Nth Order Double Play
–
FAIMS Nth Order Double Play
–
Nth Order Triple Play
–
Data Dependent NL MS3
–
Data Dependent product MS3
–
Data Dependent Zoom Map
• Data Dependent Ion Tree
• Ion Mapping
ETD Experiment Types
With the addition of the ETD module to the LTQ XL or Velos Pro mass spectrometer, two
other experiment templates are available:
• Nth Order Double Play (ETD)—This experiment is useful as a survey experiment when
you cannot determine whether the CID or ETD mode of dissociation works best. For
example, CID works best for dissociating doubly-charged ions while ETD works best for
ions of a higher charge state.
• Data Dependent NL MS3 (ETD)—Identifying sites of a specific posttranslational
modification, indicated by a specific neutral loss, might be easier by using ETD to repeat
the MS/MS experiment. ETD is also useful when there are multiple sites of modification.
For additional information, see Chapter 5, “Running ETD Experiments,” and refer to the
Instrument Setup application Help.
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ETD Module Getting Started Guide
Thermo Scientific
1 Introduction
Tuning and Calibration
Tuning and Calibration
Calibrate the mass spectrometer to ensure its mass accuracy, resolution, isolation efficiency,
and dissociation efficiency. Calibration parameters are instrument parameters whose values do
not vary with the type of experiment.
IMPORTANT Thermo Fisher Scientific recommends that you check the calibration once
a week and calibrate the mass spectrometer as needed.
Tune parameters are instrument parameters (for example, ETD optics) whose values can vary
with the type of experiment or analyte being mass analyzed. To achieve the highest sensitivity
or the lowest limits of detection for an analyte of interest, tune the mass spectrometer with the
analyte.
To tune and calibrate the MS/ETD system, perform these basic steps:
• Tune and calibrate the mass spectrometer as described in the LTQ Series Getting Started
Guide.
• Tune the optics in the ETD module as described in Chapter 3, “Tuning the ETD
Module.”
Diagnostics
To learn more about some of the mass spectrometer’s diagnostic tools and tests, refer to the
LTQ Series Getting Started Guide, LTQ Series Hardware Manual, and the Tune Plus
application Help. To learn more about the ETD module’s diagnostic tools and tests, refer to
the ETD Module Hardware Manual. (At this time, not all diagnostic tools or tests are
discussed in the product guides or Help.)
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Setting Up Thermo Foundation Instrument
Configuration
Follow these procedures to add the LTQ XL or Velos Pro mass spectrometer and the reagent
ion source (ETD module) to Thermo Foundation™ Instrument Configuration and to specify
some of their configuration options.
Contents
• Adding the Mass Spectrometer to the Instrument Configuration
• Specifying the Reagent Ion Source for the Mass Spectrometer
Adding the Mass Spectrometer to the Instrument Configuration
To control the reagent ion source and the mass spectrometer from the Xcalibur data system,
add these devices to the instrument configuration.
 To add the mass spectrometer to the list of configured devices
1. Close the Xcalibur data system and Tune Plus application.
2. On the Windows taskbar, choose Start > All Programs > Thermo Foundation x.x >
Instrument Configuration, where x.x is the installed version of Thermo Foundation, to
open the Thermo Foundation Instrument Configuration window.
Note For Xcalibur data system version 2.0.7 or earlier, choose Start > All Programs >
Xcalibur > Instrument Configuration.
3. Select the devices to control from the Xcalibur data system as follows:
a. In the Device Types list (Figure 2), select All.
b. In the Available Devices list, double-click the following:
i.
The LTQ Velos MS (not shown), LTQ XL MS (not shown), or Velos Pro MS
icon to add it to the Configured Devices list.
ii. The appropriate LC device, which for this example is the Thermo EASY-nLC™.
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Setting Up Thermo Foundation Instrument Configuration
Adding the Mass Spectrometer to the Instrument Configuration
Figure 2.
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Thermo Foundation Instrument Configuration window with configured devices
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2
Setting Up Thermo Foundation Instrument Configuration
Specifying the Reagent Ion Source for the Mass Spectrometer
Specifying the Reagent Ion Source for the Mass Spectrometer
Specify the ETD module as the reagent ion source for the mass spectrometer.
 To specify the reagent ion source
1. In the Foundation Instrument Configuration window, in the Configured Devices list,
select the mass spectrometer icon, and then click Configure to open the
Model Configuration dialog box.
2. In the left pane, select Reagent Ion Source to display the reagent ion source
configuration page (Figure 3).
Figure 3.
Velos Pro Configuration dialog box
3. Select the Reagent Ion Source Configured check box.
You must manually select this check box for an MS/ETD system.
4. (Optional) Select the Use Cooling Gas check box.
Tip To save your supply of nitrogen cooling gas, clear the Use Cooling Gas check box
and allow more time for the reagent vials to cool.
Note The cooling gas only turns on if the vial temperature exceeds 100 °C (212 °F),
regardless of the vial temperature setting you select. This document assumes that you
enabled the cooling gas feature.
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Setting Up Thermo Foundation Instrument Configuration
Specifying the Reagent Ion Source for the Mass Spectrometer
5. (Optional; strongly recommended) Select the Use Low Vial Temperature check box to
heat the vial to 90 °C instead of 108 °C.
IMPORTANT Thermo Fisher Scientific recommends that you select the lower vial
temperature setting and have your local field service engineer install the new filament
and heater block (available as of October 2011).
Although the lower vial temperature can significantly extend the lifetime of the
filament, be aware that it will likely increase the reagent ion injection time, which
increases the overall MSn ETD scan times.
The instrument configuration dialog box (Figure 3) also provides information about the
contents of the reagent vials.
6. Click OK.
A message box opens:
In order for the configuration changes to take effect, you must restart the data system
and then the LTQ.
7. Click OK.
8. Restart the data system.
9. Restart the mass spectrometer.
For instructions, refer to the LTQ Series Hardware Manual.
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Tuning the ETD Module
Follow these procedures to tune the ETD module. Tuning the optics in the reagent ion source
provides settings for optimum transmission of the reagent ions (fluoranthene).
Before starting, follow the procedure “Turning On the Reagent Ion Source” in Chapter 4,
“Daily Operation,” of the ETD Module Hardware Manual.
Note You do not need to tune and calibrate the ETD module as part of your daily
routine. However, if you use the MS/ETD system regularly, perform ETD-related
calibration at least once a month (Electron Multiplier Gain for the negative ion mode and
Reagent Ion Selection on the Check page of the Calibrate dialog box).
IMPORTANT For LTQ version 2.7 or later, the Tune Plus application saves the ETD
parameters (excluding the ETD reaction time) in an ETD-dedicated system file. This
means you can switch to another ion source probe type without losing the current ETD
parameters. In LTQ version 2.6 SP3 or earlier, the ETD parameters were saved in the tune
file associated with a specific ion source probe.
Contents
• Opening the Tune Plus Window
• Tuning the Reagent Ion Optics
• Viewing the Reagent Ion Optics Settings
• Saving the ETD Tune Parameters
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Tuning the ETD Module
Opening the Tune Plus Window
Opening the Tune Plus Window
There are several ways to open the Tune Plus window (Figure 4).
 To open the Tune Plus window
Do one of the following:
• On the Windows taskbar, choose Start > All Programs > Thermo Instruments >
LTQ > model Tune, where model is your specific MS/ETD system.
Note For LTQ Series version 2.5.0 or earlier, choose Start > All Programs >
Xcalibur > model Tune.
• In the Xcalibur application, click the Roadmap View icon, the Instrument Setup
icon, the model MS icon, and then Tune Plus.
–or–
• In the Xcalibur application, click the Roadmap View icon, the Instrument Setup
icon, and the model MS icon. Then, from the main toolbar choose model >
Start Tune Plus.
Figure 4.
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Tune Plus window for the Velos Pro/ETD system
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
Tuning the Reagent Ion Optics
This section describes the three ways to tune the ion optics within the reagent ion source:
• Automatic Tuning of the Reagent Ion Optics
Automatic tuning of the reagent ion optics is the best method for most situations. Use
manual tuning to manually optimize reagent ion optics parameters and reagent ion source
parameters that are not automatically tuned, such as CI gas pressure, electron energy, and
emission current.
• Manually Tuning the Reagent Ion Source, on page 19
With manual tuning you can observe the effects of adjusting these parameters as you
change them.
• Semi-Automatically Tuning the Reagent Ion Optics, on page 23
With semi-automatic tuning you can optimize each lens setting individually within an
optimization range and according to the selected step size.
Note After tuning the reagent ion optics, remember to save the ETD tune parameters. See
“Saving the ETD Tune Parameters” on page 25.
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Tuning the ETD Module
Tuning the Reagent Ion Optics
Automatic Tuning of the Reagent Ion Optics
 To automatically tune the reagent ion optics
1. Open the Tune Plus window (see page 12).
2. Click the On/Standby button to select the On mode.
On
Standby
Note
When on, the System LED on the mass spectrometer’s front panel turns green and the
high voltage to the electron multipliers turns on. (The status LEDs are not provided
for the Orbitrap/ETD systems.)
The following conditions can cause the MS/ETD system to remain in standby mode
even though you try to turn it on:
• Attempting to turn on the reagent ion source when the restrictor, source, and
transfer line heaters are not at their target temperatures.
• When either the mass spectrometer or the ETD module goes into standby mode.
If enabled, the reagent vial nitrogen cooling turns on if the vials are at an elevated
temperature.
Exception: If you place the MS/ETD system in standby mode, the cooling
nitrogen turns on after a 1-hour delay.
• Whenever the pressure in the mass spectrometer or the ETD module exceeds its
protection limit. If enabled, the reagent vial nitrogen cooling turns on if the vials
are at an elevated temperature.
• Whenever the reagent ions’ flux intensity becomes insufficient as determined by
the Automatic Gain Control™ (AGC) setting. When this occurs, the MS/ETD
system completes the current Xcalibur sequence step before entering standby
mode. This prevents the loss of analysis results that might be affected by the
reduced reagent ion production.
3. Click the Display Graph View button.
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
4. Set the reagent ion source as follows:
a. Choose Setup > Reagent Ion Source to open the Reagent Ion Source dialog box
(Figure 5).
Figure 5.
Reagent Ion Source dialog box (default view)
b. Select the following check boxes:
• Reagent Ion Source On
• Filament On
• View Reagent Ion Spectra
For tuning, you must manually select the Filament On check box.
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Tuning the ETD Module
Tuning the Reagent Ion Optics
c. Click OK.
Note
If the reagent vials are not at their target temperature, a message appears:
Reagent Vial NOT At Temperature! Please wait …
The System LED on the ETD module flashes green to indicate that the reagent
vial heaters are turned on but are not at their target temperatures (the other
heaters are at their target temperatures).
When the reagent vial heaters reach their target temperatures, which can take
20 minutes or more, the System LED turns solid green.
When the reagent vials reach their target temperature, the system applies voltage
to the ETD module ion optics. By selecting the Filament On check box, you
ensure that the filament turns on and the Filament LED turns solid green.
(The status LEDs are not provided for the Orbitrap/ETD systems.)
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
Figure 6 shows the reagent ion spectrum at peak m/z 202.
Figure 6.
Reagent ion spectrum for the Velos Pro/ETD system (centroid mode)
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Tuning the ETD Module
Tuning the Reagent Ion Optics
5. Click the Tune button to open the Tune dialog box (Figure 7).
Figure 7.
Tune dialog box showing the Automatic page
6. Click Start.
The system begins automatically tuning the reagent ion optics in the ion source. The
Status area displays the message “Optimization Complete” after completing automatic
tuning. This message also states the percentage change in the reagent ion signal (m/z 202)
intensity relative to the prior value.
Table 3 lists the expected reagent signal intensity in profile (not centroid) mode for the
MS/ETD systems, including Orbitrap™ systems. All values assume that the reagent ion
isolation is on; and the use of a clean reagent ion source, clean ion volume and holder,
good reagent ion optics tune, and good and recent detector gain calibration in negative
ion mode.
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
Table 3. Expected profile intensities of m/z 202
Expected profile intensities of m/z 202
Reagent vial 108 °C with
the original filament
and heater block
Reagent vial 90 °C with
the new filament
and heater blocka
(recommended)
LTQ XL/ETD
~1 × 107 or greater
~2.5 × 106 or greater
Velos Pro/ETD
~4 × 107 or greater
~1 × 107 or greater
LTQ Orbitrap XL/ETD
~3 × 106 or greater
~1 × 106 or greater
LTQ Orbitrap Velos Pro/ETD
~1 × 107 or greater
~3 × 106 or greater
LTQ Orbitrap Elite/ETD
~1 × 107 or greater
~3 × 106 or greater
ETD system
Standalone MS with ETD
Orbitrap MS with ETD
a
As of October 2011
7. Repeat the Automatic Tune procedure if the percentage change is greater than 20%.
Note This is an iterative process. At some point, the signal intensity does not improve.
Manually Tuning the Reagent Ion Source
 To manually tune the reagent ion source
1. Open the Tune Plus window (see page 12).
2. Click the Display Graph View button.
3. Set the reagent ion source as follows:
a. Choose Setup > Reagent Ion Source to open the Reagent Ion Source dialog box.
b. Select the following check boxes if they are not already selected:
• Reagent Ion Source On
• Filament On
• View Reagent Ion Spectra
c. Click OK.
Note For information about the reagent vial temperatures, see page 16.
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Tuning the ETD Module
Tuning the Reagent Ion Optics
4. Choose Setup > Reagent Ion Optics to open the Reagent Ion Optics dialog box
(Figure 8).
Observe these parameters during the tuning process.
Figure 8.
Reagent Ion Optics dialog box
5. (For Velos Pro/ETD only) Choose Setup > Trap Ion Transfer to open the Trap Reagent
Ion Transfer Optics dialog box (Figure 9).
Figure 9.
Trap Reagent Ion Transfer Optics dialog box
After automatic tuning is complete, the values in this dialog box are optimally set.
Changing these values changes the reagent ion abundance in the view reagent ion spectra
mode.
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
6. Click the Tune button to open the Tune dialog box, and then click the Manual tab
(Figure 10).
Figure 10. Tune dialog box showing the Manual page
7. Select the Reagent Ion from Vial Number check box.
The vial number corresponds to the Instrument Configuration setting (see page 9).
8. Click Start.
A plot of the reagent ion signal intensity appears in the Tune Plus window (Figure 11).
Observe:
• In the Reagent Ion Optics dialog box, the response of the reagent ion signal intensity
to changes in the lens parameters
• In the Reagent Ion Source dialog box, the emission current, electron energy, and CI
gas pressure
You can adjust these parameters to achieve the maximum reagent ion signal intensity.
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Tuning the ETD Module
Tuning the Reagent Ion Optics
Figure 11. Graph view of the reagent ion source tuning in Tune Plus (Velos Pro/ETD system)
9. When you are finished, click OK in the open dialog boxes.
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3 Tuning the ETD Module
Tuning the Reagent Ion Optics
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. Open the Tune Plus window (see page 12).
2. Click the Display Graph View button.
3. Set the reagent ion source as follows:
a. Choose Setup > Reagent Ion Source to open the Reagent Ion Source dialog box.
b. Select the following check boxes if they are not already selected:
• Reagent Ion Source On
• Filament On
• View Reagent Ion Spectra
c. Click OK.
Note For information about the reagent vial temperatures, see page 16.
4. Click the Tune button to open the Tune dialog box, and then click the Semi-Automatic
tab (Figure 12).
Figure 12. Tune dialog box showing the Semi-Automatic page
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Tuning the ETD Module
Viewing the Reagent Ion Optics Settings
5. In the What to Optimize list, select one of the following items to tune, as applicable:
•
•
•
•
•
•
•
•
•
•
Reagent Ion Lens 1 (V)
Reagent Ion Gate Lens (V)
Reagent Ion Lens 2 (V)
Reagent Ion Lens 3 (V)
Back Multipole Offset (V)
Front Section LPT Offset (V) (Velos Pro/ETD only)
Center Section LPT Offset (V) (Velos Pro/ETD only)
Back Section LPT Offset (V) (Velos Pro/ETD only)
Center Lens Offset (V) (Velos Pro/ETD only)
Back Lens (V)
6. Under Optimization Range, adjust the settings in the Start, End, and Step boxes.
7. Click Start.
Viewing the Reagent Ion Optics Settings
 To view the current reagent ion optics settings
1. Open the Tune Plus window (see page 12).
2. Choose Setup > Reagent Ion Optics to open the Reagent Ion Optics dialog box
(Figure 13).
The Reagent Ion Optics dialog box (Figure 13) shows the optimized settings from an
example tuning. Your actual optimized values depend on the level of cleanliness of the ion
source and ion volume/holder. Ensure that you reoptimize the ion source voltages and CI
gas pressure flow after 5–10 days of use, after changing the ion volume, and after cleaning
the ion source.
Figure 13. Reagent Ion Optics dialog box
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3 Tuning the ETD Module
Saving the ETD Tune Parameters
3. (For Velos Pro/ETD only) Choose Setup > Trap Ion Transfer to open the Trap Reagent
Ion Transfer Optics dialog box (Figure 14).
Figure 14. Trap Reagent Ion Transfer Optics dialog box
4. When you are finished viewing the settings in these dialog boxes, click OK in each
dialog box.
Saving the ETD Tune Parameters
Tip You must save the tune parameters while the MS/ETD system is on.
For LTQ version 2.6 and earlier, after completing the tuning, you can save the ETD Tune
parameters in a tune method file.
 To save the ETD tune parameters (LTQ version 2.6 and earlier)
1. In the Tune Plus window, click the Save button.
2. Browse to a location, and then specify a file name.
3. Click Save.
IMPORTANT For LTQ version 2.7 or later, the Tune Plus application saves the ETD
parameters (excluding the ETD reaction time) in an ETD-dedicated system file. This
means you can switch to another ion source probe type without losing the current ETD
parameters. In LTQ version 2.6 SP3 or earlier, the ETD parameters were saved in the tune
file associated with a specific ion source probe.
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Performing an ETD Infusion Experiment
Follow these procedures to perform an ETD infusion experiment using angiotensin I.
Contents
• Setting the Reagent Injection Settings
• Troubleshooting an AGC Target Error
• Obtaining an ETD Spectrum for Angiotensin I
• Optimizing the Reagent Ion Reaction Time
Setting the Reagent Injection Settings
The parameters on the Reagent page in the Injection Control dialog box (Figure 15) regulate
the number of reagent ions admitted into the ion trap of the mass spectrometer.
 To set the reagent ion injection settings
1. Open the Tune Plus window (see page 12).
2. Choose Setup > Injection Control to open the Injection Control dialog box.
3. Click the Reagent tab (Figure 15).
Figure 15. Injection Control dialog box showing the Reagent page
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Performing an ETD Infusion Experiment
Troubleshooting an AGC Target Error
The ETD reagent injection control consists of two parameters:
• Max. Inject Time (ms)—Specifies the maximum time that the system allows for
anions to be injected into the trap. The default value is 50 ms.
• AGC Target—Sets the target number of reagent anions to be injected into the trap to
perform ETD. Table 4 lists the typical AGC target ranges and the recommended
initial settings. If the reagent source and filament are on, changing the reagent target
value triggers a reagent AGC event. Normally, reagent AGC events occur when a
method starts and every 30 minutes during an acquisition.
4. Set the AGC Target value to the recommended setting specified in the following table.
Table 4. AGC target values
MS/ETD system
Typical AGC target range
Recommended setting
LTQ XL/ETD
2–4 × 105
4 × 105
Velos Pro/ETD
6 × 105 to 1.6 × 106
6 × 105
5. Click OK.
The reagent ion source injects reagent anions into the trap until the ETD AGC target is
reached. The time allowed to reach the ETD AGC target cannot exceed the maximum
injection ion time setting; the maximum injection ion time takes precedence over the AGC
target.
Troubleshooting an AGC Target Error
Two reagent parameters—maximum injection time and AGC target (Figure 15 on
page 27)—influence the ETD reaction results. The ETD (ion/ion) reaction rate increases
with the reagent AGC target setting until the trap reaches the maximum storage density for
the ions. As the trapped reagent population (proportional to the reagent AGC target setting)
reaches this level, the reagent ion cloud size grows instead of becoming more dense. This
generally occurs at above 4 × 105 for the LTQ XL/ETD and above 6 × 105 for the
Velos Pro/ETD system.
If the maximum ion injection time for the reagent stays within the set limit, the ion injection
time automatically adjusts (through AGC) to provide the appropriate number of reagent ions
for the ETD reaction as defined by the reagent target. The adjustment of the ion injection
time occurs regardless of the reagent ion abundance or of the optimal reaction time that you
set. The ETD product ion yield or spectra do not vary; low reagent ion abundance only
extends the overall scan time but does not alter the quality of the product ion spectra.
The reaction time required to yield the maximum number of product ions decreases
approximately reciprocally with the reagent target setting, while the ion injection time
required to achieve the reagent AGC target setting grows proportionally.
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Performing an ETD Infusion Experiment
Troubleshooting an AGC Target Error
For 2+, 3+, and 4+ charge state precursor ions and reagent ion profile intensities in the
neighborhood of 2.5 × 106 for the LTQ XL and 1 × 107 for the Velos Pro mass spectrometer
(see Table 3 on page 19), the reaction time is longer than the reagent injection time for all
reasonable reagent target settings. Therefore, you can minimize the overall scan time by using
a sufficiently high reagent target to yield the shortest optimal ETD reaction time. If the ETD
reagent abundance (flux) is relatively low, the reagent injection time can exceed the ETD
reaction time. Use of a lower reagent target and extending the reaction times might reduce the
overall scan cycle time and, therefore, increase the number of ETD spectra acquired per
minute. At the earliest opportunity, take the appropriate steps to restore the reagent ion
abundance (see the list on this page).
If the AGC target is not reached because of the maximum injection time limit, the system
displays an error message:
Maximum Injection time limit exceeded.
The intensity of the reagent ion source is likely to be too low. You can resolve this error several
ways:
• Increase the reagent ion transmission of the reagent ion source to the ion trap by running
the automatic tuning of the reagent ion source; see “Tuning the Reagent Ion Optics” on
page 13.
• Reoptimize the reagent carrier/CI gas flow. As the ion volume and holder become dirty
with use, the optimal pressure for the reagent ion production increases.
• Increase the emission current in the Reagent Ion Source dialog box (Figure 5 on page 15).
However, an increase in emission current might decrease the filament life.
• Increase the maximum injection time limit. This is a temporary way to eliminate the error
message. You can increase the maximum injection time limit up to the limits imposed by
the overall scan cycle time.
• Clean or change the ion volume; refer to Chapter 5, “Maintenance,” in the ETD Module
Hardware Manual. The decrease in the ion intensity might be due to a dirty ion volume.
A sufficiently contaminated ion volume causes the maximum injection time limit to be
exceeded. (After you clean or change the part, retune the reagent ion source and
reoptimize the reagent carrier/CI gas flow.)
• Clean the reagent ion source and its optics; refer to Chapter 5, “Maintenance,” in the
ETD Module Hardware Manual. The sensitivity decrease might be due to a dirty reagent
ion source, its optics, or both. A contaminated reagent ion source or its optics can also
cause the maximum injection time limit to be exceeded. (After you clean the parts, retune
the reagent ion source and reoptimize the reagent carrier/CI gas flow.)
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Performing an ETD Infusion Experiment
Obtaining an ETD Spectrum for Angiotensin I
Obtaining an ETD Spectrum for Angiotensin I
This section assumes that you are infusing angiotensin I into the LTQ XL/ETD or
Velos Pro/ETD system according to the procedures in the LTQ Series Getting Started Guide.
For instructions about preparing the angiotensin I solutions, see Appendix B, “Angiotensin I
Solutions.”
 To obtain an ETD spectrum of angiotensin I
1. Open the Tune Plus window (see page 12).
2. Click the On/Standby button to select the On mode.
On
Standby
3. Set the reagent ion source as follows:
a. Choose Setup > Reagent Ion Source to open the Reagent Ion Source dialog box
(Figure 16).
Figure 16. Reagent Ion Source dialog box (default view)
b. Select the following check boxes:
• Reagent Ion Source On
• Filament On
c. Click OK.
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Performing an ETD Infusion Experiment
Obtaining an ETD Spectrum for Angiotensin I
Note
If the reagent vials are not at their target temperature, a message appears:
Reagent Vial NOT At Temperature! Please wait …
The System LED on the ETD module flashes green to indicate that the reagent
vial heaters are turned on but are not at their target temperatures (the other
heaters are at their target temperatures).
When the reagent vial heaters reach their target temperatures, which can take
20 minutes or more, the System LED turns solid green.
When the reagent vials reach their target temperature, the system applies voltage
to the ETD module ion optics. By selecting the Filament On check box, you
ensure that the filament turns on and the Filament LED turns solid green.
(The status LEDs are not provided for the Orbitrap/ETD systems.)
4. In the Tune Plus window, click the Define Scan button to open the Define Scan dialog
box (Figure 17), and then do the following:
a. Under MSn Settings, in the n = 2 row, enter the Parent Mass (m/z) of the 3+ charge
state of angiotensin I.
The molecular weight of angiotensin I (acetate hydrate) is 1296 Da. The
mass-to-charge ratio of the parent ion [M + 3H]3+ is 433.3.
b. In the Act. Type list, select ETD.
c. In the Act. Time (ms) list, enter 50.0.
d. (Optional) To identify more dissociated ions of the precursor ions, do the following:
i.
Select the Supplemental Activation check box.
ii. For this example, in the SA Charge State box, enter 3, which is the charge state of
m/z 433.3 from the angiotensin I precursor peak.
The default range is 2–6.
For additional information, refer to the Tune Plus application Help.
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Performing an ETD Infusion Experiment
Obtaining an ETD Spectrum for Angiotensin I
Figure 17. Define Scan dialog box configured for ETD on angiotensin I
5. Click OK.
The Define Scan dialog box closes and the Tune Plus window displays the ETD MS/MS
spectrum of angiotensin I (Figure 18).
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Performing an ETD Infusion Experiment
Obtaining an ETD Spectrum for Angiotensin I
Figure 18. ETD MS/MS spectrum of angiotensin I (Velos Pro/ETD system)
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Performing an ETD Infusion Experiment
Optimizing the Reagent Ion Reaction Time
Optimizing the Reagent Ion Reaction Time
In some cases obtaining an optimized reagent ion reaction time is helpful for a specific
analyte. For 2+ peptides, the optimal reaction time is approximately 100 ms, and for 3+ ions
it is approximately 50 ms. To determine a more accurate value, follow the procedure in this
section. This section assumes that the system generates the reagent ions as described in
“Turning On the Reagent Ion Source” in the ETD Module Hardware Manual.
Note When setting up a data-dependent experiment, you can shorten the ETD reaction
time for charge states that are higher than two by selecting the Enable Charge State
Dependent ETD Time feature on the Charge State page of the Data Dependent Settings
dialog box. For additional information, refer to the Tune Plus application Help.
 To optimize the reagent ion reaction time
1. Turn on ETD activation for the analyte (angiotensin I, in this case).
2. Open the Tune Plus window (see page 12).
3. Choose Setup > Reagent Ion Source to open the Reagent Ion Source dialog box.
4. Clear the View Reagent Ion Spectra check box, and then click OK.
5. Click the Define Scan button to open the Define Scan dialog box, and then do the
following:
a. Under MSn Settings, in the Parent Mass (m/z) box, enter the mass for the analyte.
b. In the Act. Type list, select ETD.
6. In the Tune Plus window, click the Tune button to open the Tune dialog box.
For ETD tuning, the Tune dialog box has two additional tabs, Collision Energy and
Reagent Ion Reaction Time.
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Performing an ETD Infusion Experiment
Optimizing the Reagent Ion Reaction Time
7. Click the Reagent Ion Reaction Time tab (Figure 19).
Figure 19. Tune dialog box showing the Reagent Ion Reaction Time page
8. Select the TIC option.
9. Click Start.
The Tune Plus application generates a graph showing the intensity of your choice of
either the mass-to-charge ratio of the ion of interest or the product ion’s total ion current
(TIC), excluding the precursor, versus the reaction time. The Status area of the page
shows a reagent ion reaction time after the tune process is complete.
A message then prompts you to accept the optimized value. If you accept the optimized
value, the reaction time parameter for the reagent ion sets to this optimized value in the
Define Scan dialog box. Otherwise, the parameter returns to its previous value.
For this example, the reagent ion reaction time is now optimized based on the TIC
(Figure 20).
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4
Performing an ETD Infusion Experiment
Optimizing the Reagent Ion Reaction Time
Figure 20. ETD MS/MS spectrum of m/z 433.3 and the optimization graph displayed in Tune Plus (Velos Pro/ETD system)
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4
Performing an ETD Infusion Experiment
Optimizing the Reagent Ion Reaction Time
To optimize on a particular product ion, in the Tune dialog box under What to Optimize On,
select the Product Ion Mass (m/z) option (Figure 21), enter the mass-to-charge ratio of the
selected product ion, and then repeat step 7. Figure 22 shows the ion spectra and optimization
graphs for m/z 388.3.
Figure 21. Tune dialog box showing the Reagent Ion Reaction Time page (m/z 388.3)
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4
Performing an ETD Infusion Experiment
Optimizing the Reagent Ion Reaction Time
Figure 22. ETD MS/MS spectrum of m/z 433.3 and the optimization graph displayed in Tune Plus (Velos Pro/ETD system)
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5
Running ETD Experiments
This chapter describes the two ETD-specific experiment templates provided by the Xcalibur
data system. For additional information, refer to the LTQ Series Getting Started Guide. Also
included is an example for using the data dependent decision tree (DDDT) logic procedure.
Contents
• Nth Order Double Play (ETD) Experiment
• Data Dependent NL MS3 (ETD) Experiment
• Data Dependent Decision Tree Procedure
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5
Running ETD Experiments
Nth Order Double Play (ETD) Experiment
Nth Order Double Play (ETD) Experiment
 To create an Nth Order Double Play (ETD) method
1. Choose Start > All Programs > Thermo Xcalibur > Xcalibur (Figure 23).
Note For Xcalibur data system version 2.0.7 or earlier, choose Start > All Programs >
Xcalibur > Xcalibur.
Figure 23. Xcalibur Roadmap view of the Home Page
2. Click the Instrument Setup icon to open the Instrument Setup window.
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5 Running ETD Experiments
Nth Order Double Play (ETD) Experiment
3. In the left Available Devices pane, click the LTQ XL MS icon or the Velos Pro MS icon
(shown in Figure 24) to open the New Method page for the device.
Figure 24. New Method page for the Velos Pro/ETD system
4. At the bottom, under Select ETD Experiment Type, click Nth Order Double
Play (ETD) to open the Nth Order Double Play dialog box (Figure 25).
The Initialize Method with Additional ETD Scan Event check box is selected as the
default setting.
Figure 25. Nth Order Double Play dialog box
5. In the Analyze Top N Peaks box, enter the number of top peaks.
6. Click OK to open the Nth Order Double Play with ETD template.
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5
Running ETD Experiments
Nth Order Double Play (ETD) Experiment
7. Toward the bottom, clear the APCI Corona On and APPI Lamp On check boxes
(Figure 26).
Figure 26. Nth Order Double Play with ETD template
Clear these check boxes.
8. Enter the scan event settings by clicking each Scan Event Number bar.
For Scan Event 2 and Scan Event 3, the Dependent Scan check box (near the lower left
corner) and its adjacent Settings button become active.
9. (For Scan Event 2 and Scan Event 3 only) Click Settings to open the Data Dependent
Settings dialog box.
For the ETD experiment type templates, the Data Dependent Settings dialog box has
fewer pages.
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5 Running ETD Experiments
Nth Order Double Play (ETD) Experiment
10. In the Data Dependent Settings dialog box, in the Scan Event list, click Activation to
display the Activation page (Figure 27).
Figure 27. Data Dependent Settings dialog box showing the Activation parameters
11. Do the following:
a. In the Activation Type box, select ETD.
b. In the Default Charge State box, enter the default value 2.
For information about the default charge state, refer to the Instrument Setup
application Help.
c. In the Isolation Width (m/z) box, enter 2, 3, or a value between 2 and 3.
The Normalized Collision Energy and Activation Q parameters are unavailable
because they are not applicable to ETD.
d. In the Activation Time (ms) box, leave the default value or enter a different value as
described in “Optimizing the Reagent Ion Reaction Time” on page 34.
e. Select the Use Mass Range check box, and use the default range of 110.00-2000.00.
f.
Click OK.
12. In the Tune Plus window, click the Save button.
13. Browse to the drive:\Xcalibur\methods folder, and specify a file name.
14. Click Save.
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5
Running ETD Experiments
Data Dependent NL MS3 (ETD) Experiment
Data Dependent NL MS3 (ETD) Experiment
With the Data Dependent NL MS3 (ETD) experiment, the mass spectrometer performs an
ETD-activated MS/MS full scan on the precursor ion if the CID-activated MS/MS scan
indicates a predefined neutral loss (NL). This experiment type can identify and characterize
metabolites and post-translational modifications.
For additional information, refer to the Instrument Setup application Help.
 To run a Data Dependent NL MS3 (ETD) experiment
1. Choose Start > All Programs > Thermo Xcalibur > Xcalibur.
Note For Xcalibur data system version 2.0.7 or earlier, choose Start > All Programs >
Xcalibur > Xcalibur.
2. Click the Instrument Setup icon.
3. In the Available Devices pane, click the LTQ XL MS or the Velos Pro MS icon to open
the New Method page (Figure 24 on page 41).
4. At the bottom, under Select ETD Experiment Type, click Data Dependent NL
MS3 (ETD) to open the Data Dependent NL MS3 dialog box (Figure 28).
The Initialize Method to Repeat the MS2 Event Using ETD check box is selected as the
default setting.
Figure 28. Data Dependent NL MS3 dialog box
5. In the Analyze Top N Peaks box, enter the number of top peaks.
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5 Running ETD Experiments
Data Dependent NL MS3 (ETD) Experiment
6. In the Neutral Loss Masses table, do one of the following:
• Type the mass and name for the neutral loss masses to identify.
–or–
• Click Import to import a list of neutral loss masses from a tab-delimited text file or
from an .xml file.
Figure 29 shows an example of a populated neutral loss table for a single phosphorylation
modification where the ion of interest might have a charge state of 1+, 2+, or 3+.
Figure 29. Example data in the Neutral Loss Masses table
7. Click OK to open the Data Dependent NL MS3 template.
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Running ETD Experiments
Data Dependent NL MS3 (ETD) Experiment
8. Toward the bottom, clear the APCI Corona On and APPI Lamp On check boxes
(Figure 30).
Figure 30. Data Dependent NL MS3 template
Clear these check boxes.
9. Enter the scan event settings by clicking each Scan Event 1 Number bar.
For Scan Event 2 and Scan Event 3, the Dependent Scan check box (near the lower left
corner) and its adjacent Settings button become active.
10. (For Scan Event 2 and Scan Event 3 only) Click Settings to open the Data Dependent
Settings dialog box (Figure 10 on page 43).
11. In the Data Dependent Settings dialog box, in the Scan Event list, click Activation to
display the Activation page (Figure 27 on page 43).
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5 Running ETD Experiments
Data Dependent Decision Tree Procedure
12. Do the following:
a. In the Activation Type box, select ETD.
b. In the Default Charge State box, enter the default value 2.
For information about the default charge state, refer to the Instrument Setup
application Help.
c. In the Isolation Width (m/z) box, enter a value 2, 3, or a value between 2 and 3.
d. The Normalized Collision Energy and Activation Q parameters are unavailable
because they are not applicable to ETD.
e. In the Activation Time (ms) box, leave the default value or enter a different value as
described in “Optimizing the Reagent Ion Reaction Time” on page 34.
f.
Select the Use Mass Range check box, and use the default range of 110.00-2000.00.
g. Click OK.
13. In the Tune Plus window, click the Save button.
14. Browse to the drive:\Xcalibur\methods folder, and specify a file name.
15. Click Save.
Data Dependent Decision Tree Procedure
The data dependent decision tree (DDDT) procedure uses real-time logic to select the most
effective dissociation method, ETD or CID, depending on the peptide’s mass-to-charge ratio
and charge state. ETD is more effective for peptides with three or more charges, and CID is
more effective for those with one or two charges.
 To configure the data dependent decision tree procedure
1. Choose Start > All Programs > Thermo Xcalibur > Xcalibur.
Note For Xcalibur data system version 2.0.7 or earlier, choose Start > All Programs >
Xcalibur > Xcalibur.
2. Click the Instrument Setup icon.
3. In the Available Devices pane, click the LTQ XL MS or the Velos Pro MS icon to open
the New Method page (Figure 24 on page 41).
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5
Running ETD Experiments
Data Dependent Decision Tree Procedure
4. Click one of the following data-dependent experiment type icons, as applicable:
•
•
•
•
•
•
•
General MS or MSn
Data Dependent MS/MS
Data Dependent Triple Play
Nth Order Double Play
FAIMS Nth Order Double Play
Nth Order Triple Play
Nth Order Double Play (ETD)
The Instrument Setup window opens showing the selected experiment template.
5. Enter the scan event settings by clicking each Scan Event Number bar.
For Scan Event 2 and higher, the Dependent Scan check box (near the lower left corner)
is selected and its adjacent Settings button becomes active. Figure 31 shows the Nth
Order Double Play (ETD) template with Scan Event 2 selected.
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5 Running ETD Experiments
Data Dependent Decision Tree Procedure
Figure 31. Nth Order Double Play with ETD template (Scan Event 2 selected)
Dependent Scan Settings button
6. Select the scan event, other than Scan Event 1, to implement the DDDT procedure.
The DDDT procedure does not work with the first “independent” scan event.
7. Below the Source Fragmentation area, click Settings (Figure 31) to open the Data
Dependent Settings dialog box.
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5
Running ETD Experiments
Data Dependent Decision Tree Procedure
8. In the Data Dependent Settings dialog box, in the Scan Event list, click Current Scan
Event to display the Current Scan Event page (Figure 32).
Figure 32. Current Scan Event page in the Data Dependent Settings dialog box
9. Select the Use Decision Tree or Other Procedure check box to open the Procedures
dialog box (Figure 33).
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5 Running ETD Experiments
Data Dependent Decision Tree Procedure
Figure 33. Procedures dialog box for the Data Dependent Decision Tree (Velos Pro/ETD)
10. In the Parameters table, select or enter a value in each row.
For information about these parameters, refer to the Instrument Setup application Help.
11. Click OK.
12. In the Data Dependent Settings dialog box, click OK.
13. In the Tune Plus window, click the Save button.
14. Browse to the drive:\Xcalibur\methods folder, and specify a file name.
15. Click Save.
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6
Troubleshooting
The MS/ETD system uses the following consumables:
• Fluoranthene
• Filament
• Ion volume
After a period of time, you might need to replenish the ETD fluoranthene reagent, replace the
filament, and clean or replace the ion volume. As the fluoranthene is depleted, the ETD
reagent mass-to-charge peak intensity diminishes by more than 10 times within a few hours of
operation (m/z = 202 in view reagent ion spectra mode).
Periodically checking this peak intensity is a good way to monitor the ETD module
consumables. To view the ETD reagent m/z signal, follow the procedure “Turning On the
Reagent Ion Source” in the ETD Module Hardware Manual.
Tip Using the 90 °C vial temperature setting, expect the filament and ion volume to last
for at least 1000 hours of operation, and a vial of the fluoranthene reagent to last for
approximately 6 months of continuous use.
Table 3 lists some ETD module problems, their causes, and their possible solutions.
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Troubleshooting
Table 3. ETD module problems, causes, and solutions (Sheet 1 of 2)
Problem
Cause
Solution
With the emission current at the
correct level, no ions appear at
m/z 202.
The m/z 202 is outside of the
displayed mass range.
Change the displayed mass range to
include m/z 202.
With the emission current at the
correct level, the m/z 202 signal
intensity drops slowly over several
days.
The filament is sagging.
Replace the filament as described in
the ETD Module Hardware Manual.
The ion volume is contaminated.
Clean or replace the ion volume, as
described in the ETD Module
Hardware Manual, when the ETD
reagent ion injection time becomes
excessive (excessively lengthening scan
times), typically 50–100 ms.
With the emission current at the
correct level, the m/z 202 signal
significantly drops within one
hour.
The reagent vial might be empty.
Replace the fluoranthene vial as
described in the ETD Module
Hardware Manual.
With a low emission current,
there is a sudden and complete
drop of the m/z 202 signal.
The filament might have just blown
out.
Check the filament. Replace it if
necessary as described in the ETD
Module Hardware Manual.
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ETD Module Getting Started Guide
In the Reagent Ion Source dialog box
The system is displaying the front
(Figure 5 on page 15), select the View
source spectra instead of the reagent
ion spectra. After automatically tuning Reagent Ion Spectra check box.
the reagent ion source, the spectrum
view redisplays the spectra of ions from
the API source, and the View Reagent
Ion Spectra check box in the Reagent
Ion Source dialog box is clear.
Thermo Scientific
6
Troubleshooting
Table 3. ETD module problems, causes, and solutions (Sheet 2 of 2)
Problem
Cause
The m/z 202 signal is less than
~2 × 105 in profile mode.
The reagent ion source needs retuning. Retune according to the instructions in
See Table 3 on page 19 for the typical Chapter 3, “Tuning the ETD
profile mode m/z 202 signal
Module.”
intensities.
The ion volume slowly becomes
contaminated. Reoptimize the reagent
carrier/CI gas flow.
A system error message advises that The AGC target has not been reached
the maximum injection time has
within the specified time limit. The
been reached for the ETD AGC.
ion volume is contaminated from use.
Solution
For details, see “Optimizing the
Reagent Ion Reaction Time” on
page 34.
1. Clean the ion volume as described
in the ETD Module Hardware
Manual.
2. Increase the maximum injection
time limit, which is typically set to
50 ms.
For details, see “Setting the
Reagent Injection Settings” on
page 27.
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A
Fluoranthene
CAUTION AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS
By law, producers and suppliers of chemical compounds are required to provide their
customers with the most current health and safety information in the form of Material
Safety Data Sheets (MSDSs). The MSDSs must be freely available to lab personnel to
examine at any time. MSDSs describe the chemicals and summarize information on the
hazard and toxicity of specific chemical compounds. They also provide information on
the proper handling of compounds, first aid for accidental exposure, and procedures for
the remedy of spills or leaks.
Read the MSDS for each chemical you use. Store and handle all chemicals in accordance
with standard safety procedures. Always wear protective gloves and safety glasses when you
use solvents or corrosives. Also, contain waste streams, use proper ventilation, and dispose
of all laboratory reagents according to the directions in the MSDS.
Fluoranthene is used as the ETD reagent in the ETD module portion of the LTQ XL/ETD
and Velos Pro/ETD systems. Fluoranthene is potentially hazardous. Use it in accordance with
its MSDS.
The fluoranthene radical anion is generated according to the reaction shown in Figure 34.
Figure 34. ETD reagent (fluoranthene radical anion) generation from fluoranthene
e→
-•
Fluoranthene
Thermo Fisher Scientific supplies fluoranthene as a two-vial kit. One vial contains 0.15 g of
fluoranthene and the other is the required empty vial. The fluoranthene vial in the Reagent
Kit for the LTQ XL/ETD or Velos Pro/ETD system is Sigma-Aldrich Supelco™ #48535. To
obtain another fluoranthene vial, click the MSDS link:
http://www.sigmaaldrich.com/catalog/search/ProductDetail/SUPELCO/48535
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B
Angiotensin I Solutions
This appendix describes how to prepare solutions containing angiotensin I (human acetate
hydrate). Dilute a stock solution to make a test solution, which you use to demonstrate the
application of the LTQ XL/ETD or Velos Pro/ETD system and to optimize the reagent ion
reaction time.
CAUTION AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS
By law, producers and suppliers of chemical compounds are required to provide their
customers with the most current health and safety information in the form of Material
Safety Data Sheets (MSDSs). The MSDSs must be freely available to lab personnel to
examine at any time. MSDSs describe the chemicals and summarize information on the
hazard and toxicity of specific chemical compounds. They also provide information on
the proper handling of compounds, first aid for accidental exposure, and procedures for
the remedy of spills or leaks.
Read the MSDS for each chemical you use. Store and handle all chemicals in accordance
with standard safety procedures. Always wear protective gloves and safety glasses when you
use solvents or corrosives. Also, contain waste streams, use proper ventilation, and dispose
of all laboratory reagents according to the directions in the MSDS.
Angiotensin I (human acetate hydrate) is potentially hazardous. Use it in accordance with its
MSDS.
The 1 mg vial of angiotensin I in the Reagent Kit for the LTQ XL/ETD or Velos Pro/ETD
system is Sigma-Aldrich Sigma #A9650. To obtain another angiotensin I vial, click the MSDS
link:
http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A9650
The procedures in this section include other potentially hazardous chemicals:
• Glacial acetic acid
• Methanol
Handle these chemicals according to their MSDS guidelines.
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B
Angiotensin I Solutions
 To prepare an angiotensin I stock solution
1. Remove the 1 mg vial of angiotensin I from the accessory kit.
2. Add the following to the 1 mg of angiotensin I:
382 μL of water
382 μL of methanol
8 μL of glacial acetic acid
3. Mix the solution thoroughly.
 To prepare an angiotensin I sample solution
1. Pipet 100 μL of the stock solution (1 nmol/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 the 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 the solution (1 pmol/μL) thoroughly.
7. Store this sample solution (1 pmol/μL) in a refrigerator until it is needed.
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I
Index
A
AGC target
error, troubleshooting 28
ranges and recommended settings 28
angiotensin I
handling 59
sample solution, preparing 60
spectrum, obtaining 30
stock solution, preparing 60
C
calibration
description 5
frequency 5
compliance
FCC v
regulatory iii
WEEE vii
contacting us xv
cooling gas, nitrogen 9, 14
D
data dependent decision tree procedure 47
Data Dependent NL MS3 (ETD) experiment
description 4
running 44
diagnostics 5
documentation survey xvi
E
electromagnetic compatibility v
electron transfer decomposition (ETD), description 1
EMC compliance iii–iv
ETD experiments 4, 27, 39
ETD module
description 1
reagent 57
ETD tune method, saving 25
Thermo Scientific
experiments, ETD
Data Dependent NL MS3 (ETD 44
Nth Order Double Play (ETD) 40
F
FCC compliance v
figures, list of xi
filament
replacing 54
sagging 54
temperature affects 10
fluoranthene, handling 57
Foundation
See instrument configuration
I
injection reagent ion settings 27
instrument configuration 7
Instrument Setup window, New Method 41
ion volume, troubleshooting 54
L
LTQ Velos mass spectrometer, note xiii, 1
M
mass spectrometer
tuning and calibration 5
MS/ETD system
description 1
experiments 2
troubleshooting 53
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Index: N
N
V
nitrogen
cooling 9, 14
description, ETD process 2
Nth Order Double Play (ETD) experiment
description 4
running 40
vial temperature 10
W
WEEE compliance vii
X
O
optimize reagent ion reaction time 34
Xcalibur
experiment templates 2
experiments for ETD 4, 39
R
reagent ion injection settings 27
reagent ion optics
tuning
automatic 14
manual 19
semi-automatic 23
viewing 24
reagent ion reaction time, optimizing 34
reagent ion signal intensities 19
reagent ion source
reoptimizing 24
resolving low intensity 29
tuning on 11
reagent ion spectrum 17
regulatory compliance iii
S
safety standards iii
standby mode, troubleshooting 14
Supplemental Activation check box 31
survey link xvi
T
troubleshooting 53
tune method
saving 25
Tune Plus, opening 12
tuning
automatic 14
description 5
manual 19
reagent ion optics 14
semi-automatic 23
tuning, ETD parameters 11
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