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- Page 1 ETD Module Getting Started Guide for the LTQ XL and LTQ Velos MS Detectors (compatible with LTQ Orbitrap XL and LTQ Orbitrap Velos) 98000-97003 Revision C December 2010...
- Page 2 © 2010 Thermo Fisher Scientific Inc. All rights reserved. EASY-nLC, LTQ Velos, and LTQ XL are trademarks, and Xcalibur is a registered trademark of Thermo Fisher Scientific Inc. in the United States. Convectron is a registered trademark of Helix Technology Corporation. Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries.
- Page 3 Thermo Fisher Scientific or one of its authorized representatives. LTQ XL/ETD System (January 2007) EMC Directive 89/336/EEC EMC compliance has been evaluated by TÜV Rheinland of North America, Inc.
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Page 4: Fcc Compliance Statement
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... - Page 5 Notice on the Proper Use of Thermo Scientific Instruments 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.
- Page 7 WEEE Compliance This product is required to comply with the European Union’s Waste Electrical & Electronic Equipment (WEEE) Directive 2002/96/EC. It is marked with the following symbol: Thermo Fisher Scientific has contracted with one or more recycling or disposal companies in each European Union (EU) Member State, and these companies should dispose of or recycle this product.
- Page 8 Conformité DEEE Ce produit doit être conforme à la directive européenne (2002/96/EC) des Déchets d'Equipements Electriques et Electroniques (DEEE). Il est marqué par le symbole suivant: Thermo Fisher Scientific s'est associé avec une ou plusieurs compagnies de recyclage dans chaque état membre de l’union européenne et ce produit devrait être collecté...
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Page 9: Table Of Contents
Saving the ETD Tune Method ........ - Page 10 Chapter 5 Performing an ETD Infusion Experiment........33 Viewing the Injection Reagent Settings .
- Page 11 Figure 1. LTQ XL/ETD system New Method page ......3 Figure 2.
- Page 12 Data Dependent NL MS3 template ....... . . 49 Figure 38. ETD Reagent (fluoranthene radical anion) generation from fluoranthene ..53 MS/ETD System Getting Started Guide...
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Page 13: Preface
Preface The ETD Module Getting Started Guide provides information about how to set up, calibrate, and tune the LTQ XL™/ETD or LTQ Velos™/ETD system and how to acquire electron transfer dissociation (ETD) data. This guide uses a shorter description for navigating Microsoft™ Windows™ and the Xcalibur™... -
Page 14: Safety And Special Notices
Note Highlights information of general interest. Tip Highlights helpful information that can make a task easier. Table 2 lists the Caution statements that appear in the MS/ETD System Getting Started Guide. MS/ETD System Getting Started Guide Thermo Scientific... -
Page 15: Contacting Us
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 MS/ETD System Getting Started Guide... - Page 16 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 techpubs-lcms@thermofisher.com. MS/ETD System Getting Started Guide Thermo Scientific...
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Page 17: Chapter 1 Introduction
The nitrogen gas serves two functions in the ETD process. It is both a carrier gas and a CI vehicle. As a carrier gas, the nitrogen sweeps the reagent from the vial to the ion source where reagent radical anions are formed. -
Page 18: Electron Transfer Dissociation
Electron Transfer Dissociation 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. Amino acid side chains and important modifications such as phosphorylation are left intact. -
Page 19: Figure 1. Ltq Xl/Etd System New Method Page
Introduction Types of Experiments Figure 1. LTQ XL/ETD system New Method page Thermo Scientific MS/ETD System Getting Started Guide... -
Page 20: Ltq Xl And Ltq Velos Experiment Types
• Ion Mapping ETD Experiment Types With the addition of the ETD module to the LTQ XL or LTQ Velos MS detector, 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. -
Page 21: Tuning And Calibration
To tune and calibrate the LTQ XL/ETD or LTQ Velos/ETD system, perform these basic steps: • Tune and calibrate the LTQ XL or LTQ Velos MS detector as described in the LTQ Series Getting Started Guide. -
Page 23: Setting Up The Xcalibur Instrument Configuration
Specifying the Reagent Ion Source for the MS Detector Adding the MS Detector to the Xcalibur Instrument Configuration To control the ETD source and the LTQ XL or LTQ Velos MS detector from the Xcalibur data system, add these devices to the Xcalibur instrument configuration. -
Page 24: Figure 2. Thermo Foundation Instrument Configuration Window
In the Device Types list, select All. b. In the Available Devices list, double-click the LTQ XL MS or LTQ Velos MS icon to add it to the Configured Devices list (Figure MS/ETD System Getting Started Guide Thermo Scientific... -
Page 25: Figure 3. Thermo Foundation Instrument Configuration Window With Configured
Setting Up the Xcalibur Instrument Configuration Adding the MS Detector to the Xcalibur Instrument Configuration Figure 3. Thermo Foundation Instrument Configuration window with configured devices Thermo Scientific MS/ETD System Getting Started Guide... -
Page 26: Specifying The Reagent Ion Source For The Ms Detector
Setting Up the Xcalibur Instrument Configuration Specifying the Reagent Ion Source for the MS Detector Specifying the Reagent Ion Source for the MS Detector Specify the ETD module as the reagent ion source for the LTQ XL or LTQ Velos MS detector. ... -
Page 27: Figure 5. Ltq Velos Configuration Dialog Box With The Reagent Ion Source
Note The lower vial temperature can extend the lifetime of the filament, but it might increase the injection time. Figure 6. LTQ Velos Configuration dialog box with the low vial temperature option selected Thermo Scientific MS/ETD System Getting Started Guide... - Page 28 In order for the configuration changes to take effect, you will need to reboot the data system and then the LTQ. 6. Click OK. 7. Reboot the data system. 8. Reboot the LTQ XL or LTQ Velos MS detector. For instructions, refer to the LTQ Series Hardware Manual. MS/ETD System Getting Started Guide Thermo Scientific...
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Page 29: Tuning
Do one of the following to open the Tune Plus window (Figure • On the Windows taskbar, choose Start > Programs > Thermo Instruments > LTQ > Tune, where model is your specific MS/ETD system. Note For LTQ Series version 2.5.0 or earlier, choose Start > Programs > Xcalibur >... -
Page 30: Tuning The Reagent Ion Optics
Manual tuning lets you observe the effects of adjusting these parameters as you change them. • “Semi-Automatically Tuning the Reagent Ion Optics” page 22 Semi-automatic tuning lets you optimize each lens setting individually within an optimization range and according to the selected step size. MS/ETD System Getting Started Guide Thermo Scientific... -
Page 31: Automatic Tuning Of The Reagent Ion Optics
MS/ETD System goes into standby mode. Reagent vial nitrogen cooling turns on if the vials are at an elevated temperature. • Exception: If you put the LTQ XL/ETD or LTQ Velos/ETD system in standby mode, the cooling nitrogen turns on after a one hour delay. -
Page 32: Figure 8. Reagent Ion Source Dialog Box
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 not at their target temperatures (the other heaters are at their target temperatures). -
Page 33: Figure 9. Spectrum Displayed In Tune Plus
5. In the Reagent Ion Source dialog box, select the View Reagent Ion Spectra check box. Figure 9 shows the reagent spectrum at peak m/z 202. Figure 9. Spectrum displayed in Tune Plus Thermo Scientific MS/ETD System Getting Started Guide... -
Page 34: Figure 10. Automatic Page In The Tune Dialog Box
5E6 to 1E7 in profile mode when the system has been cleaned and the ion volume is new. 8. Repeat Automatic Tune if the percentage change is greater than 20%. Note This is an iterative process. At some point there will be no more improvements in signal intensity. MS/ETD System Getting Started Guide Thermo Scientific... -
Page 35: Manually Tuning The Reagent Ion Source
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 not at their target temperatures (the other heaters are at their target temperatures). -
Page 36: Figure 11. Reagent Ion Optics Dialog Box
11). Figure 11. Reagent Ion Optics dialog box 7. In the Tune dialog box (Figure 10 page 18), click the Manual tab (Figure 12). Figure 12. Manual page in the Tune dialog box MS/ETD System Getting Started Guide Thermo Scientific... -
Page 37: Figure 13. Graph View Of The Reagent Ion Source Tuning In The Tune Plus Window
(Figure 8 page Adjust these parameters to achieve the maximum reagent ion signal intensity. Figure 13. Graph view of the reagent ion source tuning in the Tune Plus window for the LTQ XL Thermo Scientific MS/ETD System Getting Started Guide... -
Page 38: Semi-Automatically Tuning The Reagent Ion Optics
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 not at their target temperatures (the other heaters are at their target temperatures). -
Page 39: Figure 14. Semi-Automatic Page In The Tune Dialog Box
• Reagent ion lens 2 (V) • Reagent ion lens 3 (V) • Back Multipole Offset (V) • Back lens (V) 8. Under Optimization Range, adjust the settings in the Start, End, and Step boxes. 9. Click Start. Thermo Scientific MS/ETD System Getting Started Guide... -
Page 40: Viewing The Reagent Ion Optics Settings
Figure 15. Reagent Ion Optics dialog box 3. Click OK. Saving the ETD Tune Method After completing the tuning, you can save the ETD Tune parameters in a tune method. To save the tune method 1. In the Tune Plus window, click the Save ) button. -
Page 41: Chapter 4 Daily Operation
Daily Operation To prepare the LTQ XL/ETD or LTQ Velos/ETD system for daily operation, follow these procedures. • Before Operating the LTQ XL/ETD or LTQ Velos/ETD System • Turning On the Reagent Ion Source • After Operating the System Before Operating the LTQ XL/ETD or LTQ Velos/ETD System Follow these procedures every day before beginning the first analysis: •... -
Page 42: Checking The Vacuum Pressure
MS/ETD System goes into standby mode. Reagent vial nitrogen cooling turns on if the vials are at an elevated temperature. • Exception: If you put the LTQ XL/ETD or LTQ Velos/ETD system in standby mode, the cooling nitrogen turns on after a one hour delay. -
Page 43: Checking The Gas Supplies
To check the gas supplies 1. Check the connections to the back of the ETD module for the two nitrogen lines. 2. Verify that the pressure of the high-purity nitrogen at the MS detector is 690 ±140 kPa (100 ±20 psi). -
Page 44: Turning On The Reagent Ion Source
For more information about gas requirements, refer to the LTQ Series Preinstallation Requirements Guide. Turning On the Reagent Ion Source Even when the ETD module is on, the reagent ion source within it stays off until you turn it on. ... - Page 45 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 not at their target temperatures (the other heaters are at their target temperatures).
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Page 46: Figure 19. Fluoranthene Radical Anion Mass Spectrum
• If the spectrum is not satisfactory, tune the reagent ion optics as described in Chapter 3, “Tuning.” 8. In the Reagent Ion Source dialog box, clear the View Reagent Ion Spectra check box. This action clears the Tune Plus spectrum view from the ETD module. MS/ETD System Getting Started Guide Thermo Scientific... -
Page 47: After Operating The System
3. Clear the Reagent Ion Source On check box. When you clear this check box, the ETD source and reagent heaters go in standby mode and the filament and vial heaters turn off. Simultaneously, a valve opens to allow nitrogen gas to cool the reagent vials. -
Page 48: Purging The Oil In The Fore Pump
Purging the Oil in the Fore Pump Purge (decontaminate) the oil in the fore pump daily to remove water and other dissolved chemicals from the pump oil. For details, refer to the manufacturer’s documentation. MS/ETD System Getting Started Guide Thermo Scientific... -
Page 49: Performing An Etd Infusion Experiment
Performing an ETD Infusion Experiment This chapter discusses the ETD infusion experiment. • 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... -
Page 50: Figure 22. Reagent Page In The Injection Control Dialog Box
ETD. The default value is 1E5. 4. Click OK or Cancel. 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 Max. Inject Time setting—the maximum injection time takes precedence over the AGC target. -
Page 51: Troubleshooting An Agc Target Error
“Tuning the Reagent Ion Optics” page • Clean or change the ion volume; refer to Chapter 6, “Maintenance,” in the ETD Module Hardware Manual. 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. -
Page 52: Figure 23. Define Scan Dialog Box
The molecular weight of Angiotensin I (acetate hydrate) is 1296 Da. The mass-to-charge ratio of the parent ion [M + 3H] is 433.0. 6. In the Act. Type list, select ETD (Figure 24). Figure 24. Activation Type list in the Define Scan dialog box... -
Page 53: Figure 25. Etd Ms/Ms Spectrum Of Angiotensin I
Performing an ETD Infusion Experiment Obtaining an ETD Spectrum for Angiotensin I 7. Click OK. The Define Scan dialog box closes and Tune Plus displays the ETD MS/MS spectrum of Angiotensin I (Figure 25). Figure 25. ETD MS/MS spectrum of Angiotensin I... -
Page 54: Optimizing The Reagent Ion Reaction Time
“Turning On the Reagent Ion Source” page 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 the instructions on page 3. Click the Define Scan (... -
Page 55: Figure 27. Reagent Ion Reaction Time Page In The Tune Dialog Box
Performing an ETD Infusion Experiment Optimizing the Reagent Ion Reaction Time 7. Click the Reagent Ion Reaction Time tab (Figure 27). Figure 27. Reagent Ion Reaction Time page in the Tune dialog box 8. Click Start. Tune Plus generates a graph showing the intensity of the m/z of interest versus reaction time. -
Page 57: Running An Xcalibur Experiment By Using Etd
Running an Xcalibur Experiment by Using ETD This chapter describes the two ETD-specific experiment templates provided by the Xcalibur data system. For more information, refer to the LTQ Series Getting Started Guide. • Nth Order Double Play (ETD) Experiment •... -
Page 58: Nth Order Double Play (Etd) Experiment
Running an Xcalibur Experiment by Using ETD Nth Order Double Play (ETD) Experiment Nth Order Double Play (ETD) Experiment To create an Nth Order Double Play (ETD) method 1. Choose Start > Programs > Thermo Xcalibur > Xcalibur (Figure 28). -
Page 59: Figure 29. Instrument Setup Window For Thermo Easy-Nlc
Running an Xcalibur Experiment by Using ETD Nth Order Double Play (ETD) Experiment 2. Click the Instrument Setup icon (Figure 29). Figure 29. Instrument Setup window for Thermo EASY-nLC Thermo Scientific MS/ETD System Getting Started Guide... -
Page 60: Figure 30. New Method Page For The Ltq Xl/Etd System
LTQ Velos MS icon to open the New Method page for the device. Figure 30. New Method page for the LTQ XL/ETD system 4. Under Select ETD Experiment Type, click Nth Order Double Play (ETD) to open this dialog box (Figure 31). -
Page 61: Figure 32. Nth Order Double Play With Etd Template
Running an Xcalibur Experiment by Using ETD Nth Order Double Play (ETD) Experiment 6. Click OK. The Instrument Setup window opens showing the Nth Order Double Play with ETD template (Figure 32). Figure 32. Nth Order Double Play with ETD template 7. -
Page 62: Figure 33. Data Dependent Settings Dialog Box
(Figure 34). The activation type is automatically set to ETD. Figure 34. Activation parameters 10. In the Default Charge State box, enter a value of 5 or more. 11. In the Isolation Width (m/z) box, enter a value between 2 and 3. -
Page 63: Data Dependent Nl Ms Experiment
(Figure 35). The Initialize Method to Repeat the MS2 Event Using ETD check box is selected as the default setting. Figure 35. Data Dependent NL MS3 dialog box 5. In the Analyze Top N Peaks box, enter the number of top peaks. -
Page 64: Figure 36. Neutral Loss Masses Table Example
Running an Xcalibur Experiment by Using ETD Data Dependent NL MS 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. • Click Import to import a list of neutral loss masses from a tab-delimited text file or from an XML file. -
Page 65: Figure 37. Data Dependent Nl Ms3 Template
10. In the left pane, under the Scan Event list, click Activation to see the activation parameters (Figure 34 page 46). The activation type is automatically set to ETD. 11. In the Default Charge State box, enter a value of 5 or more. Thermo Scientific MS/ETD System Getting Started Guide... - Page 66 Running an Xcalibur Experiment by Using ETD Data Dependent NL MS Experiment 12. In the Isolation Width (m/z) box, enter a value between 2 and 3. The Normalized Collision Energy and Activation Q parameters are unavailable because they are not applicable to ETD.
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Page 67: Chapter 7 Troubleshooting
• Fluoranthene • 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 consumables are depleted, the ETD reagent m/z peak intensity diminishes (m/z = 202 in negative polarity mode). Periodically checking this peak intensity is a good way to monitor the ETD module consumables. - Page 68 Troubleshooting Table 3. ETD module problems, causes, and solutions Problem Cause Solution With the emission current at the The m/z 202 is outside of the mass Set the starting mass lower. correct level, no ions appear at range. m/z 202.
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Page 69: Appendix A Fluoranthene
Fluoranthene Fluoranthene is used as the ETD reagent in the ETD module portion of the LTQ XL/ETD or LTQ Velos/ETD system. The fluoranthene radical anion is generated according to the reaction shown in Figure Figure 38. ETD Reagent (fluoranthene radical anion) generation from fluoranthene •... - Page 70 Fluoranthene The fluoranthene in the ETD Reagent Kit (Thermo Reagent Kit P/N 98000-62008, Thermo Fluoranthene P/N HAZMAT-00013) is Sigma/Aldrich Supelco P/N 48535. The fluoranthene MSDS is available on the following Web page: http://www.sigmaaldrich.com/catalog/search/ProductDetail/SUPELCO/48535 Thermo Fisher Scientific supplies fluoranthene as a two-vial kit. One vial contains 150 mg of fluoranthene and the other is the required empty vial.
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Page 71: Appendix B Angiotensin I Solutions
This chapter describes how to prepare 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 XL/ETD or LTQ Velos/ETD system and to optimize the reagent ion reaction time. - Page 72 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. MS/ETD System Getting Started Guide Thermo Scientific...
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Page 73: Index
Nth Order Double Play (ETD) experiment description regulatory running WEEE reagent carrier gas, checking supply Data Dependent NL MS (ETD) experiment... - Page 74 Index: V vacuum pressure, checking WEEE compliance Xcalibur experiments for ETD Xcalibur instrument configuration Xcalibur templates MS/ETD System Getting Started Guide Thermo Scientific...
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