Waters Xevo QTof MS/ nanoACQUITY UPLC System€¦ · Xevo QTof MS Operator’s Overview and Maintenance Guide (p/n 71500174502) Waters Educational Services . This guide assumes that

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Waters® Xevo® QTof MS/ nanoACQUITY UPLC® System

Customer Familiarization Guide

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NOTICE

Notice

The information in this document is subject to change without notice and must not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This guide is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from the use of this guide.

©2009 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS BOOK OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

Waters, Xevo, and nanoACQUITY UPLC are registered trademarks of Waters Corporation.

MassLynx, LockSpray, DDTC, and The Science of What’s Possible are trademarks of Waters Corporation.

All other trademarks are the sole property of their respective owners.

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Table of contents

Notice................................................................................................................. 2 Table of contents.................................................................................................. 3 Introduction......................................................................................................... 4

Safety precautions ........................................................................................... 4 Other operational assistance.............................................................................. 4

System overview.................................................................................................. 5 Important instrument concepts............................................................................... 6

Mass measurement .......................................................................................... 6 Detector (MCP) gain ......................................................................................... 7 Dead time ....................................................................................................... 7 nanoACQUITY injection modes ........................................................................... 8

Preparing the mobile phase.................................................................................... 9 Preparing the samples......................................................................................... 10 Opening MassLynx™ software and creating a project ............................................... 11 Preparing the nanoACQUITY UPLC® system ............................................................ 12

Characterizing the needle seal.......................................................................... 15 Characterizing the needle and loop volumes....................................................... 15 Connecting the inlet tubing and equilibrating the system ..................................... 15

Setting up the nanoACQUITY UPLC inlet methods.................................................... 20 Configuring and tuning the mass spectrometer ....................................................... 23

Checking the quality of the ion beam and optimizing the sprayer position............... 24 Using IntelliStart for LockSpray source setup ..................................................... 27

Mass calibration ................................................................................................. 32 Using IntelliStart to create calibration ............................................................... 32 Using IntelliStart for the calibration check.......................................................... 37

Setting up the mass spectrometer MSE methods ..................................................... 41 Acquisition and processing samples....................................................................... 44

Creating the sample list .................................................................................. 44 Acquiring the sample data ............................................................................... 45 Viewing the sample results .............................................................................. 46 Extracting specific masses by m/z value ............................................................ 48 Creating a ChroTool method ............................................................................ 48 View chromatograms created by ChroTool ......................................................... 52

Appendix................................................................................................................ 54

ACQUITY TUV detector ........................................................................................ 54

TABLE OF CONTENTS

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Introduction

The guide demonstrates the basic process of daily setup and operation, creating processing methods, and full MS scan data processing.

Once completed, the guide remains with the customer for future reference and training.

Safety precautions

The customer must understand and follow all the necessary safety precautions described in the relevant Waters® Operator’s Guides provided with the system, prior to performing this procedure.

Other operational assistance

MassLynx™ Software Online Help

Online nanoACQUITY UPLC® Console Help

Xevo QTof MS Operator’s Overview and Maintenance Guide (p/n 71500174502)

Waters Educational Services

This guide assumes that the Xevo QTof MS / nanoACQUITY UPLC system has been installed and verified to the appropriate performance level, and the system is ready to be operated. If this is not the case, refer to the Xevo QTof MS and/or nanoACQUITY UPLC installation documentation.

INTRODUCTION

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System overview

The Waters Xevo QTof MS / nanoACQUITY UPLC system is a high performance LC/MS system enabling the rapid acquisition and processing of accurate mass data from chromatographic analyses.

The basic components of the system are:

Waters Xevo QTof mass spectrometer

Waters nanoACQUITY UPLC system

Each component of the system may have differences in configuration depending on the options specified during the purchase of the system.

Ensure that the basic familiarization topics have been covered prior to completing this Customer Familiarization Guide. Refer to the appropriate sections of the Xevo QTof MS nanoACQUITY UPLC System Installation Checklist (p/n 715002121).

SYSTEM OVERVIEW

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Significant concepts

Mass measurement

There are three levels of mass measurement:

Nominal mass

Calibrated mass

nanoLockSpray™

Nominal mass

Upon initial startup, the mass axis must be nominally adjusted using a known reference ion to ensure that the placement of the ion peaks is nominally correct (for example, to within ±0.1 Da). This is required for ease of tuning and proper calibration of the instrument for accurate mass determination.

1. Place the system in System View.

2. From the MS Tune page, click System > Acquisition Settings.

3. Optimize the Veff parameter.

Calibrated mass

A reference file, containing the accurate mass details of the peaks for a known sample, is compared against the peaks obtained when an acquisition is performed on the known sample. This process creates a calibration equation which is then applied to correct the mass position of all subsequently acquired sample data.

NOTE: The frequency of calibration will vary depending on many factors, such as environmental conditions, level of vacuum, and accuracy of desired results.

The Xevo QTof MS features IntelliStart technology for simple, automated calibration creation and checking. During normal use, it is recommended that the IntelliStart calibration check process is performed as required to maintain the level of calibration accuracy.

nanoLockSpray

Temperature variations in the environment can cause drifts in measurements of a few hundred parts per million (ppm) over the course of a day. This natural drift can be compensated for by applying a single or multipoint Lock mass correction from an external reference compound.

The Xevo QTof MS incorporates an integral nanoLockSpray ion source with a second sprayer for introducing the reference compound separately to the main analyte data. A rotating baffle allows the instrument to sample the reference spray at regular intervals during the sample acquisition, ensuring consistent reference measurements are taken.

The MassLynx software uses the reference data to correct the mass position of the main analyte data automatically.

IMPORTANT INSTRUMENT CONCEPTS

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Figure 1 - nanoLockSpray source

Detector (MCP) gain

The MCP detector gain is the amount of electronic signal generated from the arrival of a single ion. Optimize the gain periodically to ensure that the mass spectrometer can efficiently detect all ion impacts.

NOTE: Using a detector voltage that is too low may result in some ions being undetected. Using a detector voltage that is too high may result in decreased detector lifetime.

Dead time

The data acquisition system for the instrument is a time to digital converter (TDC). This is an ion counting system which generates a mass spectrum by histogramming the arrival times of ions in transient memory. After the arrival and registration of an ion by the TDC there is a minimum time interval before a subsequent ion arrival can be registered. This "dead time" of the TDC is approximately 5 ns. If more than one ion arrives at the detector simultaneously, then only one count is registered.

At high ion currents, some of the ion arrivals are not registered, leading to a shift to lower mass centroids, and lower measured areas on reported peaks. MassLynx compensates for these effects by applying Digital Dead Time Correction (DDTC™), which compensates for these effects and enables accurate mass measurement and quantitation to be achieved over a large range of ion currents.

NOTE: The effectiveness of DDTC is not unlimited. It is important to keep ion counts well below these limits when performing manual calibrations or acquisitions.

The maximum peak intensity that can be corrected by DDTC corresponds to an "ions-per-push" value of 0.6. Peaks with intensities in excess of 0.6 IPP are not corrected. Peaks with IPP values of less than 0.1 do not show any dead time distortion effects, and the application of DDTC to such peaks will make little or no difference.

IMPORTANT INSTRUMENT CONCEPTS

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nanoACQUITY injection modes

The sample manager uses a fixed loop injection scheme, and has two modes of injection:

Full loop for maximum precision and column efficiency for a specific injection volume.

Partial loop for maximum speed and minimum consumption of sample.

NOTE: All mechanically driven injections result in a dispersal of the sample as a result of viscous flow.

The volumes of the loops vary slightly, so every loop is "characterized" to determine the actual volume. The sample manager Characterize menu records the measured loop volume and the nominal loop volumes. Discard the loop volume if it is smaller than expected.

Full loop mode

The sample loop is overfilled several times, ensuring that the diluted portions of the volume drawn from the sample are not injected, and that the sample loop is filled with sample solution. Because the internal surfaces of the sampling needle and sample loop are thoroughly rinsed with the sample itself, the effects of solute adsorption and solvent exchange with the needle wash and mobile phase are reduced.

NOTE: The default loop overfill factor in this mode specifies the excess volume of sample drawn when you select the full loop option. For example, for a 2 µL loop, 11.2 µL of sample is used per 2 µL full loop injection. For a 10 µL loop, 40 µL of sample is used per 10 µL full loop injection. Smaller loops are harder to fill and so require larger overfill factors.

Partial loop mode

This is best used for applications where the volume of sample is limited or when reduced cycle time is critical. In this mode the sample is bracketed with air gaps and the weak wash solvent. The weak wash solvent must match the mobile phase in order to ensure that the solutes do not begin to elute in the weak wash upon injection. This can lead to peak distortion and peak splitting in extreme cases.

NANOACQUITY INJECTION MODES

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Preparing the mobile phase

1. Prepare the following mobile phases:

Mobile Phase A1 Water with 0.1% formic acid

Water with 0.1% TFA*

Mobile Phase B1 Acetonitrile with 0.1% formic acid

Seal Wash Mobile Phase 90:10 water:acetonitrile

ASM Reference B1 GFP, 50:50 water:acetonitrile with 0.1% formic acid (200 fmol/μL or suitable concentration)

NOTE: * 0.1% TFA in water may be used as an alternative weak seal wash for the sample manager, instead of the 0.1% formic acid in water. This is application dependent and is particularly useful for proteomic based applications.

2. Place the nanoACQUITY solvent lines in the correct bottle:

Table 1 : Destination of nanoACQUITY solvent lines

Solvent Line Solvent/Mobile Phases

BSM A1 Sample Manager (weak wash) Mobile Phase A1

BSM B1 Sample Manager (strong wash) Mobile Phase B1

BSM seal wash ASM seal wash Seal Wash Mobile Phase

ASM B1 ASM Reference B1

NOTE: These mobile phases are the same as those used in the Xevo QTof MS nanoACQUITY UPLC System performance test during installation.

If no ASM is available, the fluidics available on the Xevo QTof MS may be used as an alternative to ‘ASM Reference B1’ to provide the reference solution.

WATERS® XEVO® QTOF MS NANOACQUITY UPLC® SYSTEM CUSTOMER FAMILIARIZATION GUIDE

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Preparing the samples

1. Prepare a test mixture, comprising Waters MassPREP digestion standard (MPDS) Mixture 1. This contains each of the following proteins in a 1:1 ratio. The accession number for each protein is also listed.

Bovine serum albumin (BSA) P02769

Alcohol dehydrogenase (ADH) P00330

Enolase P00924

Glycogen phosphorylase B (GPB) P00489

2. Add 1 mL of water with 0.1% formic acid to the MPDS Mixture 1 vial to provide a 1 pmol/μL stock solution.

3. Aliquot 100 μL of the MPDS Mixture 1 stock into a clean sample vial.

4. Add 900 μL water with 0.1% formic acid to provide the test solution 100 fmol/μL (equivalent to 25 fmol/μL per protein).

5. Prepare the following samples:

Calibration Sodium caesium iodide solution (1 mg/mL)

Lock Mass [Glu1]-fibrinopeptide B (200 fmol/μL in 50:50 water:acetonitrile + 0.1% formic acid)

NOTE: Theses samples are the same as those used in the Xevo QTof MS nanoACQUITY UPLC System performance test during installation. If required, the MPDS Mixture 1 (p/n 186002865) is available from Waters.


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Opening MassLynx™ software and creating a project

1. Double-click the MassLynx icon .

2. Click File > Project Wizard.

3. Click Yes to the prompt “When changing to a new project, some services are automatically closed down. Continue?”.

4. Input the Project name as TRAINING and the description as Customer Familiarization, then click Next.

Figure 2 - Create Project window

5. Select the option Create using exisiting project as template, and ensure that C:\MassLynx\Commission.PRO is the existing project, then click Finish.

Figure 3 - Create Project window

NOTE: The MassLynx project structure is described in the “Understanding Project Structure” section of the MassLynx software online help.


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Preparing the nanoACQUITY UPLC® system

The following section describes how to set up the nanoACQUITY UPLC system.

1. In the MassLynx software, click the Inlet Method icon to open the inlet method editor.

2. Click the ACQUITY Additional Status tab to display the status of the nanoACQUITY modules.

Top nanoTee nection to ping valve

contrap

Recommended workflow

Priming the syringes

Characterizing the needle seal

Characterizing the needle and loop volumes

Equilibrating the system

Setting up the inlet methods

Priming the solvent lines

Figure 4 - Inlet Method Editor


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3. Click the ACQUITY UPLC Console icon to display the nanoACQUITY UPLC Console.

Figure 5 - nanoACQUITY Additional Status window

4. Prime both the A1 and B1 lines of the binary solvent manager:

a. Click Control > Prime solvents in the binary solvent manager console window.

b. Select both A1 and B1 and specify a time of 5 minutes.

c. Click Start to begin the solvent line prime.

Figure 6 - Priming the solvent lines

NOTE: This process can be simplified by using the System Startup and Refresh System functions. Previous settings are remembered and can be automated to walk the user through the procedure.


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5. Prime the binary solvent manager seal wash lines:

a. Click Control > Prime seal wash in the binary solvent manager console window.

b. Click Yes to begin the seal wash prime.

Figure 7 - Priming the seal wash

6. Prime the B1 line for the auxillary solvent manager:

a. Click Control > Prime Solvents in the auxillary solvent manager console window.

b. Select B1 only and specify a time of 5 minutes.

c. Click Start to begin the solvent line prime.

7. Prime the sample manager syringes:

a. Click Control > Prime syringes.

b. Ensure Sample syringe and wash syringes is selected.

c. Specify a minimum of 10 cycles.

d. Click OK to begin the prime.

Figure 8 - Priming the syringes


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Characterizing the needle seal

1. Click Maintain > Characterize > Needle seal… in the Sample Manager Console window to perform the needle seal calibration procedure.

Figure 9 - Characterizing the needle seal

2. Ensure the procedure passes successfully.

Characterizing the needle and loop volumes

1. In the Sample Manager Console window, click Maintain > Characterize > Needle and loop Volumes… to perform the system volume calibration procedure.

2. Ensure the procedure passes successfully.

NOTE: Characterize the system volume whenever there is a change to the mobile phase, wash solvents, sample loop, needle, or any of the syringes.

This process can be simplified by using the System Startup and Refresh System functions. Previous settings are remembered and can be automated to walk the user through the procedure.

Connecting the inlet tubing and equilibrating the system

1. Ensure the nanoACQUITY UPLC symmetry C18 trapping column (180-μm x 20-mm, 5-µm) is connected.

2. If the symmetry trapping column is already attached, proceed to step 14 of this section.

3. Connect the trapping column assembly to port #6 of the sample manager’s inject valve.

4. Flush the trapping column with a 50:50 (A:B) mix at 10 µL/min for at least one hour to remove particulates.

5. Stop the flow and install the outlet tubing from the trapping column into the one side of the nanoTee.

6. Place pin plugs into the additional two ports of the nanoTee.


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7. In the Solvent Manager Console, click Troubleshoot > Set Pressure Diagnostics to display the Set Pressure Diagnostics dialog (Figure 10).

Figure 10 - Set Pressure Diagnostic dialog

Table 2 : Set Pressure Diagnostic tests

Test Component under

test

Measured leak rate criteria at 10,000 psi

(μL/min)

Location to plug

Position of

injection valve

Position of

trapping valve

A Flow control module 0.015 Outlet

mixing tee N/A N/A

B Flow control module

Injection valve 0.015 N/A Blocked N/A

C1

Flow control module Injection valve

Sample loop off-line Trap column with tee

0.018 Nano tee (two PEEK

plugs) Load N/A

C2


Sample loop in-line Trap column with tee

0.018 Nano tee (two PEEK

plugs) Inject N/A

D


Trap column with tee Trap (HTM) valve

0.020

Nano tee (one PEEK

plug: vent line installed)

Inject Analytical


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8. Perform test C1 (Table 2):

a. Set Inject Valve to Load.

b. Ensure the pressure is set to 0.

c. Start the flow.

d. Increase the pressure in steps of 2000 psi up to 10,000 psi, allowing the pressure to stabilize at each step before increasing to the next pressure.

e. When the system reaches the set pressure (10,000 psi), monitor the measured flow (leak criteria) of pump A and ensure it passes the test (0.018 µL/min or less).

f. Lower the set pressure in steps of 2000 psi back to 0 psi

g. Stop the flow.

9. Perform test C2 (Table 2):

a. Set Inject Valve to Inject.

b. Ensure the pressure is set to 0.

c. Start the flow.

d. Increase the pressure in steps of 2000 psi up to 10,000 psi, allowing the pressure to stabilize at each step before increasing to the next pressure.

e. When the system reaches the set pressure (10,000 psi), monitor the measured flow (leak criteria) of pump A and ensure it passes the test (0.018 µL/min or less).

f. Lower the set pressure in steps of 2000 psi back to 0 psi

g. Stop the flow.

10. Connect tubing to the nanoTee connection.

11. Attach the other end to port #1 of the trapping valve (as shown in Figure 11).

Top nanoTee connection to trapping valve

Trapping column Analytical

column

nanoTee

Figure 11 - nanoTee connections


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12. With one port of the nanoTee still plugged, perform the set pressure diagnostic Test D (Table 2) as described below:

a. Set Inject Valve to Inject.

b. Set Trapping Valve to Analytical.

c. Ensure the pressure is set to 0.

d. Start the flow.

e. Increase the pressure in steps of 2000 psi up to 10,000 psi, allowing the pressure to stabilize at each step before increasing to the next pressure.

f. When the system reaches the set pressure (10,000 psi), monitor the measured flow (leak criteria) of pump A and ensure it passes the test (0.020 µL/min or less).

g. Lower the set pressure in steps of 2000 psi back to 0 psi.

h. Stop the flow.

NOTE: If the results for Test D are significantly above the leak rate criteria, perform Test B. If Test B passes, perform Test C1. If Test C1 passes, the capillary tubing from the nanoTee to the trap valve might need replacing.

13. Remove the pin plug from the nanoTee.

14. Ensure the 75 µm x 100 mm C18 BEH analytical column is connected to the outlet of the nanoTee and placed in the column heater.

15. Prior to connecting the outlet of the analytical column to the MS, flush adequately with 15:85 (A:B) for at least one hour.

16. Connect a waste line (p/n 430001456) to port #2 of the trapping valve, and a pin-plug to port #6 (for ICOP 1.41).

17. Connect a waste line (p/n 430001456) to port #6 of the trapping valve (for pre-ICOP 1.41).

18. Place the nanoACQUITY UPLC outlet tubing into a suitable waste container.


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19. In the BSM window of the nanoACQUITY UPLC console, click Control > Set Flow....

20. Set the initial mobile phase flow conditions as shown in Figure 12.

Figure 12 - Setting the initial flow rate

21. Ensure the inlet editor status LEDs for Pump ON and OK and turn to green.

NOTE: If the LEDs do not turn to green, refer to the nanoACQUITY UPLC System Operator’s Guide (p/n 71500082502).

22. Equilibrate the column until the BSM system pressured delta value is ≤20 psi.

23. Place a vial containing mobile phase A1 diluent (blank) into position 1:1 in the sample manager.

24. Place test mixture into position 1:2 of the sample manager.


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Setting up the nanoACQUITY UPLC inlet methods

1. In the inlet method editor, click the Inlet icon , to open the binary solvent manager method editor.

2. Create an inlet method for the familiarization tests using the following parameters:

NOTE: For further details on the ACQUITY UPLC inlet methods, gradient curves and method optimization, refer to the ACQUITY UPLC system documentation and the ACQUITY UPLC online help.

a. Set the outlined parameters.

Figure 13 - Binary solvent manager settings for customer familiarization

b. Set the gradient values to those in the following table:

Table 3 : Gradient values

Time (mins) Flow (μL/min)

%A %B Curve

1 Initial 0.3 97.0 3.0 Initial

2 1.0 0.3 97.0 3.0 6

3 30.0 0.3 60.0 40.0 6

4 32.0 0.3 5.0 95.0 6

5 37.0 0.3 5.0 95.0 6

6 45.0 0.3 97.0 3.0 6


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3. Click the Browse icon to customize a mobile phase name.

4. Click the Trapping tab and set the parameters as shown in Figure 14.

Figure 14 - Binary solvent manager trapping conditions

5. Click to access the auxiliary solvent manager.

6. Enter a flow rate of 0.5 μL/min for pump B1 (Figure 15).

NOTE: If there is no ASM available, then ensure that the reference fluidics are flowing at 0.5 μL/min, and m/z 785.8 provides at least 150 counts per second.

Figure 15 - Auxiliary solvent manager Lock mass flow settings


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7. Click the Autosampler icon to open the sample manager method editor.

8. Create an autosampler method for the familiarization tests with the settings shown in Figure 16.

Figure 16 - Sample manager settings

NOTE: The sample manager advanced settings may be customized if required. For example, samples in more viscous matrices may require slower syringe draw rates.

9. If the nanoACQUITY UPLC system contains a TUV detector, set up any additional ACQUITY UPLC TUV method as appropriate:

a. In the inlet method editor, click the ACQUITY TUV icon to open the detector method editor.

b. Configure the detector method as required. See the “Appendix” for examples of TUV detector methods.

NOTE: Ensure that all fluidic connections are correct for a multi-detector system.

10. Click File > Save As in the inlet editor, and save the inlet method using the format CFG_ddmmyy (where ddmmyy = date).

11. Click the Load Method icon in the inlet editor to load the inlet method to the nanoACQUITY UPLC system.

12. Allow the column and sample temperatures to equilibrate.


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Configuring and tuning the mass spectrometer

The following section describes how to set up the Xevo QTof MS mass spectrometer.

Optimizing the source conditions

Checking the quality of the ion beam

LockSpray source setup

IntelliStart calibration

Creating the MS Method

Recommended

workflow

1. In MassLynx, click the MS Tune icon to open the MS Tune page.

NOTE: This may also be opened from the IntelliStart console screen.

2. Ensure the mass spectrometer is in Operate. If not, click to place the instrument into Operate.

3. Click File > Open and select an MS Tune file that has been configured to give the required instrument performance.

4. Click File > Save As... to save the MS Tune file with the file name CFG_DDMMYY (where DDMMYY = date).

5. Ensure Positive Ion is selected in the MS Tune page.

6. Set the following values as initial source parameters on the MS Tune page:

Capillary voltage 2.5 to 3.0 kV (tune as required)

Sample cone voltage 30 to 60 V (tune as required)

Source temperature 70 to 80 °C

Cone gas 10 L/Hr (tune as required)

Nanoflow gas 0 L/Hr (tune as required)

Purge gas 0 L/Hr (tune as required)

7. Optimize the source parameters as required in the following section.


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Checking the quality of the ion beam and optimizing the sprayer position

1. On the MS Tune page, click the Fluidics tab.

2. Select the LockSpray position for the baffle .

3. Start the Reference fluidics infusing 200 fmol/µL [Glu1]-fibrinopeptide B solution into the universal sprayer.

4. Alternatively, use the ASM of the nanoAcquity (0.5 μL/min of 200 fmol/μL [Glu1]-fibrinopeptide B solution) for introducing the Lock mass compound.

5. Adjust the stage position of the nanoflow sprayer to obtain the maximum number of counts for the m/z 785 [Glu1]-fibrinopeptide B peak (80:20 water:acetonitrile).

NOTE: This allows optimum position of the analyte sprayer to be achieved.

6. Assess the quality of the ion beam:

a. On the MS Tune page, with continuum mode data displayed, observe the peak resolution (FWHM) above the peak display.

b. Take resolution measurements at less than 200 counts/sec.

NOTE: Adjust the capillary voltage to achieve this if necessary.

c. Ensure the peak resolution is appropriate for the ion mode.

d. In continuum mode, with a 1 second scan, ensure that the intensity of the reference [Glu1]-fibrinopeptide B peak at m/z 785 is at least 150 counts/sec.

NOTE: Under these conditions, an intensity of at least 150 counts/sec on the m/z 785 ensures that there is sufficient Lock mass signal to complete the IntelliStart LockSpray source setup.

e. Click on the MS Tune page and input the acquisition parameters as shown in Figure 17.


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Figure 17 - MS tune page acquisition parameters

7. Click Start to begin the MS Tune page acquisition.

NOTE: Acquiring data from the MS Tune page is a convenient method of assessing the ion beam quality before making an acquisition using the sample list.

8. Click Chromatogram in the main MassLynx window to open the Chromatogram browser window.

9. Click File > Open… in the Chromatogram dialog box and browse to the MS_Tune_DDMMYY data file created earlier.


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Figure 18 - Opening the tuning acquisition data file dialog box

10. Ensure that the TOF MS ES+ TIC is displayed.

11. Confirm that the ion beam is stable and consistent.

12. Click Process > Combine… and specify scans 1:10.

13. Set the peak separation to 0.05 Da.

CAUTION: The peak separation value must be set to 0.05 Da for TOF MS systems. If the peak separation field is not set correctly, background ions of a similar mass may be incorporated during the spectrum combine, producing incorrect masses.

14. Click OK to open the Spectrum Browser window showing the combined spectrum (Figure 19).

Figure 19 - Example of [Glu1]-fibrinopeptide B spectrum


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Using IntelliStart for LockSpray source setup

IntelliStart can determine suitable source parameters for a specified Lock mass compound automatically to ensure sufficient Lock mass intensity is achieved during later acquisitions.

The recommended operating procedure is to run an initial LockSpray Source Setup to set and store source parameters in the Lock mass profile, and from then on, to use the “Lock mass Check” within IntelliStart to confirm suitable Lock mass performance is maintained.

1. From the main MassLynx window, open the MS console.

2. Click Configure > Configuration Mode to access the instrument configuration display (Figure 20).

Figure 20 - Instrument configuration display

3. Select the LockSpray Source Setup check box, then click Start.

4. Click Next on the initial wizard screen, and then select Lock mass Editor.

5. Complete the details for the required Lock mass.

NOTE: For the purpose of this document, the example Lock mass will be based on [Glu1]-fibrinopeptide B (GFP).


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6. Define the Lock mass parameters for [Glu1]-fibrinopeptide B (GFP) (m/z 785.8426), as shown in Figure 21.

Figure 21 - Creating the GFP Lock mass in the Lock mass Editor function

7. Click OK to display the Lock Mass Editor table (Figure 22).

Figure 22 - Lock mass Editor table


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8. Click the GFP Lock mass.

9. Ensure the [Glu1]-fibrinopeptide B Lock is selected as the sample.

10. Ensure that the sample is flowing through the LockSpray sprayer.

11. Click Next.

Figure 23 - LockSpray Source Setup dialog box

12. Select the Automatic check box, then click Next.

Figure 24 - LockSpray Source Setup Options dialog box


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13. Select Manual for the reference fluidics to be controlled from the MassLynx Tune page.

NOTE: The ‘Manual’ option is selected for the fluidics section, since the Lock mass is provided from the ASM of the nanoACQUITY.

If there is no ASM available, ensure that the reference fluidics are flowing at 0.5 μL/min.

The system is now ready to optimize the source conditions for the Lock mass profile specified.

14. Click Start.

Figure 25 - Fluidics control

The IntelliStart software automatically adjusts the source conditions. The values chosen are written automatically into the Lock mass profile, so whenever the profile is used in an acquisition, these parameters are set on the Lock mass sprayer.

NOTE: Because the values are saved into the Lock mass profile, they can not be overwritten without performing the “Using IntelliStart for LockSpray source setup” section again.


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When successfully completed, the MS Console displays the green check mark.

Figure 26 - Setup successful icon

NOTE: The completed Lock mass profile is available for selection in the “LockSpray” window when creating new MS methods.


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Mass calibration

To ensure consistent mass measurements, the software requires that the mass axis is calibrated so that the m/z values for sample peaks can be accurately determined. To achieve this, a solution of known references peaks is infused, and the software determines the differences between the expected mass of the reference peaks and the observed locations of those peaks along the mass axis. A calibration curve can then be produced so that any mass can be correctly determined.

The calibration process can be partially or completely automated using the IntelliStart options available; manual, assisted or automatic.

IntelliStart can also automatically check the quality of a calibration to ensure it is still producing acceptable results.

The recommended typical operating procedure is to run a calibration check, and only if that result is unacceptable, to create a new calibration.

Using IntelliStart to create a calibration

1. Ensure the baffle is set to the Sample position.

2. Connect the sample fluidics to the universal sprayer and set the flow.

3. Open the MS console, and click Configure > Configuration Mode to access the instrument configuration display (Figure 27).

Figure 27 - Configuration mode display

4. Select the Create Calibration check box, then click Start.


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5. Click Next on the initial wizard screen to access the Create Calibration display (Figure 28).

6. Select the IdentityE_ExpressionE calibration profile from the drop-down list.

NOTE: The red triangle indicates the calibration profile is defined, but has not yet been calibrated.

7. Click Next.

Figure 28 - Create calibration display

8. Select the Positive ion, Display Report and Make the Calibration Profile Active check boxes.


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9. Click Next to access the Data Acquisition Options display (Figure 29).

NOTE: The data acquisition options are automatically populated by IntelliStart.

10. Click Next to access the Fluidics display (Figure 30).

Figure 29 - Data acquisition options

11. Select the Manual check box for the reference fludics to be controlled from the MassLynx Tune page.

Figure 30 - Fluidics control


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12. Click Next to access the Acceptance Criteria display (Figure 31).

NOTE: Use the default acceptance criteria for automatic calibration as shown.

13. Click Next.

Figure 31 - Acceptance criteria

14. Ensure there is calibrant sodium caesium iodide in vial C, then click Start.

NOTE: When starting an automatic or assisted calibration, IntelliStart will automatically load the blank calibration Uncal.cal before acquiring any new data.


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The IntelliStart window displays the current status of the calibration process. The automatic steps are as follows:

Removing any previous calibration by loading Uncal.cal

Performing intensity check – if required, the instrument will use the DRE lens to achieve a suitable intensity on the most intense peak in the calibrant sample

Acquiring data – once sufficient data is obtained on the least intense peak as defined in the calibrant reference file, the peak picking application attempts to create the calibration.

Assisted Calibrations – launches the peak picking window and requires the user to manually accept the calibration.

The generated report displays the result of the calibration check (Figure 32). A successful check (with an RMS result within the acceptance criteria set above) displays a green tick in the header. Further details of the calibration check data are provided underneath.

Figure 32 - IntelliStart calibration report

The IntelliStart display indicates the calibration has completed successfully.

Figure 33 - Successful calibration result


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Using IntelliStart for the calibration check

Use the IntelliStart automatic calibration check to demonstrate the quality of an automatic calibration. This compares the mass accuracy of the acquired data to a known reference file.

1. Open the MS console, and click Configure > Normal Mode.

2. Select the Check Calibration check box, then click Start.

3. Click Next on the initial screen to access the Check Calibration display (Figure 34).

4. Select the IdentityE_ExpressionE calibration profile from the drop-down list.

NOTE: Only those calibration profiles that have already been calibrated (shown by a green tick) can be checked through IntelliStart. Manual calibrations (.cal files made in Custom mode), or defined profiles not yet calibrated (shown by a red triangle), cannot be checked using IntelliStart.

5. Click Next.

Figure 34 - Check Calibration display


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6. Select the Positive ion and Display Report check boxes (Figure 35).

7. Click Next.

Figure 35 - Calibration Options display

8. Select the Acquire Data with LockMass and Automatic check boxes.

NOTE: Where the Lock mass is by the ASM, select Manual.

9. Select GFP from the drop-down list.

10. Click Next.

Figure 36 - LockMass Options display


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11. Set the Calibration Check Acceptance Criteria to 3.0 ppm RMS residual mass error and click Next.

Figure 37 - Acceptance Criteria display

NOTE: IntelliStart is now ready to check the calibration.

12. Ensure the samples required are in the appropriate fluidics vials as described on screen.

13. Click Start.


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The IntelliStart window displays the current status of the calibration check process. The automatic steps are as follows:

Performing intensity check – if required, the instrument uses the DRE lens to achieve a suitable intensity (IPP) on the sample.

Acquiring sample and Lock mass data – once sufficient data is obtained, Lock mass corrected data is presented in the IntelliStart report.

The generated report displays the result of the calibration check (Figure 38). A successful check (with an RMS result within the acceptance criteria set above) displays a green tick in the header. Further details of the calibration check data are provided underneath.

Figure 38 - IntelliStart calibration report

The IntelliStart display indicates the calibration has completed successfully.

Figure 39 - Successful calibration result


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Setting up the mass spectrometer MSE methods

1. In the main MassLynx window, click the MS Method icon to open the MS method editor.

NOTE: The MS method settings are the acquisition parameters that will be used during the acquisition of data from the sample list.

2. Click MSE Continuum, then File > New to create an MSE based method.

Figure 40 - Selection of MSE method in MS method editor

3. Click MSE Continuum to open the MSE method parameters.

4. For each tab, complete the following information:

Table 4 : Entry data for MSE method

Tab Values

Acquisition

Start time = 0 End time = 45 Source = ES

Positive polarity Analyzer in V Mode

TOF MS Start = 50 Da End = 1990 Da

Scan time = 0.6 sec

Collision Energy Low energy = 6 V Ramp high energy from 15 to 35 V

Cone Voltage * Override clear

NOTE: * Cone voltage used is that defined in Tune page.

Alternatively, the Lock mass parameters can be derived from the in-source fragmentation test.


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5. Click LockSpray in the MS method editor.

6. Create a LockSpray method to enable LockSpray scans to be acquired over the course of the MSE acquisitions:

7. Select the Acquire LockSpray - Do not apply correction check box.

8. Select GFP *from the LockSpray Reference Compound drop-down list.

9. Set the required parameters:

Interval 60 seconds

Scans to Average 3

Mass Window ±0.5 Da

NOTE: * Previously prepared [Glu1]-fibrinopeptide B reference solution.

10. Click More to view additional source parameters.

Figure 41 - LockSpray acquisition settings


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NOTE: The values provided are based on those derived from the Setup wizard.

Figure 42 - Additional source settings

11. Click File > Save As… to save the MSE method using an appropriate filename; for example, CFG_ddmmyy.exp (where ddmmyy = date).


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Acquisition and processing samples

The following section describes the acquisition and processing of samples.

Creating the sample list

Acquiring the sample data

Viewing the sample results

Recommended workflow

Creating the sample list

1. Click File > New... on the MassLynx sample list, to create a new blank sample list.

2. Double-click in the File Name column of row 1, and type the filename as Blank 1.

3. Double-click in the File Text column of the same sample, and set the file text as Blank Injection.

4. Right-click in the MS File column, click Browse... and select the CFG_DDMMYY.exp system precision MS method created earlier.

5. Right-click in the Inlet File column, click Browse... and select the CFG_DDMMYY inlet method created earlier.

6. Set the vial position as 1:1 in the Bottle column.

7. Set 2 µL in the Inject Volume column.

8. Right-click anywhere on row 1 of the sample list, select Add, specify a value of 6, then click OK.

9. Edit the list to resemble the sample list below (Figure 43).

10. Save the sample list as CFG_DDMMYY.spl, where DDMMYY = date.

Figure 43 - Example sample list

NOTE: If a new analytical column is being used for the following acquisitions, then optimal chromatography will be achieved for the latter runs of the sample list.


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Acquiring the sample data

1. Connect the nanoACQUITY UPLC column outlet tubing to the Xevo QTof MS analyte nanosprayer, and allow the baseline to stabilize.

2. Ensure that the Lock mass fluidics have enough sample for the entire sample list acquisition.

3. Start the Lock mass infusion of GFP to the reference probe.

NOTE: If Lock mass is being provided by the ASM, ensure a flow of 0.5 μL/min.

4. If the fluidics are being used, perform the following procedure to set the Method Events to ensure that automatic refill of the vial takes place:

a. In the main MassLynx window, click the MS Method icon to open the MS method editor.

b. Click Method Events.

c. Select Initial Condition from the Time/Mins drop-down list, then click Add.

d. Select Refill from the Event drop-down list, then click Change.

e. Select Refill if Required from the Action drop-down list, then click Change.

f. Select LockSpray from the System menu, then click Change.

g. Select the Enable check box.

h. Click OK to confirm and exit the Method Events dialog box.

Figure 44 - Refilling Lock mass on the Method Events dialog box


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5. Alternatively, when using an auxiliary solvent manager, perform the following steps:

a. Access the auxillary solvent manager for provision of the [Glu1]-fibrinopeptide B Lock mass.

b. Enter a suitable flow rate (typically 0.3 to 0.5 μL/min).

c. Enter this into the appropriate nanoUPLC methods.

6. Highlight all seven samples within the sample list.

7. Click Run > Start.

8. Select the Acquire Sample Data check box; ensure other boxes are clear.

9. Click OK to perform the analysis.

10. To view the chromatogram data during the acquisition:

a. Open the Chromatogram Data Browser window.

b. Click the icon to enable the real-time update.

c. Click the icon to maximize the view if the whole of the TOF MS ES+ TIC is not visible.

11. Allow the sample list acquisition to complete.

Viewing the sample results

1. In the Chromatogram Data Browser window, click File > Open.

2. Navigate to, and select, the Test Mixture 1 data file (Figure 45).

3. Click OK.

Figure 45 - Browsing for the Test Mixture 1 data file


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4. Ensure that the Chromatogram View window updates to include the Test Mixture 1 TOF MS ES+ BPI (Figure 46).

Figure 46 - Example chromatogram for Test Mixture 1

NOTE: To view the displayed chromatogram in BPI mode, click Display > TIC and select the BPI Chromatogram check box for Function 1 (Figure 47).

Figure 47 - Displaying the chromatogram in BPI format


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Extracting specific masses by m/z value

Specific masses can be extracted from the chromatogram:

1. Click Display > Mass.

2. Enter the mass of interest in the Description (m/z) field.

NOTE: When entering more than one mass, separate each mass with a comma.

Figure 48 - Extracting specific masses from the chromatogram

Creating a ChroTool method

The MassLynx ChroTool may also be used to extract specific masses from the sample:

1. In the Chromatogram window, click the ChroTool icon to open the ChroTool window (Figure 49).

NOTE: The ChroTool window may also be accessed by clicking on Display > ChroTool. If the button is not visible, add it to the toolbar using Display > Customise toolbar.

2. Click File > New to set up a new method file.

Figure 49 - ChroTool window


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3. Click the Add Group button.


Name group name

Comment any supportive text

Range Use current check box selected

5. Click OK.

Figure 50 - Add Group dialog box


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6. Click the Add Chromatogram icon to access the Chromatogram Details dialog box (Figure 51).


a. Enter the name of the chromatogram to be displayed.

b. Select the type of chromatogram from the drop-down list (for example, Mass/Channel for an extracted mass chromatogram).

c. Select the TIC function number from the drop-down list (for example, 1 for analyte data on a LockSpray system).

d. For a mass chromatogram, enter the m/z value into the description field.

e. Click OK.

Figure 51 - Chromatogram Details dialog box

8. Repeat step 7 for each mass to be extracted.

NOTE: As an example, peptides with their respective masses (from the MPDS Mix 1 sample) are listed in Table 5. A completed ChroTool window for them is shown in Figure 52.


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Table 5 : Example peptides extracted from the raw data

Peak Name m/z

Glycogen phosphorylase B (GPB) 639.77

Enolase 644.86

Bovine serum albumin (BSA) 740.40

Alcohol dehyrogenase (ADH) 507.30

Figure 52 - ChroTool window with entries

9. Click File > Save As and save the method file under an appropriate filename.


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View chromatograms created by ChroTool

1. Access a chromatogram window.

2. Click the ChroTool icon , to access the ChroTool window.

3. Click the icon next to the group name to extract the listed chromatogram.

Figure 53 - Chromatogram for an individual compound

4. Click the icon next to other entries to view their ChroTool chromatograms.

Figure 54 - Chromatograms for multiple compunds

5. Click and drag across a peak of interest to magnify it.

Figure 55 - Example of a magnified peak


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6. To provide a spectrum, right-click and drag across the width of the chromatographic peak to combine the peak spectra.

Figure 56 - Example of magnified peak spectra

7. To display the peak mass to four decimal places, click Display > Peak Annotation in the Spectrum Browser window and set the value as appropriate.

NOTE: Customers with access to ProteinLynx Global Server™ (PLGS) will now be able to process their data to allow for protein identifications.


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Appendix

This section contains an example inlet method for the addition of an ACQUITY TUV detector.

ACQUITY TUV detector

Figure 57 - Example ACQUITY TUV detector method parameters

APPENDIX

Documents

Waters Xevo QTof MS/ nanoACQUITY UPLC System€¦ · Xevo QTof MS Operator’s Overview and Maintenance Guide (p/n 71500174502) Waters Educational Services . This guide assumes that