NanoLC Ultra System for TripleTOF 5600, QTRAP 5500 and 4000 QTRAP

NanoLC Ultra System for TripleTOF 5600, QTRAP 5500 and 4000 QTRAP
NanoLC Ultra® System
for TripleTOF™ 5600, QTRAP® 5500 and 4000 QTRAP® Systems
System Integration Test
Document Number: D5030175 A
Release Date: March 2012
This document is provided to customers who have purchased
AB Sciex equipment to use in the operation of such AB Sciex
equipment. This document is copyright protected and any reproduction
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manufacturers' products as supplied by AB Sciex for incorporation into
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AB Sciex makes no warranties or representations as to the fitness of
this equipment for any particular purpose and assumes no
responsibility or contingent liability, including indirect or consequential
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described herein, or for any adverse circumstances arising therefrom.
For research use only. Not for use in diagnostic procedures.
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© 2012 AB SCIEX.
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Contents
Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
General Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Symbols and Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Safety Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Qualified Personnel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Equipment Use and Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Mains Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Instrument Disposal (Waste Electrical and Electronic Equipment) . . . . . . . . .10
Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Additional Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Chapter 1 LC/MS System Configuration Test—
AB SCIEX TripleTOF™ 5600 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Create the Methods and Batch for the System Functional Test . . . . . . . . . . . . .14
Create the Acquisition Methods and Batch—Functional Test . . . . . . . . . . . . .14
Create the LC Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Create the Acquisition Method in the Analyst® TF Software . . . . . . . . . . . . . .21
Create the Methods and Batch for the System Performance Test . . . . . . . . . . .25
Create the Acquisition Methods and Batch—Performance Tests . . . . . . . . . .25
Create the LC Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Specify the Acquisition Method in the Analyst TF Software . . . . . . . . . . . . . .31
Prepare the System for Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Prepare the Solution and Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Condition the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Verify System Readiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36
Perform the System Functional Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
Perform the System Performance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Troubleshoot Peak Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Create a Backup of the EKSettings.reg File . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
Test Results—TripleTOF 5600 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Chapter 2 LC/MS System Configuration Test—
AB SCIEX QTRAP® 5500 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Create the Methods and Batch for the System Functional Test . . . . . . . . . . . . .52
Create the Acquisition Methods and Batch—Functional Test . . . . . . . . . . . . .52
Create the LC Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
Create the Acquisition Method in the Analyst® Software . . . . . . . . . . . . . . . .59
Create the Methods and Batch for the System Performance Test . . . . . . . . . . .63
Create the Acquisition Methods and Batch—Performance Tests . . . . . . . . . .63
Create the LC Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
Specify the Acquisition Method in the Analyst Software . . . . . . . . . . . . . . . . .69
Prepare the System for Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Prepare the Solution and Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
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Contents
Condition the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
Verify System Readiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
Perform the System Functional Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Perform the System Performance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
Troubleshoot Peak Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
Create a Backup of the EKSettings.reg File . . . . . . . . . . . . . . . . . . . . . . . . . . . .82
Test Results—QTRAP 5500 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
Chapter 3 LC/MS System Configuration Test—
AB SCIEX 4000 QTRAP® System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Create the Methods and Batch for the System Functional Test . . . . . . . . . . . . .86
Create the Acquisition Method and Batch—Functional Test . . . . . . . . . . . . . .86
Create the LC Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88
Create the Acquisition Method in the Analyst® Software . . . . . . . . . . . . . . . .93
Create the Methods and Batch for the System Performance Test . . . . . . . . . . .97
Create the Acquisition Methods and Batch—Performance Tests . . . . . . . . . .97
Create the LC Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98
Create the Acquisition Method in the Analyst Software . . . . . . . . . . . . . . . . .103
Prepare the System for Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
Prepare the Solution and Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
Condition the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108
Verify System Readiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108
Perform the System Functional Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110
Perform the System Performance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112
Troubleshoot Peak Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114
Create a Backup of the EKSettings.reg File . . . . . . . . . . . . . . . . . . . . . . . . . . .116
Test Results—4000 QTRAP Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117
Appendix A System Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119
Prepare the [Glu1]-Fibrinopeptide B Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . .119
Edit the Calibration Reference Table for [Glu1]-Fibrinopeptide B . . . . . . . . . . .120
Calibrate the TOF MS Scan Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
Calibrate the TOF MS/MS for High Sensitivity and High Resolution
Product Ion Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122
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Foreword
This foreword contains general safety-related information, describes the symbols and
conventions used in the documentation, and provides regulatory compliance information. It also
describes potential hazards and associated warnings for the system, and the precautions that
should be taken to minimize the hazards. In addition to this foreword, refer to the Site Planning
Guide for site requirements.
General Safety Information
Before operating any instrument, become familiar with its operation and with the potential
hazards. To prevent personal injury or instrument damage, read, understand, and obey all safety
precautions. Warnings in this document and labels on the device are shown with international
symbols. Failure to heed these warnings could result in serious injury.
This safety information is intended to supplement federal, state or provincial, and local
environmental health and safety (EHS) regulations. The information provided covers instrumentrelated safety with regard to the operation of the device. It does not cover every safety procedure
that should be practised. Ultimately, you and your organization are responsible for compliance
with federal, state or provincial, and local EHS regulations and for maintaining a safe laboratory
environment.
For more information, refer to the appropriate laboratory reference material and standard
operating procedures.
Symbols and Conventions
The following conventions may be used throughout the guide.
DANGER! Danger signifies an action which leads to severe injury or death.
WARNING! Personal Injury Hazard: A warning indicates an operation that could
cause personal injury if precautions are not followed.
WARNING! Electric Shock Hazard: This symbol indicates a warning of electrical
shock hazard. Read the warning and follow all precautions before performing any
operation described in the guide. Failure to do so can result in serious injury.
WARNING! Burn Hazard: This symbol indicates a warning of potential burns
from hot surfaces. Read the warning and follow all precautions before performing
any operation described in the guide. Failure to do so can result in serious injury.
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Foreword
WARNING! Biohazard: This symbol indicates a warning of biohazardous
materials. Read the warning and follow all precautions before performing any
operation described in the guide. Failure to do so can result in serious injury.
Caution: A caution indicates an operation that could cause damage to the instrument or
loss of data if precautions are not followed.
Tip! Provides useful information that helps apply the techniques and procedures in
the text for a specific need, and provides shortcuts, but is not essential to the completion
of a procedure.
i
Note: A note emphasizes significant information in a procedure or description.
Safety Instructions
The following safety instructions apply to the NanoLC Ultra system:
WARNING! Potential Operator Injury: Use of this equipment in a manner not
approved by the manufacturer may inhibit its safety protection.
Caution: Changes or modifications to this unit not expressly approved by the
manufacturer could void the instrument warranty and render the system inoperable.
WARNING! Electrical Shock Hazard: Only use fuses of the type and current
rating specified. Do not use repaired fuses or by-pass the fuse holder.
WARNING! Electrical Shock Hazard: The supplied power cord must be used with
a power outlet containing a protective ground contact.
WARNING! Biohazard: When replacing tubing or fittings on the ekspert microLC
200 system, exposure to solvents may occur. It is therefore recommended that
appropriate safety procedures be followed and personal protective equipment be
used, according to the applicable Material Safety Data Sheets supplied by the
solvent vendor.
WARNING! Electrical Shock Hazard: Do not change the external or internal
grounding connections. Tampering with or disabling these connections could
create a safety hazard and/or damage the system. The instrument, as shipped, is
properly grounded in accordance with normal safety regulations.
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Foreword
WARNING! Potential System Damage: Do not turn the system on if you suspect
that it has incurred any kind of electrical damage. Instead, disconnect the power
cord and evaluate the system.
WARNING! Potential System Damage: Electrical damage may have occurred if
any part of the system shows visible signs of damage, exposure to liquids or of
having been transported under severe stress.
WARNING! Electrical Shock Hazard: Continue to exercise caution as capacitors
inside the system may still be charged even after the system has been turned off.
WARNING! Electrical Shock Hazard: Disconnect power cords from the power
supply before attempting any type of maintenance.
WARNING! Electrical Shock Hazard: The combination of the pump and
autosampler with a LC/MS system may require additional safety measures as
described by AB SCIEX. See the mass spectrometer Safety Guide for instructions
for the safe grounding on the LC/MS system.
WARNING! Electrical Shock Hazard: Use a grounding cable connected between
the injection valve's sample loop and an appropriate grounding point at the
LC/MS source. This supplementary grounding will reinforce the safety
configuration specified by AB SCIEX.
WARNING! Potential System Damage: Damage can result if the system is stored
for prolonged periods under extreme conditions (for example, subjected to heat,
water, etc.).
WARNING! Environmental Hazard: Do not allow flammable and/or toxic solvents
to accumulate. Follow a regulated, approved waste disposal program. Never
dispose of flammable and/or toxic solvents into a municipal sewage system.
WARNING! Potential System Damage: To avoid damaging electrical parts, do not
disconnect an electrical assembly while power is applied to the system. Once the
power is turned off, wait approximately 30 seconds before disconnecting an
assembly.
WARNING! Potential System Damage: The system contains a number of
sensitive electronic components that may be damaged if exposed to excessive
line voltage fluctuations and/or power surges.
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Foreword
WARNING! Potential Operator Injury: To avoid injury during operation, keep
hands and loose objects away from the autosampler arm and syringe assembly.
WARNING! Potential Operator Injury: Use caution when working with any
polymeric tubing under pressure:
– Always wear proper eye protection when near pressurized polymer tubing.
– Do not use polymer tubing that has been severely stressed or kinked.
– Do not use polymer tubing, in particular PEEK or DuPont Tefzel tubing, with
tetrahydrofuran (THF), dimethylsulfoxide (DMSO), chlorinated organic solvents,
concentrated mineral acids, such as nitric, phosphoric or sulfuric acids, or any
related compounds.
WARNING! Puncture Hazard: Do not operate the autosampler without the safety
shield properly installed.
Caution: Potential System Damage: An on-board lithium battery maintains the
autosampler firmware when the instrument is turned off. It should only be replaced by a
factory-authorized service engineer.
Caution: Potential Data Corruption: When you use the HTC-xt PAL autosampler for
chromatographic analyses and observe a change in the retention of a particular
compound, the resolution between two compounds or peak shapes, immediately
determine the reason for the changes. Do not rely on the analytical results until the cause
of the change is determined.
Qualified Personnel
After installing the system, the FSE (Field Service Employee) uses the Customer Familiarization
Checklist to train the customer on system operation, cleaning, and basic maintenance. Only
AB SCIEX trained personnel shall operate and maintain the equipment. Equipment installation
and service shall only be conducted by AB SCIEX Field Service Employees. Contact an AB
SCIEX FSE for more information.
Equipment Use and Modification
Use the system indoors in a laboratory that complies with the environmental conditions
recommended in the system Site Planning Guide. If the system is used in an environment or in a
manner not prescribed by AB SCIEX, the protection provided by the equipment can be impaired.
Unauthorized modification or operation of the system may cause personal injury and equipment
damage, and may void the warranty. Contact an AB SCIEX representative for more information
on servicing the system.
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Foreword
Mains Supply
WARNING! Electrical Shock Hazard: Use only qualified personnel for the
installation of all electrical supplies and fixtures, and make sure that all
installations adhere to local regulations.
For information on system electrical specifications, refer to the Site Planning Guide.
Protective Conductor
The mains supply should include a correctly installed protective earth conductor that must be
installed or checked by a qualified electrician before connecting the instrument.
Do not intentionally interrupt the protective conductor. Any interruption of the protective
conductor is likely to make the installation dangerous.
Environmental Conditions
Use qualified personnel for the installation of electrical mains, heating, ventilation, and plumbing
supplies and fixtures. Make sure that all installations follow local bylaws and biohazard
regulations. For more information about the required environmental conditions for the system,
refer to the Site Planning Guide for the instrument.
WARNING! Explosion Hazard: The instrument is not designed for operation in an
explosive environment. Do not operate the instrument in an environment
containing explosive gases.
WARNING! Asphyxiation Hazard: The use of instruments without adequate
ventilation to outside air may constitute a health hazard. In addition, certain
procedures required during the operation of the instrument may cause gases to
be discharged into the exhaust stream; under these conditions, inadequate
ventilation may result in serious injury. Take extreme care to vent exhaust gases
properly.
WARNING! Toxic Chemical Hazard: Make sure that the source exhaust system is
properly connected, particularly if samples containing toxic or highly volatile
chemicals or solvents are being analyzed. A minimum 20% positive air flow into
the laboratory is required.
WARNING! Biohazard: This instrument or any part is not intended to act as a
biological containment safety cabinet. For biohazardous material use, always
apply local regulations for hazard assessment, control, and handling.
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Foreword
Instrument Disposal (Waste Electrical and Electronic
Equipment)
Do not dispose of system components or subassemblies, including computer parts, as unsorted
municipal waste. Follow local municipal waste ordinances for proper disposal provisions to
reduce the environmental impact of WEEE (waste, electrical, and electronic equipment). To
make sure that you safely dispose of this equipment, contact an FSE for instructions.
European Union customers: Contact a local AB SCIEX Customer Service office for
complimentary equipment pick-up and recycling.
Regulatory Compliance
This system complies with the standards and regulations listed in this section. Applicable labels
have been affixed to the system.
Canada
•
Safety—CSA 61010-1
Europe
•
Low Voltage Directive 2006 / 95 / EC
•
Electromagnetic Compatibility—61326-1 EN 55011 Class A, EMC Directive 2004 /
108 / EC
•
Safety—EN 61010-1
For more information on EU compliance, see the Declaration of Conformance included with the
system.
International
•
Electromagnetic Compatibility—CISPR 11 Class A, IEC 61326-1
•
Safety—IEC 61010-1
United States
•
Safety—UL 61010-1
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Foreword
Additional Documentation
•
NanoLC Ultra® System Operator’s Manual—Printed and electronic copies are
included with the system
•
Eksigent® Control Software User Guide—installed with the Eksigent control software
•
Analyst® Software Getting Started Guide—installed with the Analyst software
Technical Support
AB SCIEX and its representatives maintain a staff of fully-trained service and technical
specialists located throughout the world. They can answer questions about the instrument or any
technical issues that may arise. For more information, visit the web site at www.absciex.com.
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1
LC/MS System Configuration Test—
AB SCIEX TripleTOF™ 5600 System
This chapter describes the steps for preparing and performing LC/MS system configuration tests
for the NanoLC Ultra® system configured with the cHiPLC® Nanoflex system (or external
ChromXP column) and the AB SCIEX TripleTOF™ 5600 instrument.
Note: The tests in this chapter are written for Gradient 2 as the low-flow channel. If this
is not true for your system (for example, if you have a 1D or 1D+ system), then make
appropriate changes throughout the tests.
The tests in this chapter are divided as follows:
•
Fast test to condition the column and determine the functional status of the system.
See Create the Methods and Batch for the System Functional Test for details.
Perform these tests after completing the NanoSpray® ion source infusion tests in
order to first confirm the spray performance of the tip. Refer to the NanoSpray® Ion
Source Operator Guide for more information.
•
Longer test to determine the performance level of the instrument for proteomics
applications such as protein identification and quantification. See Create the
Methods and Batch for the System Performance Test for details.
These tests can be used as a measure of the NanoLC Ultra system performance in
isolation of performance of the other components. Results from these tests can
become the baseline performance for the system and can be performed regularly
and used as a system quality control test in the future.
Approximate time required:
1. Create the methods and batch: 45 minutes
2. Prepare the system for testing: 3-4 hours
3. Perform the test.
i. System functional test: 90 minutes
ii. System performance test: 180 minutes
Recommended solvents can be ordered from VWR:
•
Burdick and Jackson acetonitrile with 0.1% formic acid, P/N BJLC441-1.0
•
Burdick and Jackson water with 0.1% formic acid, P/N BJLC452-1.0
Required materials for a Nanoflex system installation:
•
Reverse phase cHiPLC column (75 µm x 15 cm ChromXP C18-CL 3 µm 120 Å)
•
cHiPLC trap (200 µm x 0.5 mm ChromXP C18-CL 3 µm 120 Å)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: Make sure that the Nanoflex system is in the Load position before beginning
these tests.
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Required materials for an external column installation:
•
Reversed phase ChromXP nanoLC column (75 µm ID x 15 cm, ChromXP C18 3 µm
120 Å, P/N 805-00120)
•
ChromXP nanoLC Trap column (350 µm ID x 0.5 mm, ChromXP C18 3 µm 120 Å,
P/N 5016752)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: After successfully completing the tests, create a backup of the EKSettings.reg
file. See Create a Backup of the EKSettings.reg File for more information.
Create the Methods and Batch for the System
Functional Test
This section describes a test for the NanoLC Ultra system to condition the column and determine
the functional status.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications. If the NanoLC system has been idle for two weeks or more, then
calibrate the system. Refer to the appendix, System Calibration, for more information.
Note: The steps in this section do not constitute a NanoLC Ultra system performance
test. See Create the Methods and Batch for the System Performance Test.
The expected test duration is 30 minutes using the NanoSpray® ion source. Repeat the test until
you have consistent peak shape and intensity (approximately 90 minutes).
Create the Acquisition Methods and Batch—Functional Test
Note: Use Gradient 2 for the autosampler method. Gradient 2 is the nanoflow module
for LC configuration.
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler status window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 1-1.
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Figure 1-1
Autosampler Settings dialog—trap-elute configuration (Nanoflex system)
Figure 1-2
Autosampler Settings dialog—trap-elute configuration (column)
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LC/MS System Configuration Test— AB SCIEX TripleTOF™ 5600 System
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj Nanoflex
trap elute or AS2 10uLloop 1uLinj Ch2Valve Trap, depending on the installation.
5. Click Save.
Create the LC Methods
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffer mixtures are shown in Table 1-1.
Table 1-1 Typical Buffer Mixtures
.
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent® Control Software
1. Make sure that Loading Pump is selected as the channel (top, right corner of
window).
2. Click LC Methods.
3. In the Name field, type Load Pump 2 min Trap Wash, and then click Save.
4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 1-3.
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Figure 1-3
LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 1-4.
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Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
Figure 1-4
LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Gradient Method in the Eksigent Control Software
For the analytical gradient (typically, the Gradient 2 pump with the nanoflow module), create the
gradient method.
1. Make sure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If the installation includes a Nanoflex system, then in the Name field, type CH2
15min 400nLmin nanoflex trap, and click Save.
Or
If the installation includes an external column, then in the Name field, type CH2
15min 400nLmin trap, and click Save.
4. On the Gradient Table tab, specify the method as shown in Figure 1-5 or Figure 1-7,
depending on the installation.
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Figure 1-5
LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown verify the correct switching of the Nanoflex valve.
The signals at Time 0 will move the Nanoflex valve to the Inject position and
the signals at Time 15 will move the Nanoflex valve back to the Load
position.
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Figure 1-6
LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 1-7.
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Figure 1-7
LC Method Settings dialog—Run Conditions tab (Nanoflex
system)
6. Click Save.
Create the Acquisition Method in the Analyst® TF Software
1. In the Acquire section of the Navigation toolbar, click the Method Wizard button to
open the wizard.
2. On the Choose Methods tab of the Method Wizard window, specify the MS
method.
•
In the Choose MS Method list, select ToF MS + Hi Sensitivity Product Ion
(+) method.
3. If an autosampler and LC method has already been associated with a previous
acquisition method, in the Choose LC Method list, select an existing .dam method.
4. If an LC method has not been used before, then select No LC Method.
Tip! A method can be defined later in this test. See Add LC Information to the
Acquisition Method.
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Figure 1-8
Method Wizard window—Choose Methods tab
5. In the Save Method As field, type System Functional Test and press Enter.
6. Click Next.
7. On Ion Source Parameters tab, specify the source conditions that were determined
during the NanoSpray® ion source infusion test. Refer to the NanoSpray® Ion
Source Operator Guide for more information.
Figure 1-9
Method Wizard window—Ion Source Parameters tab
8. Click Next.
9. On the TOF MS - MS/MS tab, specify the acquisition parameters for the
TripleTOF 5600 instrument.
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i.
Enter the scan information for the TOF MS experiment: In the Start TOF Mass
(Da) field and the Stop TOF Mass (Da) field, specify the mass range to 400
and 1800, respectively. The TOF Accumulation Time is 0.250 seconds.
ii. Enter the scan information for the TOF MS/MS experiment: In the MS2 Start
Mass (Da) and MS2 Stop Mass (Da) fields, specify the mass range to 100
and 1800, respectively. The MS2 Accumulation Time is 0.5 seconds.
iii. The total Cycle Time is automatically calculated from the times specified.
iv. In the Mass Spec Acquisition Duration field, specify the Duration Time for
the entire LC/MS run.
This value depends on the length of time of the analytical gradient. In this
example, it is 14 minutes.
Figure 1-10 Method Wizard window—TOF MS - MS/MS tab
10. In the table at the bottom of the tab, define the precursor ion for the product ion scan.
Note: As this method is a looped MS and MS/MS method, the precursor
ion must be defined.
•
For the Beta-Galactosidase digest, a good peptide to fragment for testing is
the 729.3652 ion.
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•
In the table, specify the m/z of this peptide along with the collision energy (45)
and a CES of 3 V.
•
The Declustering Potential is 70.
11. Click Finish to save the method.
Add LC Information to the Acquisition Method
If the autosampler and pump method information was not specified above, add it now.
Note: The Q1 Isolation window is automatically set to Unit. This is the correct setting for
this test.
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
2. Click Eksigent AS2 and then select the autosampler method, AS2 10uLloop 1uLinj
nanoflex trap elute.ini or AS2 10uLloop 1uLinj trap elute.ini, depending on the
installation.
Figure 1-11 Software Application Properties tab—autosampler filename
3. Click Eksigent Gradient 1, and then select the gradient 2 pump method, CH2 15min
400nLmin nanoflex trap.ini or CH2 15min 400nLmin trap.ini, depending on the
installation.
4. Click Eksigent Loading Pump, and then select the loading pump method, Load
Pump 2 min Trap Wash.ini.
5. Save the method as “System Functional Test”.
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Note: This method can also function as the LC auto calibration method
when using Beta-Galactosidase as a calibrant.
Create the Methods and Batch for the System
Performance Test
This section provides tests for the NanoLC Ultra system that indicate of the performance level of
the instrument for proteomics applications such as protein identification and quantification.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications. If the NanoLC system has been idle for two weeks or more, then
calibrate the system. Refer to the appendix, System Calibration, for more information.
The expected test duration is 60 minutes using the NanoSpray ion source. Repeat the test until
you have consistent peak shape and intensity (approximately 180 minutes).
Create the Acquisition Methods and Batch—
Performance Tests
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 1-12.
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Figure 1-12 Autosampler Settings dialog—trap-elute configuration (Nanoflex system or
column installation)
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj trap elute
or AS2 10uLloop 1uLinj Ch2Valve Trap, depending on the installation.
5. Click Save.
Create the LC Method
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffer mixtures are shown in Table 1-2.
Table 1-2 Typical Buffer Mixtures
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent Control Software
1. Make sure that Loading Pump is selected as the channel (top, right corner of
window).
2. Click LC Methods.
3. In the Name field, type Load Pump 10min Trap Wash, and then click Save.
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4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 1-13.
Figure 1-13 LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 1-14.
Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
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Figure 1-14 LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Gradient Method in the Eksigent Control Software
For the analytical gradient (typically on the Gradient 2 pump with the nanoflow module), create
the gradient method.
1. Make sure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If this is a Nanoflex system installation, then in the Name field, type CH2 45min
300nL/min Nanoflex trap, and click Save.
Or
If this is a column installation, then in the Name field, type CH2 45min 300nLmin
column trap, and click Save
4. On the Gradient Table tab, specify the method, as shown in Figure 1-15.
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Figure 1-15 LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown verify the correct switching of the Nanoflex valve. The signals
at Time 0 will move the Nanoflex valve to the Inject position and the signals at Time 45
will move the Nanoflex valve back to the Load position.
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Figure 1-16 LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 1-17.
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Figure 1-17 LC Method Settings dialog—Run Conditions tab
6. Click Save.
Specify the Acquisition Method in the Analyst TF Software
1. In the Acquire section of the Navigation toolbar, click the Method Wizard button to
open the wizard.
2. On the Choose Methods tab of the Method Wizard window, specify the MS
method.
•
In the Choose MS Method list, select ToF MS + Hi Sensitivity Product Ion
(+) method.
3. If an autosampler and LC method has already been associated with a previous
acquisition method, in the Choose LC Method list, select the existing .dam method.
4. If an LC method has not been used before, then select No LC Method.
Tip! Any method can be selected as it can be edited after creation of this
acquisition method.
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Figure 1-18 Method Wizard window—Choose Methods tab
5. In the Save Method As field, type System Performance Test for the acquisition
method and press Enter.
6. Click Next.
7. On Ion Source Parameters tab, specify the source conditions that were determined
during the NanoSpray ion source infusion test. Refer to the NanoSpray® Ion Source
Operator Guide for more information.
Figure 1-19 Method Wizard window—Ion Source Parameters tab
8. Click Next.
9. On the TOF MS - MS/MS tab, specify the acquisition parameters for the
TripleTOF 5600 system.
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i.
Enter the scan information for the TOF MS experiment: In the Start TOF Mass
(Da) and Stop TOF Mass (Da) fields, specify the mass range to 400 and
1800, respectively. The TOF Accumulation Time is 0.250 seconds.
ii. Enter the scan information for the TOF MS/MS experiment: In the MS2 Start
Mass (Da) and MS2 Stop Mass (Da) fields, specify the mass range to 100
and 1800, respectively. The MS2 Accumulation Time is 0.5 seconds.
iii. The total Cycle Time is automatically calculated from the times specified.
iv. In the Mass Spec Acquisition Duration field, specify the Duration Time for
the entire LC/MS run.
This value depends on the length of time of the analytical gradient. In this
example, it is 44 minutes.
Figure 1-20 Method Wizard window—TOF MS - MS/MS tab
10. In the table at the bottom of the tab, define precursor ion as 729.3652. for the
product ion scan.
11. Specify the m/z of the peptide with the collision energy (45) and a CES of 3 V.
12. Click Finish to save the method.
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Add LC Information to the Acquisition Method
If the autosampler and pump method information was not specified, add it now.
a
Note: The Q1 Isolation window is automatically set to Unit. This is the correct setting
for this test.
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
2. Click Eksigent AS2 and then select the autosampler method, AS2 10µLinj nanoflex
trap elute.ini or AS2 10µLinj trap.ini, depending on the installation.
Figure 1-21 Software Application Properties—Eksigent AS2
3. Click Eksigent Gradient 2 and then select the gradient pump method, CH2 45min
300nLmin Nanoflex trap.ini or CH2 45min 300nLmin trap.ini, depending on the
installation.
4. Click Eksigent Loading Pump and then select the loading pump method, Load
Pump 10 min Trap Wash.ini.
5. Save the method as “System Performance Test”.
Note: This method can also function as the LC auto calibration method when using
Beta-Galactosidase as a calibrant.
Prepare the System for Testing
Plumb the system in trap-elute configuration to perform the pre-column desalting workflow.
Prepare the Solution and Dilution
Prepare the Beta-Galactosidase stock solution from the Beta-Galactosidase vial provided in the
LC/MS Peptide/Protein Mass Standards Kit as described below. This will produce a stock
solution of 1 pmol/µL.
1. Add 625.0 µL of Buffer A (100% water:0.1% formic acid) to the Beta-Galactosidase
vial.
2. Vortex the vial for at least 30 seconds.
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3. Using a centrifuge, spin the vial to bring the liquid down to the bottom of the vial
before opening.
4. Repeat these steps to confirm dissolution.
5. Aliquot the stock solution (1 pmol/µL concentration) into 50 µL volumes and freeze
for future use.
Note: Solutions can be stored at 4°C for up to 3 days after thawing.
Prepare the Dilution for Functional Evaluation
1. Combine 40 µL of Buffer A (100% water:0.1% formic acid) with 10 µL of the BetaGalactosidase protein digest stock solution in a clean vial. A 1 µL injection of a
200 fmol/µL solution will be performed.
2. Vortex the vial for at least 30 seconds to properly mix the solution. This is a
1/5 dilution and will give a final concentration of 200 fmol/µL.
3. Transfer the solution to the autosampler vial and make sure there is no bubble on the
bottom of the vial.
Prepare the Dilution for Performance Evaluation
1. Prepare the solution as described above. A 1 µL injection of a 25 fmol/µL solution
will be performed.
2. Prepare 400 µL of the working solution of Beta-Galactosidase.
•
Combine 390 µL of Buffer A (100% water:0.1% formic acid) with 10 µL of the
Beta-Galactosidase protein digest stock solution in a clean vial.
•
Vortex the solution for at least 30 seconds to properly mix the solution. This is
a 1/40 dilution and will give a final concentration of 25 fmol/µL.
•
Transfer the solution to the autosampler vial and make sure there is no bubble
on the bottom of the vial.
Condition the System
A trap and column typically require 2 to 3 runs with 200 fmol of protein digest for conditioning.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test.
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Verify System Readiness
Make sure the NanoLC Ultra system is meeting performance specifications.
1. Connect effluent from the NanoLC Ultra system to the NanoSpray ion source and
verify that the spray is stable by monitoring the background signal in the Analyst TF
software.
2. Equilibrate the LC/MS system with the starting conditions of the method outlined
above.
3. Make sure the spray is stable.
4. Double-click Manual Tune in the left Navigation bar.
5. Enter the key parameters in Table 1-3, and then click Start to begin acquisition.
Table 1-3 Key Parameters
Parameter
Value
MS
Scan type
TOF MS
Polarity
Positive
Start Mass
400
Stop Mass
1000
Run Time
2 min
Source/Gas**
Curtain Gas (CUR)
20-25
IonSpray Voltage (IS)
2100-2400 V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150°C
Compound
Declustering Potential (DP)
70
Figure 1-22 Analyst TOF MS scan for background noise
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Figure 1-23 Unstable spray induced by air bubbles
6. Make sure that the spray is still stable by monitoring the background signal with a
TOF MS scan.
•
Stable spray appears as shown in Figure 1-22.
•
Unstable spray appears as shown in Figure 1-23 (typical unstable spray
induced by air bubbles).
•
If the spray is not stable, retune the NanoSpray ion source by infusion. Refer
to the NanoSpray® Ion Source Operator Guide for more information.
Perform the System Functional Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst TF Software
1. Double-click Build Acquisition Batch in the left Navigation bar.
2. Build the acquisition batch, as shown in Figure 1-24.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
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Figure 1-24 Add Samples dialog—System Functional Test
3. Click OK.
4. Save the data file as TT5600 system LC BGal functional status check <date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results in the PeakView Software
1. Open the data file and double-click the magenta arrow to display the individual TICs.
2. Right-click the TIC of the TOF MS and select Remove all Traces Except Active to
display the overlaid TOF MS TIC.
3. Extract the TOF MS peak XICs for the target peptides.
i. On the Show menu, click Extract Ions Using.
ii. Specify the masses and extraction width for the peptides as shown in
Figure 1-25. Only the masses and widths are required.
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Figure 1-25 Specify XIC Ranges dialog
4. Click OK.
The XICs for each peptide are generated.
Note: The XIC can be used to evaluate the NanoLC Ultra LC/MS peak
retention times and shapes. Peak widths, retention times, and XIC width
vary from LC to LC system depending on transfer line volumes, column type
used, column age, and more.
5. In the XIC pane, click and drag across the range of the more intense peaks, as
shown in Figure 1-26.
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Figure 1-26 TOF MS XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra trap elute
run (200 fmol on column)
Note: This test is not a specification. Use this example to confirm injection
and peak separation and shape only.
6. Make sure the peaks have good separation and shape.
i.
In the PeakView software, on the Window menu, click Graph Selection
Window.
ii. To collect the Peak Area for each XIC, click each (color-keyed) line in the
upper left.
The retention time appears on the top of the peak selected. The bottom line of
the Graph Selection Info displays the Peak Area.
Measure the Performance of the TOF MS/MS Scan
1. In the main TIC pane, extract individual TICs by double-clicking the magenta arrow.
2. Select the MS/MS scan, and then select Remove all Traces Except Active to
display the pane shown at the bottom of Figure 1-27.
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Figure 1-27 TOF MS/MS XIC of m/z 729.3652 peak
Note: This XIC is not a specification. Use this example to verify the TOF
MS/MS scan.
3. Click and drag across the peak and double-click the peak to display the underlying
MS/MS spectra.
4. Make sure the MS/MS spectra looks as shown in Figure 1-27 with fragment ions
across the whole mass range.
5. Repeat the acquisition until you have consistent peak shape and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
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Perform the System Performance Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst TF Software
1. Double-click Build Acquisition Batch in the left Navigation bar.
2. Build the acquisition batch, as shown in Figure 1-28.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
Figure 1-28 Add Sample dialog—System Performance Test
3. Click OK.
4. Save the data file as TT5600 system LC BGal Performance Test <date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results in the PeakView software
1. Open the data file and double-click the magenta arrow to display the individual TICs.
2. Right-click the TIC of the TOF MS and select Remove all Traces Except Active to
display the overlaid TOF MS TIC.
3. Extract the TOF MS peak XICs for the target peptides.
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i. On the Show menu, click Extract Ions Using.
ii. Specify the masses and extraction width for the peptides as shown in
Figure 1-25. Only the masses and widths are required.
4. Click OK.
The XICs for each peptide are generated. The peak areas of the extracted peaks
can be used to evaluate the NanoLC Ultra LC/MS sensitivity and the peak retention
times can shapes can be used to evaluate the chromatography.
Note: The XIC can be used to evaluate the NanoLC Ultra LC/MS peak
retention times and shapes. Peak widths, retention times, and XIC width
vary from LC to LC system depending on transfer line volumes, column type
used, column age, and more.
5. In the XIC pane, click and drag across the range of the more intense peaks, as
shown in Figure 1-29.
6. On the Window menu, click Graph Selection Window.
7. Collect the peak area value for each XIC.
Figure 1-29 TOF MS XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra trap elute
run (25 fmol on column)
Note: This test is not a specification. Use this example to confirm injection
and peak separation and shape only. Refer to the peak area specification in
the Test Results—TripleTOF 5600 Instruments section of this chapter.
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8. Make sure the peaks have good separation and shape using the PeakView software.
i.
In the PeakView software, on the Window menu, click Graph Selection
Window.
ii. Note the retention times of the chosen peaks.
This will vary with each system. Time of elution of the first is about 14 to 16
minutes. The longer elution time indicates you need to minimize LC dead
volume.
Note: Most of the XICs obtained should have peak widths of ~0.17 minute
half height on average and have peak intensities similar to that shown in
Figure 1-29. Some peaks will be narrower and some will be broader.
Measure the Performance of the TOF MS/MS Scan
1. In the main TIC pane, extract individual TICs by double-clicking the magenta arrow.
2. Select the MS/MS scan, and then select Remove all Traces Except Active to
display the pane shown in Figure 1-30.
Figure 1-30 TOF MS/MS XIC of m/z 729.3652 peak
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Note: This XIC is not a specification. Use this example to verify the TOF
MS/MS scan.
3. Click and drag across the peak and double-click the peak to display the underlying
MS/MS spectra.
4. Make sure the MS/MS spectra has fragment ion across the whole mass range. Refer
to Figure 1-25.
Note: This test is not a specification. Use this example to confirm injection
and peak shape only.
5. Repeat the acquisition until you have consistent peak shape, retention time, and
peak intensity (a minimum of 3 times).
For new columns, this may require that you repeat the acquisition 10 or more times
in order to obtain consistent peak shape, retention time, and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
6. Record the results for each acquisition.
7. Record the average peak area of the acquisitions in the section, Test Results—
TripleTOF 5600 Instruments on page 50.
8. Make sure that the average peak area meets the minimum requirements specified in
the section, Test Results—TripleTOF 5600 Instruments on page 50.
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Troubleshoot Peak Problems
This section provides information for troubleshooting peak related problems such as broad or
tailing peak widths, lack of separation between peaks, and low peak area.
Peak widths are too broad or are tailing
•
Inspect all connections in the flow path to verify that there are no dead volumes.
•
Look at connections post-column and around trap column. A small increase in peak
width is often seen when a trap column is used.
Caution: Potential Instrument Damage: If using the Nanoflex system and problems
persist, do not attempt to troubleshoot the fittings connected to the chip.
No separation between the peaks
•
Make sure that both pumps are delivering the correct amount of solvent.
•
Make sure that the pressure spike upon injection is not too severe in the high-flow
channel (less than 300 psi change in pressure).
•
Large pressure change upon injection suggests an air bubble has been introduced to
the sample loop or is present in the trap column plumbing.
Note: The overall separation of the chromatography itself will often be less than direct
injection. Components that elute comparably on the trap and analytical column will not
re-resolve on the analytical column and, as a result, spread out or bunch together.
Peak intensity or peak area is too low
•
Verify the performance of the mass spectrometer and the ion source spray using the
infusion tests in the Nanospray® Ion Source Operator Guide.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test. A trap typically requires 2 to 3 runs with 250
fmol to 500 fmol of protein digest on the column for conditioning.
•
Verify that the correct amount of sample has been withdrawn from the autosampler
vial.
•
Perform a direct injection with a protein digest on the analytical column to determine
if the problem is related to the trap.
•
If the first LC peak does not elute for a long time, inspect the system for dead volume
before the trap.
•
If the early eluting peaks are not visible or are very low in intensity, this could mean
that trapping efficiency is low. Replace the trap.
Tip! Minimize tubing length wherever possible and make sure all tubing for the
nanoflow path has an inner diameter of approximately 25 µm i.d.
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•
If the late eluting peaks are not visible or are very low in intensity, this is usually a
sign that the column is getting old. In rare cases, it could mean that the BetaGalactosidase standard is degraded. See Figure 1-31 and Figure 1-32 for an
example of a scan with 10 compounds and an older column.
Figure 1-31 Specify XIC Ranges dialog
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Figure 1-32 Extraction of all peaks—Late eluting peaks not present
•
Always monitor the column and trap pressure over time; increasing pressure may
indicate increasing blockage; probably at the Nanospray ion source tip. If, when the
connection between the column and the ion source head is unfastened and the
pressure changes quickly, then the tip is getting clogged and should be changed.
•
For better long-term column lifetime, verify that there is at least a 30% drop in
pressure observed during the high organic flush of the column. Increase the duration
of the high organic flush until a good pressure change is observed. This time might
increase for the trap column configuration relative to the direct injection
configuration.
•
Figure 1-33 shows a minimal pressure change upon injection and a 30% pressure
decrease during the high organic flush.
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Figure 1-33 Good pressure profile for a direct injection NanoLC Ultra
system run
Create a Backup of the EKSettings.reg File
The EKSettings.reg file can be used to re-establish the system settings derived on installation if
they are lost. Create a copy of the REG file upon completion of these tests.
1. In the Eksigent control software, on the System menu, click Instrument
Configuration.
2. Click Export Settings.
A backup of the REG file is created.
3. Navigate to the system settings folder (for example, C:\Program Files\Eksigent
NanoLC\settings).
4. Copy the previous_settings.reg file to another location, separate from the host
computer.
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Test Results—TripleTOF 5600 Instruments
For TripleTOF 5600 instruments, complete this table with the results from the ion from the BetaGalactosidase digest solution stock. Make sure that the peak area is within specification.
Most of the XICs obtained should have peak widths of ~0.17 minute half height on average and
have peak intensities similar to that shown in Figure 1-29. Some peaks will be narrower and
some will be broader Additionally, verify that the peaks elute within 5 minutes of each other.
Beta-Gal Lot Number: ___________________________________
Table 1-4 LC/MS Specification Test—TripleTOF 5600 Instruments
Q1
Peptide ID
Spec.
(peak area)
503.2368
YSQQQLMETSHR
1.0E+05
542.2645
GDFQFNISR
1.0E+05
671.3379
VDEDQPFPAVPK
2.0E+05
714.8469
DWENPGVTQLNR
5.0E+04
729.3652
APLDNDIGVSEATR
2.0E+05
Specification Passed?
Actual

Notes
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LC/MS System Configuration Test—
AB SCIEX QTRAP® 5500 System
This chapter describes the steps for preparing and performing LC/MS system configuration tests
for the NanoLC Ultra® system configured with the cHiPLC® Nanoflex system (or external
ChromXP column) and the AB SCIEX QTRAP® 5500 instrument.
Note: The tests in this chapter are written for Gradient 2 as the low-flow channel. If this
is not true for your system (for example, if you have a 1D or 1D+ system), then make the
appropriate changes throughout the tests.
The tests in this chapter are divided as follows:
•
Fast test to condition the column and determine the functional status of the system.
See Create the Methods and Batch for the System Functional Test for details.
Perform these tests after completing the NanoSpray® ion source infusion tests in
order to first confirm the spray performance of the tip. Refer to the NanoSpray® Ion
Source Operator Guide for more information.
•
Longer test to determine the performance level of the instrument for proteomics
applications such as protein identification and quantification. See Create the
Methods and Batch for the System Performance Test for details.
These tests can be used as a measure of the NanoLC Ultra system performance in
isolation of performance of the other components. Results from these tests can
become the baseline performance for the system and can be performed regularly
and used as a system quality control test in the future.
Approximate time required:
1. Create the methods and batch: 45 minutes
2. Prepare the system for testing: 3-4 hours
3. Perform the test.
i. System functional test: 90 minutes
ii. System performance test: 180 minutes
Recommended solvents can be ordered from VWR:
•
Burdick and Jackson acetonitrile with 0.1% formic acid, P/N BJLC441-1.0
•
Burdick and Jackson water with 0.1% formic acid, P/N BJLC452-1.0
Required materials for a Nanoflex system installation:
•
Reverse phase cHiPLC column (75 µm x 15 cm ChromXP C18-CL 3 µm 120 Å)
•
cHiPLC trap (200 µm x 0.5 mm ChromXP C18-CL 3 µm 120 Å)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: Ensure that the Nanoflex system is in the Load position before beginning these
tests.
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Required materials for an external column installation:
•
Reversed phase ChromXP nanoLC column (75 µm ID x 15 cm, ChromXP C18 3 µm
120 Å, P/N 805-00120)
•
ChromXP nanoLC Trap column (350 µm ID x 0.5 mm, ChromXP C18 3 µm 120 Å,
P/N 5016752)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: After successfully completing the tests, create a backup of the EKSettings.reg
file. See Create a Backup of the EKSettings.reg File for more information.
Create the Methods and Batch for the System
Functional Test
This section describes a test for the NanoLC Ultra system to condition the column and determine
the functional status.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications.
Note: The steps in this section do not constitute a NanoLC Ultra system performance
test. See Create the Methods and Batch for the System Performance Test.
The expected test duration is 30 minutes using the NanoSpray® ion source. Repeat the test until
you have consistent peak shape and intensity (approximately 90 minutes).
Create the Acquisition Methods and Batch—Functional Test
Note: Use Gradient 2 for the autosampler method. Gradient 2 is the nanoflow module
for LC configuration.
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler status window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 2-1 or Figure 2-2.
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Figure 2-1
Autosampler Settings dialog—trap-elute configuration (Nanoflex system)
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Figure 2-2
Autosampler Settings dialog—trap-elute configuration (column)
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj Nanoflex
Trap (Nanoflex system installation) or AS2 10uLloop 1uLinj Ch2Valve Trap (column
installation), and then click Save.
Create the LC Methods
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffer mixtures are shown in Table 2-1.
Table 2-1 Typical Buffer Mixtures
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent® Control Software
1. Ensure that Loading Pump is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. In the Name field, type Load Pump 2 min Trap Wash, and then click Save.
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4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 2-3.
Figure 2-3
LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 2-4.
Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
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Figure 2-4
LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Gradient Method in the Eksigent Control Software
For the analytical gradient (typically on the Gradient 2 pump with the nanoflow module), create
the gradient method.
1. Ensure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If the installation includes a Nanoflex system, then in the Name field, type CH2
15min 400nLmin nanoflex trap, and click Save.
Or
If the installation includes an external column, then in the Name field, type CH2
15min 400nLmin trap, and click Save.
4. On the Gradient Table tab, specify the method as shown in Figure 2-5 or Figure 2-6,
depending on the installation.
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Figure 2-5
LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown verify the correct switching of the Nanoflex valve.
The signals at Time 0 will move the Nanoflex valve to the Inject position and
the signals at Time 15 will move the Nanoflex valve back to the Load
position.
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Figure 2-6
LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 2-7.
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Figure 2-7
LC Method Settings dialog—Run Conditions tab (Nanoflex
system)
6. Click Save.
Create the Acquisition Method in the Analyst® Software
1. Double-click Build Acquisition Method on the left Navigation bar to create an
acquisition method.
2. Specify the key parameters, as shown in Table 2-2.
Note: The acquisition time should be shorter than the LC run time.
Table 2-2 Key Parameters
Parameter
Value
MS
Scan Type
MRM Scan
Polarity
Positive
Q1/Q3 Masses and CE
See Table 2-3.
Acquisition time
14 min
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Table 2-2 Key Parameters (Continued)
Parameter
Value
Advanced MS
Q1 Resolution
Unit
Q3 Resolution
Unit
Curtain Gas (CUR)
20
CAD Gas
HIGH
IonSpray Voltage (IS)
2300V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150°C
Declustering Potential (DP)
70
** Source/Gas parameters may vary between systems and spray tip. Determine the
best value for the system you are working with. Make sure the spray tip position is
optimized before creating the acquisition method.
3. Enter the MRM transitions from Table 2-3.
Note: In the Analyst MRM transition table, verify that the additional CE
(collision energy) column is added to the table view by right-clicking the
table and selecting CE from the menu that appears.
Table 2-3 MRM Transitions for Beta-Galactosidase
Q1
Q3
Dwell
ID
CE
503.2
760.3
50
BG_YSQQQLMETSHR
27
542.3
636.4
50
BG_GDFQFNISR
26
671.3
755.5
50
BG_VDEDQPFPAVPK
33
714.9
884.5
50
BG_DWENPGVTQLNR
32
729.4
832.5
50
BG_APLDNDIGVSEATR
48
Add LC Information to the Acquisition Method
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
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Figure 2-8
Acquisition Method Properties tab—synchronization mode
2. Click Eksigent AS2 and then select the autosampler method, AS2 10uLloop 1uLinj
Nanoflex Trap elute.ini or AS2 10uLloop 1uLinj Trap elute.ini, depending on the
installation.
Figure 2-9
Software Application Properties tab—autosampler filename
3. Click Eksigent Gradient 2, and then select the gradient 2 pump method, CH2 15min
400nL min nanoflex trap.ini or CH2 15min 400nL min trap.ini, depending on the
installation.
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Figure 2-10 Software Application Properties tab—gradient 2 filename
4. Click Eksigent Loading Pump, and then select the loading pump method, Load
Pump 2min Trap Wash.ini.
Figure 2-11 Software Application Properties tab—loading pump filename
5. Save the method as “System Functional Test”.
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Create the Methods and Batch for the System
Performance Test
This section provides tests for the NanoLC Ultra system that indicate of the performance level of
the instrument for proteomics applications such as protein identification and quantification.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications. If the NanoLC system has been idle for two weeks or more, then
calibrate the system. Refer to the appendix, System Calibration, for more information.
The expected test duration is 60 minutes using the NanoSpray ion source. Repeat the test until
you have consistent peak shape and intensity (approximately 180 minutes).
Create the Acquisition Methods and Batch—
Performance Tests
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 2-12 or Figure 2-13, depending on the installation.
Figure 2-12 Autosampler Settings dialog—trap-elute configuration (Nanoflex system)
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Figure 2-13 Autosampler Settings dialog—trap-elute configuration (column)
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj Nanoflex
Trap or AS2 10uLloop 1uLinj Ch2Valve Trap, depending on the installation.
5. Click Save.
Create the LC Methods
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffers are shown in Table 2-4.
Table 2-4 Typical Buffer Mixtures
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent Control Software
1. Ensure that Loading Pump is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. In the Name field, type Load Pump 10 min Trap Wash, and then click Save.
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4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 2-14.
Figure 2-14 LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 2-15.
Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
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Figure 2-15 LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Gradient Method in the Eksigent Control Software
For the analytical gradient (typically on the Gradient 2 pump with the nanoflow module), create
the gradient method.
1. Ensure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If this is a Nanoflex system installation, then in the Name field, type CH2 45min
300nLmin Nanoflex trap, and click Save.
Or
If this is a column installation, then in the Name field, type CH2 45min 300nLmin
column trap, and click Save.
4. On the Gradient Table tab, specify the method, as shown in Figure 2-16 or Figure 217, depending on the installation.
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Figure 2-16 LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown above verify the correct switching of the Nanoflex
valve. The signals at Time 0 will move the Nanoflex valve to the Inject
position and the signals at Time 45 will move the Nanoflex valve back to the
Load position.
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Figure 2-17 LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 2-18.
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Figure 2-18 LC Method Settings dialog—Run Conditions tab (Nanoflex
system)
6. Click Save.
Specify the Acquisition Method in the Analyst Software
1. Create the acquisition method. See Table 2-5 for details.
Note: The acquisition time should be shorter than the LC run time.
Table 2-5 Key Parameters
Parameter
Value
MS
Scan Type
MRM Scan
Polarity
Positive
MCA
Off
Q1/Q3 Masses and CE
See Table 2-6.
Acquisition time
40 min
Advanced MS
Q1 Resolution
Unit
Q3 Resolution
Unit
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Table 2-5 Key Parameters (Continued)
Parameter
Value
Source/Gas**
Curtain Gas (CUR)
20
CAD Gas
HIGH
IonSpray Voltage (IS)
2300V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150°C
Compound
Declustering Potential (DP)
70
** Source/Gas parameters may vary between systems and spray tip. Determine the
best value for the system you are working with. Ensure the spray tip position is
optimized before creating the acquisition method.
2. Enter the MRM transitions from Table 2-6.
Note: In the Analyst MRM transition table, verify that the additional CE
(collision energy) column is added to the table view by right-clicking the
table and selecting CE from the menu.
Table 2-6 MRM Transitions for Beta-Galactosidase
Q1
Q3
Dwell
ID
CE
433.9
723.4
50
BG_ELNYGPHQWR
30
450.7
524.3
50
BG_FNDDFSR
28
503.2
760.3
50
BG_YSQQQLMETSHR
27
528.9
855.4
50
BG_RDWENPGVTQLNR
25
542.3
636.4
50
BG_GDFQFNISR
26
550.3
871.4
50
BG_IDPNAWVER
27
567.1
932.5
50
BG_DVSLLHKPTTQISDFHVATR
30
607.9
685.4
50
BG_ITDSLAVVLQR
39
671.3
755.5
50
BG_VDEDQPFPAVPK
33
697.9
821.5
50
BG_LPSEFDLSAFLR
35
714.9
884.5
50
BG_DWENPGVTQLNR
32
729.4
832.5
50
BG_APLDNDIGVSEATR
48
871.9
915.5
50
BG_LSGQTIEVTSEYLFR
40
879.4
664.3
50
BG_VNWLGLGPQENYPDR
40
3. Save the method as “System Performance Test”.
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Add LC Information to the Acquisition Method
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
Figure 2-19 Acquisition Method Properties tab—synchronization mode
2. Click Eksigent AS2 and then select the autosampler method, AS2 10µLinj Nanoflex
trap elute.ini or AS2 10µLinj trap elute.ini, depending on the installation.
Figure 2-20 Software Application Properties tab—autosampler method
3. Click Eksigent Gradient 2 and then select the gradient pump method, CH2 45min
300nLmin Nanoflex trap.ini or CH2 45min 300nLmin column trap.ini, depending on
the installation.
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Figure 2-21 Software Application Properties tab—gradient 2 method
4. Click Eksigent Loading Pump and then select the loading pump method, Load
Pump 10 min Trap Wash.ini.
Figure 2-22 Software Application Properties tab—loading pump method
5. Save the method as “System Performance Test”.
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Prepare the System for Testing
Plumb the system in trap-elute configuration to perform the pre-column desalting workflow.
Prepare the Solution and Dilution
Prepare the Beta-Galactosidase stock solution from the Beta-Galactosidase vial provided in the
LC/MS Peptide/Protein Mass Standards Kit as described below. This will produce a stock
solution of 1 pmol/µL.
1. Add 625.0 µL of Buffer A (100% water:0.1% formic acid) to the Beta-Galactosidase
vial.
2. Vortex the vial for at least 30 seconds.
3. Using a centrifuge, spin the vial to bring the liquid down to the bottom of the vial
before opening.
4. Repeat step 2 and step 3 to confirm dissolution.
5. Aliquot the stock solution (1 pmol/µL concentration) into 50 µL volumes and freeze
for future use.
Note: Solutions can be stored at 4°C for up to 3 days after thawing.
Prepare the Dilution for Functional Evaluation
1. Combine 40 µL of Buffer A (100% water:0.1% formic acid) with 10 µL of the BetaGalactosidase protein digest stock solution in a clean vial. A 1 µL injection of a
200 fmol/µL solution will be performed.
2. Vortex the vial for at least 30 seconds to properly mix the solution. This is a
1/5 dilution and will give a final concentration of 200 fmol/µL.
3. Transfer the solution to the autosampler vial and make sure there is no bubble on the
bottom of the vial.
Prepare the Dilution for Performance Evaluation
1. Prepare the solution as described above. A 1 µL injection of a 10 fmol/µL solution
will be performed.
2. Prepare 500 µL of the working solution of Beta-Galactosidase.
•
Combine 495 µL of Buffer A (100 water:0.1% formic acid) with 5 µL of the
Beta-Galactosidase protein digest stock solution in a clean vial.
•
Vortex the vial for at least 30 seconds to properly mix the solution. This is a
1/100 dilution and will give a final concentration of 10 fmol/µL.
•
Transfer the solution to the autosampler vial and make sure there is no bubble
on the bottom of the vial.
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Condition the System
A trap and column typically require 2 to 3 runs with 200 fmol of protein digest for conditioning.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test.
Verify System Readiness
Ensure the NanoLC Ultra system is meeting performance specifications.
1. Connect effluent from the NanoLC Ultra system to the NanoSpray ion source and
verify that the spray is stable by monitoring the background signal in the Analyst
software.
2. Equilibrate the LC/MS system with the starting conditions of the method outlined
above.
3. Ensure the spray is stable.
4. Double-click Manual Tune in the left Navigation bar.
5. Enter the key parameters from Table 2-7 and then click Start to begin acquisition.
Table 2-7 Key Parameters
Parameter
Value
MS
Scan type
Q1 Scan
Polarity
Positive
MCA
Off
Start Mass
400
Stop Mass
1000
Run Time
2 min
Source/Gas
Curtain Gas (CUR)
20-25
IonSpray Voltage (IS)
2100-2400 V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150ºC
Compound
Declustering Potential
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Figure 2-23 Q1 MS scan for background noise
Figure 2-24 Unstable spray induced by air bubbles
6. Ensure that the spray is still stable by monitoring the background signal with a
Q1 MS scan.
•
Stable spray appears as shown in Figure 2-23.
•
Unstable spray appears as shown in Figure 2-24 (typical unstable spray
induced by air bubbles).
•
If the spray is not stable, retune the NanoSpray ion source by infusion. Refer
to the NanoSpray® Ion Source Operator Guide for more information.
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Perform the System Functional Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst Software
1. Double-click Build Acquisition Batch in the left Navigation bar.
2. Build the acquisition batch, as shown in Figure 2-25.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
Figure 2-25 Add Samples dialog—System Functional Test
3. Click OK.
4. Save the data file as QTRAP 5500 system LC MRM BGal functional status check
<date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results
1. After the experiment has finished, open the sample from the data file in the Explore
window.
Figure 2-26 shows typical data for the instrument.
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Figure 2-26 MRM XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra
pre-column desalting run (200 fmol on column)
2. Repeat the acquisition until you have consistent peak shape and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
Note: This test is not a specification. Use this example to confirm injection and peak
shape of each MRM transition.
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LC/MS System Configuration Test— AB SCIEX QTRAP® 5500 System
Perform the System Performance Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst Software
1. Double-click Build Acquisition Batch in left Navigation bar.
2. Build the acquisition batch, as shown in Figure 2-27.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
Figure 2-27 Add Sample dialog—system performance test
3. Click OK.
4. Save the data file as 5500 QTRAP system LC MRM BGal Performance Test <date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results
1. After the experiment has finished, open the sample from the data file in the Explore
window.
2. Right-click the TIC for the MRM experiment, and then click Extract Ions.
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3. In the Extract Ions dialog, select the 5 MRMs from Table 2-8, and then click OK.
Figure 2-28 shows typical data for the instrument.
Table 2-8 MRM Transitions for Beta-Galactosidase
Q1
Q3
ID
503.2
760.3
BG_YSQQQLMETSHR
542.3
636.4
BG_GDFQFNISR
671.3
755.5
BG_VDEDQPFPAVPK
714.9
884.5
BG_DWENPGVTQLNR
729.4
832.5
BG_APLDNDIGVSEATR
4. Record the peak areas of the specified MRM transitions in the section, Test
Results—QTRAP 5500 Instruments on page 83.
Note: Most XICs should have peak widths of no more than 0.2 minute half
height. Some peaks will be narrower and some broader.
Figure 2-28 MRM XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra
pre-column desalting run (10 fmol on column)
5. Record the retention times of the chosen peaks. This will vary with each system.
Time of elution of the first peak (approximately 14 -16 minutes, as shown in Figure 228) indicates dead volume of the system. Minimize dead volume where possible.
6. Repeat the acquisition until you have consistent peak shape, retention time, and
peak intensity (a minimum of 3 times).
For new columns, this may require that you repeat the acquisition 10 or more times
in order to obtain consistent peak shape, retention time, and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
7. Record the results for each acquisition.
8. Record the average peak area of the acquisitions in the section, Test Results—
QTRAP 5500 Instruments on page 83.
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9. Ensure that the average peak area meets the minimum requirement specified in the
section, Test Results—QTRAP 5500 Instruments on page 83.
Troubleshoot Peak Problems
This section provides information for troubleshooting peak related problems such as broad or
tailing peak widths, lack of separation between peaks, and low peak area.
Peak widths are too broad or are tailing
•
Inspect all connections in the flow path to verify that there are no dead volumes.
•
Look at connections post-column and around trap column. A small increase in peak
width is often seen when a trap column is used.
Caution: Potential Instrument Damage: If using the Nanoflex system and problems
persist, do not attempt to troubleshoot the fittings connected to the chip.
No separation between the peaks
•
Ensure that both pumps are delivering the correct amount of solvent.
•
Ensure that the pressure spike upon injection is not too severe in the high-flow
channel (less than 300 psi change in pressure).
•
Large pressure change upon injection suggests an air bubble has been introduced to
the sample loop or is present in the trap column plumbing.
Note: The overall separation of the chromatography itself will often be less than direct
injection. Components that elute comparably on the trap and analytical column will not
re-resolve on the analytical column and, as a result, spread out or bunch together.
Peak intensity or peak area is too low
•
Verify the performance of the mass spectrometer and the ion source spray using the
infusion tests in the Nanospray® Ion Source Operator Guide.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test. A trap typically requires 2 to 3 runs with
200 fmol of protein digest on the column for conditioning.
•
Verify that the correct amount of sample has been withdrawn from the autosampler
vial.
•
Perform a direct injection with a protein digest on the analytical column to determine
if the problem is related to the trap.
•
If the first LC peak does not elute for a long time, inspect the system for dead volume
before the trap.
•
If the early eluting peaks are not visible or are very low in intensity, this could mean
that trapping efficiency is low. Replace the trap.
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Tip! Minimize tubing length wherever possible and make sure all tubing for the
nanoflow path has an inner diameter of approximately 25 µm i.d.
•
If the late eluting peaks are not visible or are very low in intensity, this is usually a
sign that the column is getting old. In rare cases, it could mean that the BetaGalactosidase standard is degraded. See Figure 2-29 for an example of a scan with
an older column.
Figure 2-29 Extraction of all peaks—late eluting peaks not present
•
Always monitor the column and trap pressure over time; increasing pressure often
indicates increasing blockage, probably at the Nanospray ion source tip. If when the
connection between the column and the ion source head is unfastened and the
pressure changes quickly, this means that the tip is getting clogged and should be
changed.
•
For better long-term column lifetime, verify that there is at least a 30% change in
pressure observed during the high organic flush of the column. Increase the duration
of the high organic flush until a good pressure change is observed. This time might
increase for the trap column configuration relative to the direct injection
configuration.
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•
Figure 2-30 shows a minimal pressure change upon injection and a 30% pressure
decrease during the high organic flush.
Figure 2-30 Good pressure profile for a direct injection NanoLC Ultra
system run
Create a Backup of the EKSettings.reg File
The EKSettings.reg file can be used to re-establish the system settings derived on installation if
they are lost. Create a copy of the REG file upon completion of these tests.
1. In the Eksigent control software, on the System menu, click Instrument
Configuration.
2. Click Export Settings.
A backup of the REG file is created.
3. Navigate to the system settings folder (for example, C:\Program Files\Eksigent
NanoLC\settings).
4. Copy the previous_settings.reg file to another location, separate from the host
computer.
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Test Results—QTRAP 5500 Instruments
For QTRAP 5500 instruments, complete this table with the results from five of the peptides from
the Beta-Galactosidase digest solution stock. Ensure that the peak area is below specification.
Most XICs should have peak widths of no more than 0.2 minute half height. Some peaks will be
narrower and some broader. Additionally, verify that the peaks elute within 5 minutes of each
other.
Beta-Gal Lot Number: ___________________________________
Table 2-9 LC/MS Specification Test—QTRAP 5500 Instruments
Q1
Q3
Dwell Peptide ID
CE
Spec.
(peak
area)
503.2
760.3
50
BG_YSQQQLMETSHR
27
2.0E+04
542.3
636.4
50
BG_GDFQFNISR
26
5.0E+05
671.3
755.5
50
BG_VDEDQPFPAVPK
33
5.0E+05
714.9
884.5
50
BG_DWENPGVTQLNR
32
7.0E+04
729.4
832.5
50
BG_APLDNDIGVSEATR 48
2.0E+05
Actual
Specification Passed?

Notes
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3
LC/MS System Configuration Test—
AB SCIEX 4000 QTRAP® System
This chapter describes the steps for preparing and performing LC/MS system configuration tests
for the NanoLC Ultra® system configured with the cHiPLC® Nanoflex system (or external
ChromXP column) and the AB SCIEX 4000 QTRAP® system.
Note: The tests in this chapter are written for Gradient 2 as the low-flow channel. If this
is not true for your system (for example, if you have a 1D or 1D+ system), then make the
appropriate changes throughout the tests.
The tests in this chapter are divided as follows:
•
Fast test to condition the column and determine the functional status of the system.
See Create the Methods and Batch for the System Functional Test for details.
Perform these tests after completing the NanoSpray® ion source infusion tests in
order to first confirm the spray performance of the tip. Refer to the NanoSpray® Ion
Source Operator Guide for more information.
•
Longer test to determine the performance level of the instrument for proteomics
applications such as protein identification and quantification. See Create the
Methods and Batch for the System Performance Test for details.
These tests can be used as a measure of the NanoLC Ultra system performance in
isolation of performance of the other components. Results from these initial tests can
become the baseline performance for the system and can be performed regularly
and used as a system quality control test in the future.
Approximate time required:
1. Create the methods and batch: 45 minutes
2. Prepare the system for testing: 3-4 hours
3. Perform the test.
i. System functional test: 90 minutes
ii. System performance test: 180 minutes
Recommended solvents can be ordered from VWR:
•
Burdick and Jackson acetonitrile with 0.1% formic acid, P/N BJLC441-1.0
•
Burdick and Jackson water with 0.1% formic acid, P/N BJLC452-1.0
Required materials for a Nanoflex system installation:
•
Reverse phase cHiPLC column (75 µm x 15 cm ChromXP C18-CL 3 µm 120 Å)
•
cHiPLC trap (200 µm x 0.5 mm ChromXP C18-CL 3 µm 120 Å)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: Ensure that the Nanoflex system is in the Load position before beginning these
tests.
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Required materials for an external column installation:
•
Reversed phase ChromXP nanoLC column (75 µm ID x 15 cm, ChromXP C18 3 µm
120 Å, P/N 805-00120)
•
ChromXP nanoLC Trap column (350 µm ID x 0.5 mm, ChromXP C18 3 µm 120 Å,
P/N 5016752)
•
LC/MS Peptide/Protein Mass Standards Kit (P/N 4368624)
Note: After successfully completing the tests, create a backup of the EKSettings.reg
file. See Create a Backup of the EKSettings.reg File for more information.
Create the Methods and Batch for the System
Functional Test
This section describes a test for the NanoLC Ultra system to condition the column and determine
the functional status.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications.
\
Note: The steps in this section do not constitute a NanoLC Ultra system performance
test. See Create the Methods and Batch for the System Performance Test.
The expected test duration is 30 minutes using the NanoSpray® ion source. Repeat the test until
you have consistent peak shape and intensity (approximately 90 minutes).
Create the Acquisition Method and Batch—Functional Test
Note: Use Gradient 2 for the autosampler method. Gradient 2 is the nanoflow module
for LC configuration.
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler status window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 3-1 or Figure 3-2, depending on the installation.
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Figure 3-1
Autosampler Settings dialog—trap-elute configuration (Nanoflex system)
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Figure 3-2
Autosampler Settings dialog—trap-elute configuration (column)
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj Nanoflex
trap elute or AS2 10uLloop 1uLinj Ch2Valve Trap, depending on the installation.
5. Click Save.
Create the LC Methods
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffer mixtures are shown in Table 3-1.
Table 3-1 Typical Buffer Mixtures
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent® Control Software
1. Ensure that Loading Pump is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. In the Name field, type Load Pump 2 min Trap Wash, and then click Save.
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4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 3-3.
Figure 3-3
LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 3-4.
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Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
Figure 3-4
LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Gradient Method in the Eksigent Control Software
For the analytical gradient (typically, the Gradient 2 pump with the nanoflow module), create the
gradient method.
1. Ensure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If the installation includes a Nanoflex system, then in the Name field, type CH2
15min 400nLmin nanoflex trap, and click Save.
Or
If the installation includes an external column, then in the Name field, type CH2
15min 400nLmin trap, and click Save.
4. On the Gradient Table tab, specify the method as shown in Figure 3-5 or Figure 3-6,
depending on the installation.
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Figure 3-5
LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown verify the correct switching of the Nanoflex valve.
The signals at Time 0 will move the Nanoflex valve to the Inject position and
the signals at Time 15 will move the Nanoflex valve back to the Load
position.
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Figure 3-6
LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 3-7.
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Figure 3-7
LC Method Settings dialog—Run Conditions tab (Nanoflex
system)
6. Click Save.
Create the Acquisition Method in the Analyst® Software
1. Double-click Build Acquisition Method on the left Navigation bar to create an
acquisition method.
2. Specify the key parameters, as shown in Table 3-2.
Note: The acquisition time should be shorter than the LC run time.
Table 3-2 Key Parameters
Parameter
Value
MS
Scan Type
MRM Scan
Polarity
Positive
Q1/Q3 Masses and CE
See Table 3-3.
Acquisition time
14 min
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Table 3-2 Key Parameters (Continued)
Parameter
Value
Advanced MS
Q1 Resolution
Unit
Q3 Resolution
Unit
Source/Gas**
Curtain Gas (CUR)
20
CAD Gas
HIGH
IonSpray Voltage (IS)
2300V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150°C
Compound
Declustering Potential (DP)
70
** Source/Gas parameters may vary between systems and spray tip. Determine the
best value for the system you are working with. Ensure the spray tip position is
optimized before creating the acquisition method.
3. Enter the MRM transitions from Table 3-3.
Note: In the Analyst MRM transition table, verify that the additional CE
(collision energy) column is added to the table view by right-clicking the
table and selecting CE from the menu that displays.
Table 3-3 MRM Transitions for Beta-Galactosidase
Q1
Q3
Dwell
ID
CE
503.2
760.3
50
BG_YSQQQLMETSHR
27
542.3
636.4
50
BG_GDFQFNISR
26
671.3
755.5
50
BG_VDEDQPFPAVPK
33
714.9
884.5
50
BG_DWENPGVTQLNR
32
729.4
832.5
50
BG_APLDNDIGVSEATR
48
Add LC Information to the Acquisition Method
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
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Figure 3-8
Acquisition Method Properties tab—synchronization mode
2. Click Eksigent AS2 and then select the autosampler method, AS2 10uLloop 1uLinj
Nanoflex trap elute.ini or AS2 10uLloop 1uLinj Ch2Valve Trap.ini, depending on the
installation.
Figure 3-9
Software Application Properties tab—autosampler filename
3. Click Eksigent Gradient 2, and then select the gradient 2 pump method, CH2 15min
400nL min nanoflex trap.ini or CH2 15min 400nL min column trap.ini.
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Figure 3-10 Software Application Properties tab—gradient 2 filename
4. Click Eksigent Loading Pump, and then select the loading pump method, Load
Pump 2 min Trap Wash.ini.
Figure 3-11 Software Application Properties tab—loading pump filename
5. Save the method as “System Functional Test”.
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Create the Methods and Batch for the System
Performance Test
This section provides tests for the NanoLC Ultra system that indicate the performance level of the
instrument for proteomics applications such as protein identification and quantification.
Perform these tests when the mass spectrometer is known to be operating well and meeting
performance specifications.
The expected duration of the performance tests is 60 minutes using the NanoSpray ion source.
Repeat the test until you have consistent peak shape and peak intensity (approximately 180
minutes).
Create the Acquisition Methods and Batch—
Performance Tests
1. Plumb the autosampler valve with a 10 µL sample loop.
2. In the AS1/AS2 Autosampler window, click Method Editor.
3. Create the autosampler method for a trap-elute configuration, as shown
in Figure 3-12.
Figure 3-12 Autosampler Settings dialog—trap-elute configuration (Nanoflex system)
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Figure 3-13 Autosampler Settings dialog—trap-elute configuration (Nanoflex system or
column installation)
4. In the Name field, specify the name of this method as AS2 10uLloop 1uLinj trap elute
or AS2 10uLloop 1uLinj Ch2Valve Trap, depending on the installation.
5. Click Save.
Create the LC Methods
The aqueous channel for each pump (Channel A) will be filled with Buffer A. The organic channel
(Channel B) will be filled with Buffer B. For the Loading Pump, Buffer A is always used. Typical
buffers are shown in Table 3-4.
Table 3-4 Typical Buffer Mixtures
Buffer
Mixture
Channel
Buffer A
100% water:0.1% formic acid
Channel A
Buffer B
100% acetonitrile:0.1% formic acid
Channel B
In the method below, the loading pump will be the pump with the microflow module.
Create the Pump Method in the Eksigent Control Software
1. Ensure that Loading Pump is selected as the channel (top, right corner of window).
2. Click LC Methods.
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3. In the Name field, type Load Pump 10 min Trap Wash, and then click Save.
4. On the Gradient Table tab, revise the method for the loading pump (the loading
pump will be the pump with the microflow module), as shown in Figure 3-14.
Figure 3-14 LC Method Settings dialog—Gradient Table tab
5. On the Run Conditions tab, specify conditions as shown in Figure 3-15.
Note: For a trap-elute configuration, the Sample Injection method should
be Standard.
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Figure 3-15 LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create a Gradient Method in the Eksigent Control Software
For the analytical gradient (typically on the Gradient 2 pump with the nanoflow module), create
the gradient method.
1. Ensure that Gradient 2 is selected as the channel (top, right corner of window).
2. Click LC Methods.
3. If this is a Nanoflex system installation, then in the Name field, type CH2 45min
300nLmin Nanoflex trap, and click Save.
Or
If this is a column installation, then in the Name field, type CH2 45min 300nLmin
column trap, and click Save
4. On the Gradient Table tab, specify the method, as shown in Figure 3-16 or Figure 317, depending on the installation.
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Figure 3-16 LC Method Settings dialog—Gradient Table tab (Nanoflex
system)
Note: The events shown verify the correct switching of the Nanoflex valve.
The signals at Time 0 will move the Nanoflex valve to the Inject position and
the signals at Time 45 will move the Nanoflex valve back to the Load
position.
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Figure 3-17 LC Method Settings dialog—Gradient Table tab (column)
5. On the Run Conditions tab, specify the method as shown in Figure 3-18.
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Figure 3-18 LC Method Settings dialog—Run Conditions tab
6. Click Save.
Create the Acquisition Method in the Analyst Software
1. Create the acquisition method. See Table 3-5 for details.
Note: The acquisition time should be shorter than the LC run time.
Table 3-5 Key Parameters
Parameter
Value
MS
Scan Type
MRM Scan
Polarity
Positive
MCA
Off
Q1/Q3 Masses and CE
See Table 3-6.
Acquisition time
40 min
Advanced MS
Q1 Resolution
Unit
Q3 Resolution
Unit
Source/Gas**
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Table 3-5 Key Parameters (Continued)
Parameter
Value
Curtain Gas (CUR)
20
CAD Gas
HIGH
IonSpray Voltage (IS)
2300V
Ion Source Gas 1 (GS1)
3-15
Interface Heater
150°C
Compound
Declustering Potential (DP)
70
** Source/Gas parameters may vary between systems and spray tip. Determine the
best value for the system you are working with. Ensure the spray tip position is
optimized before creating the acquisition method.
2. Enter the MRM transitions from Table 3-6.
Note: In the Analyst MRM transition table, verify that the additional CE
(collision energy) column is added to the table view by right-clicking the
table and selecting CE from the menu.
Table 3-6 MRM Transitions for Beta-Galactosidase
Q1
Q3
Dwell
ID
CE
433.9
723.3
50
BG_ELNYGPHQWR
30
450.7
524.2
50
BG_FNDDFSR
28
503.2
760.3
50
BG_YSQQQLMETSHR
27
528.9
855.4
50
BG_RDWENPGVTQLNR
25
542.3
636.4
50
BG_GDFQFNISR
26
550.3
871.4
50
BG_IDPNAWVER
27
567.1
932.5
50
BG_DVSLLHKPTTQISDFHVATR
30
607.9
685.4
50
BG_ITDSLAVVLQR
39
671.3
755.5
50
BG_VDEDQPFPAVPK
33
697.9
821.5
50
BG_LPSEFDLSAFLR
35
714.9
884.5
50
BG_DWENPGVTQLNR
32
729.4
832.5
50
BG_APLDNDIGVSEATR
48
871.9
915.5
50
BG_LSGQTIEVTSEYLFR
40
879.4
664.3
50
BG_VNWLGLGPQENYPDR
40
3. Save the method as “System Performance Test”.
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Add LC Information to the Acquisition Method
1. Click Acquisition Method in the left pane, and then select LC Sync as the
Synchronization Mode.
Figure 3-19 Acquisition Method Properties tab—synchronization mode
2. Click Eksigent AS2 and then select the autosampler method, AS2 10µLinj Nanoflex
trap.ini or AS2 10µLinj trap.ini, depending on the installation.
Figure 3-20 Software Application Properties tab—autosampler method
3. Click Eksigent Gradient 2 and then select the gradient pump method, CH2 45min
300nLmin Nanoflex trap.ini or CH2 45min 300nLmin column trap.ini, depending on
the installation.
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Figure 3-21 Software Application Properties tab—gradient 2 method
4. Click Eksigent Loading Pump and then select the loading pump method, Load
Pump 10 min Trap Wash.ini.
Figure 3-22 Software Application Properties tab—loading pump method
5. Save the method as “System Performance Test”.
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Prepare the System for Testing
Plumb the system in trap-elute configuration to perform the pre-column desalting workflow.
Prepare the Solution and Dilution
Prepare the Beta-Galactosidase stock solution from the Beta-Galactosidase vial provided in the
LC/MS Peptide/Protein Mass Standards Kit as described below. This will produce a stock
solution of 1 pmol/µL.
1. Add 625.0 µL of Buffer A (100% water:0.1% formic acid) to the Beta-Galactosidase
vial.
2. Vortex the vial for at least 30 seconds.
3. Using a centrifuge, spin the vial to bring the liquid down to the bottom of the vial
before opening.
4. Repeat step 2 and step 3 to confirm dissolution.
5. Aliquot the stock solution (1 pmol/µL concentration) into 50 µL volumes and freeze
for future use.
Note: Solutions can be stored at 4°C for up to 3 days after thawing.
Prepare the Dilution for the Functional Evaluation
1. Combine 40 µL of Buffer A (100% water:0.1% formic acid) with 10 µL of the BetaGalactosidase protein digest stock solution in a clean vial. A 1 µL injection of a
200 fmol/µL solution will be performed.
2. Vortex the vial for at least 30 seconds to properly mix the solution. This is a
1/5 dilution and will give a final concentration of 200 fmol/µL.
3. Transfer the solution to the autosampler vial and make sure there is no bubble on the
bottom of the vial.
Prepare the Dilution for the Performance Evaluation
1. Prepare the solution as described above. A 1 µL injection of a 50 fmol/µL solution
will be performed.
2. Prepare 200 µL of the working solution of Beta-Galactosidase.
•
Combine 190 µL of Buffer A (100% water:0.1% formic acid) with 10 µL of the
Beta-Galactosidase protein digest stock solution in a clean vial.
•
Vortex the solution for at least 30 seconds to properly mix the solution. This is
a 1/20 dilution and will give a final concentration of 50 fmol/µL.
•
Transfer the solution to the autosampler vial and make sure there is no bubble
on the bottom of the vial.
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Condition the System
A trap and column typically require 2 to 3 runs with 200 fmol of protein digest for conditioning.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test.
Verify System Readiness
Ensure the NanoLC Ultra system is meeting performance specifications.
1. Connect effluent from the NanoLC Ultra system to the NanoSpray ion source and
verify that the spray is stable by monitoring the background signal in the Analyst
software.
2. Equilibrate the LC/MS system with the starting conditions of the method outlined
above.
3. Ensure the spray is stable.
4. Double-click Manual Tune in the left Navigation bar.
5. Enter the key parameters from Table 3-7 and then click Start to begin acquisition.
Table 3-7 Key Parameters
Parameter
Value
MS
Scan type
Q1 Scan
Polarity
Positive
MCA
Off
Start Mass
400
Stop Mass
1000
Run Time
2 min
Source/Gas
Curtain Gas (CUR)
20-25
IonSpray Voltage (IS)
2100-2400 V
Ion Source Gas 1 (GS1)
2-15
Interface Heater
150ºC
Compound
Declustering Potential
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Figure 3-23 Q1 MS scan for background noise
Figure 3-24 Typical TIC of an unstable spray induced by air bubbles
6. Ensure that the spray is still stable by monitoring the background signal with a
Q1 MS scan.
•
Stable spray appears as shown in Figure 3-23.
•
Unstable spray appears as shown in Figure 3-24 (typical unstable spray
induced by air bubbles).
•
If the spray is not stable, retune the NanoSpray ion source by infusion. Refer
to the NanoSpray® Ion Source Operator Guide for more information.
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Perform the System Functional Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst Software
1. Double-click Build Acquisition Batch in the left Navigation bar.
2. Build the acquisition batch, as shown in Figure 3-25.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
Figure 3-25 Add Samples dialog—System Functional Test
3. Click OK.
4. Save the data file as QTRAP 4000 system LC MRM BGal functional status check
<date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results
1. After the experiment has finished, open the sample from the data file in the Explore
window.
Figure 3-26 shows typical data for the instrument.
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Figure 3-26 MRM XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra
pre-column desalting run (200 fmol on column)
2. Repeat the acquisition until you have consistent peak shape and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
Note: This test is not a specification. Use this example to confirm injection and peak
shape of each MRM transition.
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Perform the System Performance Test
Create the LC/MS acquisition batch, run the batch and then verify the results.
Create the LC/MS Acquisition Batch in the Analyst Software
1. Double-click Build Acquisition Batch in left Navigation bar.
2. Build the acquisition batch, as shown in Figure 3-27.
i. In the Acquisition group, select the acquisition method from the list.
ii. Click Add Set, and then click Add Samples.
Figure 3-27 Add Sample dialog—System Performance Test
3. Click OK.
4. Save the data file as 4000 QTRAP system LC MRM BGal Performance Test <date>.
5. On the Location tab, specify the location of the Beta-Galactosidase sample in the
autosampler.
Run the Batch
1. On the Submit tab, click Submit.
2. In the View menu, click Sample Queue.
3. In the Acquire menu, click Start Sample.
Verify the Results
1. After the experiment has finished, open the sample from the data file in the Explore
window.
2. Right-click the TIC for the MRM experiment, and then click Extract Ions.
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3. In the Extract Ions dialog, select the 5 MRMs from Table 3-8, and then click OK.
Figure 3-28 shows typical data for the instrument.
Table 3-8 MRM Transitions for Beta-Galactosidase
Q1
Q3
ID
503.2
760.3
BG_YSQQQLMETSHR
542.3
636.4
BG_GDFQFNISR
671.3
755.5
BG_VDEDQPFPAVPK
714.9
884.5
BG_DWENPGVTQLNR
729.4
832.5
BG_APLDNDIGVSEATR
4. Record the peak areas of the specified MRM transitions in the section Test Results—
4000 QTRAP Instruments on page 117.
Note: Most XICs should have peak widths of no more 0.2 minute half
height. Some peaks will be narrower and some broader.
Figure 3-28 MRM XICs of 5 peptides from Beta-Galactosidase NanoLC Ultra
pre-column desalting run (10 fmol on column)
5. Record the retention times of the chosen peaks. This will vary with each system.
Time of elution of the first peak (approximately 14 to 16 minutes, as shown in
Figure 3-28) indicates dead volume of the system. Minimize dead volume where
possible.
6. Repeat the acquisition until you have consistent peak shape, retention time, and
peak intensity (a minimum of 3 times).
For new columns, this may require that you repeat the acquisition 10 or more times
in order to obtain consistent peak shape, retention time, and peak intensity. If
required, refer to Troubleshoot Peak Problems for more information.
7. Record the results for each acquisition.
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8. Record the average peak area of the acquisitions in the section, Test Results—4000
QTRAP Instruments on page 117.
9. Ensure that the average peak area meets the minimum requirement specified in the
section.
Troubleshoot Peak Problems
This section provides information for troubleshooting peak related problems such as broad or
tailing peak widths, lack of separation between peaks, and low peak area.
Peak widths are too broad or are tailing
•
Inspect all connections in the flow path to verify that there are no dead volumes.
•
Look at connections post-column and around trap column. A small increase in peak
width is often seen when a trap column is used.
Caution: Potential Instrument Damage: If using the Nanoflex system and problems
persist, do not attempt to troubleshoot the fittings connected to the chip.
No separation between the peaks
•
Ensure that both pumps are delivering the correct amount of solvent.
•
Ensure that the pressure spike upon injection is not too severe in the high-flow
channel (less than 300 psi change in pressure).
•
Large pressure change upon injection suggests an air bubble has been introduced to
the sample loop or is present in the trap column plumbing.
Note: The overall separation of the chromatography itself will often be less than direct
injection. Components that elute comparably on the trap and analytical column will not
re-resolve on the analytical column and, as a result, spread out or bunch together.
Peak intensity or peak area is too low
•
Verify the performance of the mass spectrometer and the ion source spray using the
infusion tests in the NanoSpray® Ion Source Operator Guide.
•
Verify that the trap and analytical column are well conditioned with protein digest
injections before performing this test. A trap typically requires 2 to 3 runs with
200 fmol of protein digest on the column for conditioning.
•
Verify that the correct amount of sample has been withdrawn from the autosampler
vial.
•
Perform a direct injection with a protein digest on the analytical column to determine
if the problem is related to the trap.
•
If the first LC peak does not elute for a long time, inspect the system for dead volume
before the trap.
•
If the early eluting peaks are not visible or are very low in intensity, this could mean
that trapping efficiency is low. Replace the trap.
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Tip! Minimize tubing length wherever possible and make sure all tubing for the
nanoflow path has an inner diameter of approximately 25 µm i.d.
•
If the late eluting peaks are not visible or are very low in intensity, this is usually a
sign that the column is getting old. In rare cases, it could mean that the BetaGalactosidase standard is degraded. See Figure 3-29 for an example of a scan with
an older column.
Figure 3-29 Extraction of all peaks—late eluting peaks not present
•
Always monitor the column and trap pressure over time; increasing pressure may
indicate increasing blockage, probably at the NanoSpray ion source tip. If, when the
connection between the column and the ion source head is unfastened and the
pressure changes quickly, then the tip is getting clogged and should be changed.
•
For better long-term column lifetime, verify that there is at least a 30% drop in
pressure observed during the high organic flush of the column. Increase the duration
of the high organic flush until a good pressure change is observed. This time might
increase for the trap column configuration relative to the direct injection
configuration.
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•
Figure 3-30 shows a minimal pressure change upon injection and a 30% pressure
decrease during the high organic flush.
Figure 3-30 Good pressure profile for a direct injection NanoLC Ultra
system run
Create a Backup of the EKSettings.reg File
The EKSettings.reg file can be used to re-establish the system settings derived on installation if
they are lost. Create a copy of the REG file upon completion of these tests.
1. In the Eksigent control software, on the System menu, click Instrument
Configuration.
2. Click Export Settings.
A backup of the REG file is created.
3. Navigate to the system settings folder (for example, C:\Program Files\Eksigent
NanoLC\settings).
4. Copy the previous_settings.reg file to another location, separate from the host
computer.
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Test Results—4000 QTRAP Instruments
For 4000 QTRAP instruments, complete this table with the results from five of the peptides from
the Beta-Galactosidase digest solution stock. Ensure that the peak area is below specification.
Most XICs should have peak widths of no more 0.2 minute half height. Some peaks will be
narrower and some broader. Additionally, verify that the peaks elute within 5 minutes of each
other.
Beta-Gal Lot Number: ___________________________________
Table 3-9 LC/MS Specification Test—4000 QTRAP Instruments
Q1
Q3
Dwell Peptide ID
CE
Spec.
(peak
area)
503.2
760.3
50
BG_YSQQQLMETSHR
27
2.0E+04
542.3
636.4
50
BG_GDFQFNISR
26
5.0E+05
671.3
755.5
50
BG_VDEDQPFPAVPK
33
5.0E+05
714.9
884.5
50
BG_DWENPGVTQLNR
32
7.0E+04
729.4
832.5
50
BG_APLDNDIGVSEATR
48
2.0E+05
Actual
Specification Passed?

Notes
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A
System Calibration
If the NanoLC system has been idle for two weeks or more, then calibrate the system using the
[Glu1]-Fibrinopeptide B, included in the LC/MS Peptide/Protein Mass Standards Kit
(P/N 4368624).
:
Note: Do not infuse the tuning solution and then the [Glu1]-Fibrinopeptide B solution.
Use Mobile Phase A to thoroughly flush the lines between running tests to avoid
clogging the NanoSpray® tips.
Prepare the [Glu1]-Fibrinopeptide B Dilution
Note: Prepare the solution just before calibrating the system.
1. Add 1200 µL of Standard Diluent (0.1% formic acid, 10% acetonitrile) to the glass
amber vial containing 0.1 mg [Glu1]-Fibrinopeptide B.
2. Cover the vial tightly, shake it, and then vortex it for at least 2 minutes, to make sure
that the peptide is fully dissolved.
This produces a stock solution with a concentration of approximately 50 pmol/µL.
Note: Peptide concentration may vary depending on the total peptide
content and peptide purity of the standard solution. See the Certificate of
Analysis provided by the vendor.
3. Aliquot 5 × 200 µL of the stock solution into clean tubes (included in the LC/MS
Peptide/Protein Mass Standards kit) to store for future use.
4. Put 50 µL of the stock solution into a clean tube (included in the LC/MS Peptide/
Protein Mass Standards kit), and then add 450 µL of Standard Diluent.
5. Vortex the tube for 30 seconds.
This is a 1:10 dilution, providing 500 µL of a 5 pmol/µL solution.
6. Put 50 µL of the 5 pmol/µL solution into another clean tube and add 450 µL of
Standard Diluent.
7. Vortex the tube for 30 seconds.
This is a 1:10 dilution, providing 500 µL of a 500 fmol/µL solution.
8. Aliquot 50 µL of the 500 fmol/µL solution into a clean 500 µL solution tube.
9. Add 450 µL of Standard Diluent.
10. Vortex the tube for 30 seconds.
This is the final 1:10 dilution, providing 500 µL of the final 50 fmol/µL solution, to be
used for the infusion test.
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11. Store the stock solution aliquots (50 pmol/µL solutions) and the diluted stock
solutions (500 fmol/µL solution) of [Glu1]-Fibrinopeptide B in the freezer at
–20°C.
Edit the Calibration Reference Table for
[Glu1]-Fibrinopeptide B
1. Select the Tune and Calibrate sidebar.
2. Click Tools >Settings >Tuning Options.
3. In the Calibration tab click the Reference button.
4. In the Reference Table Editor, select [Glu1]-Fibrinopeptide B in the Name list.
5. Add the masses in Figure A-1 to the Reference Ions for TOF MS Calibration table
(left side of window).
6. Click OK.
Figure A-1 [Glu1]-Fibrinopeptide B reference table
7. Click OK in the Tuning Options menu.
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Calibrate the TOF MS Scan Mode
1. In the MS Method window, specify the method parameters.
Table A-1 TOF MS Method Parameters
Parameter
Value
MS Parameters
Scan type
TOF MS
Accumulation time (sec)
1.0
Polarity
Positive
TOF masses (Da)
400 to 1800
Duration (min)
0.5
Advanced MS Parameters
MCA
Off
Auto Adjust with mass
On
Q1 Transmission Window
Default (with Auto-adjust)
Pulsar Frequency
Default (with Auto-adjust)
Time bins to sum
4
Settling time
Default
Pause between mass ranges
Default
Syringe Pump Method Parameters
Flow rate (µL/min)
0.5
Syringe Size (µL)
100 Gastight (1.46 mm)
Source/Gas Parameters
Ion Source Gas 1 (GS1)
2
Curtain Gas (CUR)
25
Interface Heater Temperature
(IHT) (°C)
150
IonSpray Voltage Floating
(ISVF)
2300
Compound-Dependent Parameters
Collision energy (CE) (V)
35
Compound Parameters
Declustering Potential
100
2. Ensure spray is stable and click Acquire and acquire at least 30 seconds of scan
data.
3. In the TIC of +TOF MS window (lower left) highlight 30 seconds of TIC signal to
average.
4. Double-click the highlighted area.
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5. In the window that appears (bottom), right-click and select Re-Calibrate TOF from
the menu that appears.
6. In the TOF Calibration window, select [Glu1]-Fibrinopeptide B on the Reference
Table list.
7. Ensure proper experimental masses have been identified from the infusion spectrum
and match up with the reference table theoretical masses.
8. Review the Average Error value displayed to the right of the Calculate New
Calibrations button.
9. Click Calculate New Calibrations and check the Average Error value has dropped
to <2 ppm.
10. In the Calibration Values area, click Calibrate Spectrum.
11. In the Save Current Calibration area, select the Set as Instrument Default and
Overwrite Current File check boxes.
12. Click Entire File to save new calibration for the TOF MS mode.
13. Click Close.
Calibrate the TOF MS/MS for High Sensitivity and
High Resolution Product Ion Modes
Complete this procedure first for high-sensitivity mode and then repeat it for high-resolution
mode.
1. In the MS Method window, create a method from the parameters in Table A-2 below.
Table A-2 TOF MS Method Parameters
Parameter
Value
MS Parameters
Scan type
Product Ion
Product of
785.8
Accumulation time (sec)
Polarity
TOF masses (Da)
1.0
Positive
100 to 1800
High sensitivity
On
Duration (min)
0.5
Advanced MS Parameters
MCA
Off
Auto Adjust with mass
On
Q1 Transmission Window
Default (with Auto-adjust)
Pulsar Frequency
Default (with Auto-adjust)
Time bins to sum
4
Settling time
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Table A-2 TOF MS Method Parameters (Continued)
Parameter
Pause between mass ranges
Value
Default
Source/Gas Parameters
Ion Source Gas 1 (GS1)
as optimized
Curtain Gas (CUR)
as optimized
Interface Heater Temperature
(IHT) (°C)
IonSpray Voltage Floating
(ISVF)
150
as optimized
Compound-Dependent Parameters
Collision energy (CE) (V)
45
Resolution Parameters
Q1 resolution
Unit
2. Ensure the spray is stable.
3. Click Acquire and acquire at least 30 seconds of scan data.
4. In the TIC of +TOF MS window (lower left), highlight 30 seconds of TIC signal to
average
5. Double-click the area you highlighted.
6. In the window that appears (at the bottom of the screen), right-click and choose ReCalibrate TOF from the menu that appears.
7. In the TOF Calibration window, select [Glu1]-fibrinopeptide B on the Reference
Table list.
8. Ensure proper experimental masses have been identified from the infusion spectrum
and match up with the reference table theoretical masses. Review the Average
Error value displayed to the right of the Calculate New Calibrations button.
9. Click Calculate New Calibrations.
10. Verify that the Average Error value has dropped to <2 ppm.
11. In the Calibration Values area, click Calibrate Spectrum.
12. In the Save Current Calibration area, select the Set as Instrument Default box
and Overwrite Current File box.
13. Click Entire File to save new calibration for the TOF MS/MS high-sensitivity product
ion mode.
14. Click Close.
15. Repeat this acquisition in high-resolution mode. Refer to the parameters in Table A-3
below for more information.
16. Ensure spray is stable and click Acquire and acquire at least 30 seconds of scan
data.
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17. In the TIC of +TOF MS window (lower left) highlight 30 seconds of TIC signal to
average and double-click Calibrate TOF using the [Glu1]-fibrinopeptide B reference
table described above.
Table A-3 TOF MS Method Parameters
Parameter
Value
MS Parameters
Scan type
Product Ion
Product of
785.8
Accumulation time (sec)
1.0
Polarity
Positive
TOF masses (Da)
100 to 1800
High resolution
On
Duration (min)
0.5
Advanced MS Parameters
MCA
Off
Auto Adjust with mass
On
Q1 Transmission Window
Default (with Auto-adjust)
Pulsar Frequency
Default (with Auto-adjust)
Time bins to sum
4
Settling time
Default
Pause between mass ranges
Default
Source/Gas Parameters
Ion Source Gas 1 (GS1)
as optimized
Curtain Gas (CUR)
as optimized
Interface Heater Temperature
(IHT) (°C)
IonSpray Voltage Floating
(ISVF)
150
as optimized
Compound-Dependent Parameters
Collision energy (CE) (V)
45
Resolution Parameters
Q1 resolution
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