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Transcript of Infusion MS/MS ALL Workflow; TripleTOF™ 5600 System for Lipid Analysis Brigitte Simons, Sr....
![Page 1: Infusion MS/MS ALL Workflow; TripleTOF™ 5600 System for Lipid Analysis Brigitte Simons, Sr. Applications and Sales Specialist at AB SCIEX.](https://reader031.fdocuments.net/reader031/viewer/2022020102/56649d1c5503460f949f1a3d/html5/thumbnails/1.jpg)
Infusion MS/MSALL Workflow; TripleTOF™ 5600 System for Lipid Analysis
Brigitte Simons,Sr. Applications and Sales Specialist at AB SCIEX
![Page 2: Infusion MS/MS ALL Workflow; TripleTOF™ 5600 System for Lipid Analysis Brigitte Simons, Sr. Applications and Sales Specialist at AB SCIEX.](https://reader031.fdocuments.net/reader031/viewer/2022020102/56649d1c5503460f949f1a3d/html5/thumbnails/2.jpg)
2 © 2011 AB SCIEX
Infusion MS/MSALL Workflow On 5600Basic Methodology
1. Lipid species confirmation is best carried out by high resolution MS/MS acquisition – MS is not sufficient as precursor ion information alone is compromised by high matrix interferences and isotopic overlap of many lipid species (across many classes) in a small mass range.
2. MS/MS triggered by IDA vs MS/MS collected without MS dependencea. MS/MS collected without a dependence on MS survey information provides a an
easy acquisition technique that can be applied generically to any experiment of desired polarity, ionization technique, or mass range.
b. Every precursor ion is captured by MS/MS and nothing is missed - CE is applied throughout a sweep to accumulate optimum ion current through many product ions.
c. MS/MS spectra can result in very rich signal intensities even if the precursor ion has a very low signal intensity
d. These files sizes are very small yet contain a very high # of MS/MS spectra per cycle
3. Quantification is carried out by complementary product ion information from the MS/MS spectrum and IS correction can be applied
4. This technique is supported by LipidView™ Software v1.1 for automated identification and quantitation of lipids using comprehensive library searching and MarkerView™ Software for PCA analysis and other statistical outputs
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3 © 2010 AB SCIEX
EASY BUTTON
Infusion MS/MSALL WorkflowWhat’s the Benefit From this Feature?
− No method development and highly reproducible technique
− Can be applied to acquisitions of any ionization mode (ESI, APCI, Positive, Negative, etc)
− Everything is sampled by high resolution, you don’t miss anything!
− Can be carried out in < 3 min
− Is applicable to direct infusion, flow injection and directed lipid class LC techniques
− An MS technique that is scalable and represents ease of high throughput
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4 © 2010 AB SCIEX
Tested and Published Quantitative TechniqueWhere to find content for customers
− AB SCIEX Technical Note– https://na10.salesforce.com/sfc/#version?selectedDocumentId=069F0000000N72q
− Publication in Metabolites 2012, 2(1), 195-213; doi:10.3390/metabo2010195– http://www.mdpi.com/2218-1989/2/1/195/
− ASMS 2012 Posters:
Lipidomics Analysis of a Subset of Human Serum Samples from the Dallas Heart Study Jeff Mcdonald1; Brigitte Simons2; Stefan Thibodeaux3; Jennifer Krone2; Phillip Sanders3
1UT Southwestern Medical Center, Dallas, TX; 2AB SCIEX, Concord, ON; 3Eli Lilly, Indianapolis, IN
Information Independent MS/MS Data Collection of All Precursors Using Time-of-Flight Mass Spectrometry. Brigitte Simons; Eva Duchoslav ; Lyle Burton; Tanya Gamble; Ron Bonner
AB SCIEX, Concord, CANADA
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5 © 2011 AB SCIEX
Lipid Maps ConsortiumUncovering The Human Plasma Lipidome
Quehenberger et al, J Lipid Res. 2010 Nov;51(11):3299-305.
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6 © 2011 AB SCIEX
Lipid Maps ConsortiumUncovering The Human Plasma Lipidome
− Complete quantitative analysis of over 580 lipids using MRM and PIS/NL on 4000 QTRAP® Systems
− Synthetic lipid internal standards available from Avanti Polar Lipids, developed on QTRAP® 5500 Systems
− Complete list of MRM methods available in Suppl. Methods for each of 8 lipid classes
− Lead to the reviews on the future of MS techniques for lipidomics− Ion mobility MS− Time-of-flight MS for quant/qual− Tissue imaging
Applications of mass spectrometry to lipids and membranes.Harkewicz R, Dennis EA.Annu Rev Biochem. 2011 Jun 7;80:301-25.
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7 © 2010 AB SCIEX
Lipid Analysis by Direct Infusion on the TripleTOF™ 5600 System
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8 © 2011 AB SCIEX
Q1 Q2
Fast Q1 precursor selection step-wise through mass range
CID Fragmentation Direct infusion, flow injection, and lipid-class targeted LC techniques
MS/MSALL Information Independent Data CollectionProduct Ions from Every Precursor
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9 © 2011 AB SCIEX
Time (one cycle)
Pre
curs
or
mas
s m
/z
MS/MSALL Product Ions from Every Precursor In Order
− How to calculate cycle time?– Mass range– Step size to cover
mass range– TOF MS/MS
accumulation time (~50 – 100 ms)
– Looping in a TOF MS scan (~ 200 ms)
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10 © 2011 AB SCIEX
Acquisition Set-upMS/MSALL
− Install MS/MSall mode Script from this location:− C:\\program files\Analyst\scripts
− Restart Analyst Service
− Turn on MS/Msall mode from Scripts Menu in Analyst TF− Restart Analyst Service
− Important: using the .poap txt file
− The ordered acquisition of MS/MS is carried out by inclusion list triggered IDA whereby the MS/MS is exclusively dictated by ordered masses in the inclusion list
User creates this inclusion list and names the file to exactly the .dam file name.txt
─ Inclusion list is created by excel and contains 3 columns: m/z, # of MS scans, # of cycles
─ It provides the appropriate mass defect as you increase m/z
─ It can be modified to provide a larger step or cover a different mass range
─ IMPORTANT: .poap file must have the same name as the acquisition method (.dam) file name and be saved to the Acquisition Methods folder in the Analyst project
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11 © 2010 AB SCIEX
− A generic acquisition technique using Information Independent Logic and collecting 1000 MS/MS/cycle
− Inclusion-based list depicting a mass at every 1 Da step
− User indicates how frequent to acquire MS scan and how many cycles to complete.
− CE is always a sweep; 50 ± 30 eV to capture all fragments
User creates this inclusion list and names the file to exactly the .dam file name.txt
Acquisition ParametersMS/MSALL
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12 © 2010 AB SCIEX
MS/MS of Every Precursor Ordered in Time Looking at the Raw Data in PeakView Software
High Resolution MS/MS of every mass
Total Ion Chromatogram
− Open Analyst TF raw data file displaying the TIC
− SHOW>LC-MS Contour Plot
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13 © 2010 AB SCIEX
MS/MS of Every Precursor Ordered in Time Mining the Raw Data in PeakView Software
− PROCESS < Fragment and Neutral Loss Filter
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14 © 2011 AB SCIEX
MS/MSALL Fragment and Neutral Loss Filter in PeakView Software
TAG 52:2 + NH4
TAG 50:2 + NH4
TAG 50:1 + NH4
TAG 54:3 + NH4
neutral loss filter = 245.20
neutral loss filter = 273.20
neutral loss filter = 299.20
neutral loss filter = 325.20
− Open MS/MS all data file in TIC mode
− PROCESS> Fragment and Neutral Loss Filter
− Precursor ion, fragment ion, and neutral loss filters
− Set threshold and mass tolerance.
TAG Profiling
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15 © 2011 AB SCIEX
Precursors of 184.0733 m/z
Precursors of 264.235 m/z
Precursors of neutral loss 141.0 m/z
MS/MSALL Fragment and Neutral Loss Filter
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16 © 2010 AB SCIEX
PeakView’s IDA Explore ModeNegative Mode TOF MS and MS/MS
Rat brain extracts (# 2) ~ 0.4 ng/mL
Resolution 28 793
Resolution 29 932
29 360 resolution
Ordered MS/MS view High Resolution MS/MS of 846.7 m/z
High Resolution TOF MS
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17 © 2011 AB SCIEX
Formula FinderAccurate Mass Tools in PeakView™ Software
IDA explore
- product ion of 936.6 m/z
FA 22:6
- XIC of 936.6 m/z
Formula Finder Results from MS/MS spectrum
PC 44:12 + AcO-
1
2
3
4
MH- -CH3
Flow of Lipid Identification from LC-MS/MS WorkflowUse of Lipid Catalogue and Calculators Ultilities for Fragment Interpretation
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18 © 2011 AB SCIEX
Lipid Analysis by Direct Infusion Using LipidView™ Software
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19 © 2010 AB SCIEX
Software Solutions for Automated Lipid AnalysisEnabling the Identification and Quantitation of Lipid MS Data
Software Highlights:– More than 50 lipid classes containing 25,000+
lipid species represented in a lipid fragments database.
– More than 600 characteristic lipid fragments lists are included.
– Lipid catalogue and lipid calculator utilities are included.
– Support of lipid bioinformatics in medium sample throughput with a seamless link to multivariate analysis tools in MarkerView™ software.
– Building of target screening methods from identification results, with special focus on accurate mass data.
– Semi-quantitative profiling with flexible use of internal standards.
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20 © 2011 AB SCIEX
Lipid Database for Accurate Mass
− Collection of building blocks of lipid molecules and their arrangements within lipid classes and lipid categories.
− Information on lipid collision-induced dissociation (CID) fragmentation.
− Isotope correction factor.
− Headgroup (HGS), neutral loss, and long change base (LCB) specific scan types.
− Fatty acids (FA).
− Detailed fragmentation information for each lipid species in both polarities.
− Common adduct forms.
Lipid Catalogue Utility
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21 © 2011 AB SCIEX
Lipid Database for Accurate Mass
− Prediction of lipid molecules based on the experimental m/z in either positive or negative polarity within lipid classes and lipid categories.
− Information on formula composition and lipid class.
− Mass accuracy and common species can be used to refine the search result.
− Fatty acids (FA) and double bonds.
− Common adduct forms.
Lipid Calculator Utility
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22 © 2011 AB SCIEX
MS/MSALL Automated Lipid Identification and Response Correction in LipidView™ Software
− Automated lipid identification and relative quantification
− Lipid class profiles (semi-quantitative) to measure ratios between samples
brain liver
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23 © 2011 AB SCIEX
− Mean and % CV measurements across samples instantly calculated for each lipid
− Excellent reproducibility is seen for lipid-class specific internal standards
MS/MSALL Product Ions from Every Precursor In Order
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24 © 2011 AB SCIEX
Table 1. A summary of the number of confirmed and common unique molecular lipid species identified by LipidView™ Software across the lipid extracts using MS/MSALL workflow.Lipid Extract Acquisition Mode # of
Glycerophospholipids identifications
# of Glycerol lipid identifications
# of Sphingolipid identifications
# of Sterol Lipid identifications
LiverNegative mode MS/MSALL 800 3 0* 0*
Positive mode MS/MSALL 152 125 40 12
BrainNegative mode MS/MSALL 1147 0* 0* 0*
Positive mode MS/MSALL 239 112 119 3
TotalsLiver (total = 1132)Brain (total = 1620)
9521386
128112
40119
123
n=5 replicates analyzed per extract; only confirmed and common lipid species reported0* = not included in search
MS/MSALL Automated Lipid Identification and Response Correction in LipidView™ Software
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25 © 2011 AB SCIEX
Linking Lipid Data to Statistical AnalysisReporting and Exporting Lipid Profiling Data
Through this function, you can export all data to MarkerView Software, as peak intensities or areas
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26 © 2011 AB SCIEX
Principal Component AnalysisMultivariate Analysis Tools in MarkerView™ Software
Scores Plot Loadings Plot
− Scores plot provides a visual distribution of the features and degree of discrimination between the sample groups
− Loadings plot shows the variables (lipids) that lead to sample clustering
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27 © 2011 AB SCIEX
Principal Component AnalysisMultivariate Analysis Tools in MarkerView™ Software
Differential Profiling t-test Log Plot
− Extraction of lipids from the data that show significant variation can be profiled across the samples
− Log-fold change is visualized in t-test of liver lipids plotted against brain