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Infusion MS/MSALL Workflow; TripleTOF™ 5600 System for Lipid Analysis
Brigitte Simons,Sr. Applications and Sales Specialist at AB SCIEX
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
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
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
5 © 2011 AB SCIEX
Lipid Maps ConsortiumUncovering The Human Plasma Lipidome
Quehenberger et al, J Lipid Res. 2010 Nov;51(11):3299-305.
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.
7 © 2010 AB SCIEX
Lipid Analysis by Direct Infusion on the TripleTOF™ 5600 System
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
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)
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
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
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
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
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
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
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
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
18 © 2011 AB SCIEX
Lipid Analysis by Direct Infusion Using LipidView™ Software
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.
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
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
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
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
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
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
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
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