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

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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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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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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

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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

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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