Discovery Metabolomics - Quantitative Profiling of the Metabolome using TripleTOF Technology

Transcription

1 ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. Discovery Metabolomics - Quantitative Profiling of the Metabolome using TripleTOF Technology Baljit Ubhi Ph.D ASMS Baltimore, June 2014

2 What is Metabolomics? Also sometimes referred to as metabonomics or metabolic profiling Measurement of low molecular weight metabolites (molecular weight 2000 Daltons) in biological fluids or tissue extracts. Univariate and multivariate statistical data analyses Functional end-point of physiology and pathophysiology Metabolomics is an attractive tool as it reveals information closest to the phenotypic level Direct mirror of environmental influences Nutrition Disease monitoring Exercise Drug Metabolism RNA Transcriptomics DNA Genomics METABOLITE Metabolomics PROTEIN Proteomics AB SCIEX

3 Importance of Metabolomics Drug Discovery Metabolomics not only plays a role in detection and diagnosis of disease and also as both a predictive and pharmacodynamic marker of drug efficacy. Toxicology and Safety Assessment Metabolic profiling can identify physiological transformation due to toxic abuse or exposure Basic Research and Biomarker Discovery Metabolites are key analytes for effecting phenotype changes resulting from genetic manipulations or system perturbations Nutrigenomics Connects the omics concepts of proteomics, genomics, metabolomics and transcriptomics to human nutrition Other applications include the food industry, agrochemical industry, environmental toxicology and biofuels AB SCIEX

4 Workflow Strategies for Metabolomics Targeted vs. Non-Targeted Analysis Non-Targeted Analysis MS MS/MS Targeted Analysis MRM MS/MS Advantages: Detects Any Species Disadvantages: Lower Dynamic Range and Sensitivity ID Step Required Sophisticated Informatics Advantages: High Sensitivity, Throughput and Dynamic Range High Quant Quality Simplified Informatics Disadvantages: Detects Specific Species Upfront Method Development AB SCIEX

5 AB SCIEX TripleTOF System Sensitivity In MS and MS/MS modes Speed 10 ms accumulation times 100 MS/MS scans per second in IDA Resolution High resolution ~30,000 High sensitivity ~15,000 Mass Accuracy 1-2 ppm RMS (external calibration) Dynamic Range ~ 3-4 orders AB SCIEX

6 AB SCIEX TripleTOF System Ion compression optics Accelerator TOF Analyzer QJet Ion Guide Q0 High Pressure Cell High Frequency Q1 40 GHz Multichannel TDC Detector 30kHz Accelerator LINAC collision cell 15 kv Acceleration voltage Front end based on the AB SCIEX Triple Quad 4500 and 5500 system Two-stage reflectron Industry-Leading Ion Sources AB SCIEX

7 Metabolomics Strategies TripleTOF System Single injection acquisition MS and MS/MS data Non-targeted data acquisition Generic MS setup Data Dependent Acquisition Targeted data acquisition Target specific species MRM-like Scheduled MRM HR Workflow Targeted processing Hypothesis Driven Biochemical Pathway Approach Non-targeted processing Non Targeted Global Metabolite Profile Targeted processing AB SCIEX

8 Metabolomics Strategies TripleTOF System Single injection acquisition MS and MS/MS data Non-targeted data acquisition Generic MS setup Data Dependent Acquisition Targeted data acquisition Target specific species MRM-like Scheduled MRM HR Workflow Targeted processing Hypothesis Driven Biochemical Pathway Approach Non-targeted processing Non Targeted Global Metabolite Profile Targeted processing AB SCIEX

9 ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. GLOBAL METABOLOMICS Non-Targeted Data Acquisition Non-targeted Data Processing

10 Untargeted Metabolomics Workflow LipidView Software Mulitvariate Data Analysis Identify and confirm the metabolites/lipids MS and MS/MS Find differential features Simplified statistics FormulaFinder predicts elemental composition And assigns MS/MS fragmentation MasterView Software allows automated link to ChemSpider for structural information AB SCIEX

11 High Data Acquisition Speed Single Injection Workflow Total cycle time < 0.9 s Survey Scan (TOF MS) IDA Criteria Intensity (cps) ** * * m/z TOF MS scan Selection Criteria (Intensity, DBS) Identify potential metabolites based on accurate mass Extracted Ion Chromatogram (XIC) Quantitative CH3 H Dependent Scan (TOF MS/MS) Top 15 precursors selected for MS/MS Intensity (cps) O H NH O N H H O O CH3 CH3 CH3 CH3 m/z TOF MS/MS scan Confirm metabolites based on spectral match or common NL or PI Qualitative AB SCIEX

12 MarkerView Software Data Visualization Software Metabolomic and protein profiling workflows Peak extraction, alignment and filtered data processing Multivariate data analysis (PCA/PCA-DA scores and loadings plots) Univariate data analysis Principal Component Variable Grouping (PCVG) Profile plots of differential ions including RT information Link to raw data in the same software Post-analysis reports allow you to easily track your work and record potential biomarkers AB SCIEX

13 Principal Component Analysis (PCA-DA) Scores Plot Find the Differences Zucker rat model to study effects of obesity, diabetes and cardiovascular diseases Three Phenotypes Lean lacks mutation to induce diabetic state and obesity Fatty Diabetic and shows mild weight gain on normal diet Obese Mutation creating early and substantial weight gain and obesity when on High Fat diet Tight clustering of QC samples highlights data reproducibility (remove before final processing) Lean Pooled QCs Obese Fatty AB SCIEX

14 PCA-DA - Loadings Plot Variables Responsible for Differences in Scores m/z / retention time pairs are the variables from the TOF MS data Higher concentration in Obese Higher concentration In Fatty AB SCIEX

15 Principle Component Variable Grouping (PCVG) Find Related Variables by Degree of Difference Only removing discrete common variables Removing a larger proportion of common variables leaving the ions with largest difference AB SCIEX

16 MasterView Software for Comparative Screening Sample Control AB SCIEX Compare a Sample vs Control

17 Confident Identification of Features of Interest AB SCIEX

18 MasterView Software Review Formula Finder Results Click Formula for automatic link to Chemspider Sample Sample MS MSMS Control MS Control MSMS AB SCIEX

19 MasterView Software Review ChemSpider Results AB SCIEX

20 IDA Explorer 7435 MSMS Spectra AB SCIEX

21 FormulaFinder Tool in PeakView Software Predict Possible Formula Rank #4 based on MS XIC of LPC 18:1 Isotope Pattern of LPC 18:1 Isotopic Pattern AB SCIEX

22 PeakView Software - Confirmation of Potential Structure Differential Feature m/z Rank #1 based on MS/MS IDA Explorer MSMS of LPC 18:1 Structure from ChemSpider AB SCIEX

23 ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. GLOBAL LIPIDOMICS Non-Targeted Data Acquisition Non-targeted Data Processing

24 Approaches to Statistical Data Analysis Two analysis paths from the same dataset Acquired Dataset Process in LipidView Software Process directly in MarkerView Software Import into MarkerView Software with lipid IDs Import Interest List into MasterView Software to identify Global Lipidomics Approach Global Metabolomics Approach AB SCIEX

25 LipidView Software 58 lipid classes, ~28000 lipid species represented in a lipid fragments database >600 characteristic lipid fragments lists Lipid catalogue, lipid calculator utilities Identification, qualitative, relative quantitation and targeted analysis of lipids Support of lipid bioinformatics in medium sample throughput <96 samples in a set processed Seamless link to MarkerView Software Capabilities to process AB SCIEX TripleTOF system, AB SCIEX Triple Quad system, QTRAP system and QSTAR system data AB SCIEX

26 LipidView Software Lipid Class Profile (All) AB SCIEX

27 LipidView Software Lipid Class Profile (LPC and PC) AB SCIEX

28 LipidView Software Lipid Class Profile (Acylcarnitines) AB SCIEX

29 LipidView Software Lipid Class Profile (Ceramides) AB SCIEX

30 LipidView Software Results Table Identify and Quantify a Large Number of Lipids Lipid ID s AB SCIEX Export Results to MarkerView Software for Statistical Data Analysis

31 PCA Analysis in MarkerView Software Find Differences between Lipid Species Scores plot Each m/z / RT pair now has a lipid ID as opposed to a m/z. Loadings plot AB SCIEX

32 Other Statistical Tools T-tests and Volcano Plots AB SCIEX Highly significant ions based on p-values from t tests

33 Metabolite Visualisation Genedata Expressionist AB SCIEX

34 Correlation Network - Genedata Expressionist AB SCIEX

35 LC-MS Solutions for Discovery Metabolomics TripleTOF System has faster acquisition with no loss of sensitivity or resolution: o o o High resolution MS for accurate mass MS-based identification High speed, high resolution MS/MS for definitive confirmation of metabolites acquired simultaneously, without a need to resample Single injection workflow for higher productivity MarkerView Software MS feature extraction and alignment across large sample sets for multivariate data analysis. MasterView Software for known and unknown metabolite features and comparative profiling of samples o o Powerful tools to ID confirmation, FormulaFinder, Structural Interpretation tool and links to ChemSpider searches Confirm identity by searching MS/MS against metabolite libraries such as MassBank AB SCIEX

36 Acknowledgments Jeff Patrick David Alonso Lorne Fell Joe Binkley Christina Nieh Christie Hunter Fadi Abdi Aaron Hudson David Cox Lyle Burton Jean Baptiste Vincendet Brigitte Simons Jeremiah Tipton Joe Shambaugh Anthony Taylor Tamas Rujan Jens Heofkens Alessio Ceroni Peter Haberl AB SCIEX

37 Trademarks/Licensing For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. All rights reserved. Information subject to change without notice AB SCIEX AB SCIEX

38 ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. Questions and Answers