Metabolomics: From Samples to Softwares

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1 Metabolomics: From Samples to Softwares Sumit Shah, B.Pharm., Ph.D. Metabolomics Applications Scientist, Agilent Technologies

2 What is Metabolomics? Metabolomics is the comparative analysis of the chemical fingerprints (metabolite profiles) generated in a biological system Metabolites are the small molecule end-products of all the biological/cellular processes occurring in a biological system (cell/tissue/organ/organism) Four major bio-molecule classes: sugars, lipids, amino acids, nucleic acids Human Metabolome Database*: 2180 metabolites (v1.0) vs metabolites (v3.5) Small molecule profiling as applied to Life Sciences Applications! * 2

3 Bio/Pharma Life Sciences Clinical/Diagnostics Environment Forensics/Toxicology Petrochemicals Agriculture Nutrition Consumer 3

4 Agilent s Commitment to Metabolomics LC-MS GC-MS ICP-MS Capillary Electrophoresis NMR 4

5 Challenges of Metabolomics Total Work Flow Sample Preparation Plan and design the experiment Collect and prepare many samples Acquire Data Analyze samples Use GC/MS or LC/MS or NMR to analyze samples Analyze Data Mine data for compounds Analyze data using multivariate software Identify Compounds Identify compounds using EI library matching (GC/MS) or database or MS/MS Library matching (LC/MS) Pathway Analysis Choose pathway database and species Analyze differential data and visualize the results on pathways 5

6 What is the Biological Objective? Experimental Design Metabolomics or Multi-omics (genomics and/or proteomics) Global profiling or Targeted approach Snap-shot view or flux/dynamic analysis What are the Biological Samples? Nature or origin (human/animal/plant/microbe..) Type (bio-fluid/tissue/cell..) Sample limited? Statistics? Pooled sample for initial profiling or Replicates (if so, how many) Biological and/or Technical replicates Normalization with internal standard/s 6

7 Global vs. Targeted Metabolomics Global Profiling Profiling of all metabolites: Discovery based approach Naïve data mining with few assumptions Track metabolites using mass and retention time Finds unexpected compounds Metabolites need to be identified and confirmed after analysis Pathway analysis and Biological interpretation Targeted Profiling Targeted profiling either based on biological or chemical relevance of the metabolites Targeted data mining uses metabolite database Track metabolites using metabolite name More selective and sensitive than untargeted analysis Metabolites need to be confirmed after analysis Pathway analysis and Biological interpretation One analysis does not preclude doing the other 7

8 Sample Handling and Processing Sample Collection Technical expertise, protocol standardization, quenching Sample Containers Glass or plastic Sample Storage Temperature, length, metabolite stability Reagents and Labware Sample Prep Dilute-n-Shoot or Ultra-filtration (molecular weight cut-offs) Protein crash (solvent, ph, temperature) Cell/Tissue lysis LLE fractionation or SPE-based enrichment 8

9 References for Metabolomics Sample Prep 9

10 Chromatography Metabolome/Small Molecule Extract Nucleo(s/t)ides Sugars Amino Acids Lipids Polars or Hydrophilics Reverse Phase (with Ion Pairing) Mixed Mode Aqueous Normal Phase HILIC Hydrophobics Reverse Phase Normal Phase 10

11 Impact of Chromatography: Human Urine Metabolomics C18 +ESI Mixed Mode +ESI HILIC +ESI 11

12 Agilent 1290 Infinity UHPLC Binary Pump Next Generation Technology ml/min ml/min Low Dead volume: 10 µl Low Mixing Noise High RT precision Automatic purge Automatic correction of micro leaks 12

13 1260 Infinity Bio-Inert Quaternary LC System Metal cladded PEEK capillary True Bio-Inert Solution for Small as well as Large Molecule Analysis Complete metal-free sample contacting surfaces throughout the system Mobile phase wetted parts are iron and steel free in solvent delivery unit ml/min High salt tolerance (2M) and wide ph range (1-13, short tem 14) Quaternary pump with inert solvent and column selection valves Novel capillary and connection design 13

14 High Resolution Accurate Mass 6550 Q-TOF Comprehensive Performance Enhancements Mass Resolution >40, spectra /sec MS and 33 spectra/sec MS/MS 5 orders of linear dynamic range Mass Accuracy <1 ppm MS; <2 ppm MS/MS Unrivalled sensitivity New- Ion Beam Compression and Excellent isotopic fidelity Shaping (resolution + sensitivity) Dual Agilent JetStream orthogonal spray (robust and stable mass calibration) Longer Invar flight tube (resolution + stable mass accuracy) New embedded processor (50 spectra/sec) New- ifunnel (10X sensitivity gain) 4 GHz digitizer + ADC detector (resolution, mass accuracy, dynamic range) Hexapole axial focusing collision cell (faster MS/MS spectra) 14

15 Ultra High Definition Accurate Mass ifunnel 6550 Q-TOF Agilent Jet Stream Thermal confinement of ESI plume Efficient desolvation to create gas phase ions Creates an ion rich zone Hexabore Capillary 6 capillary inlets Samples 6 times more ion rich gas from the source Captures the majority of the gas from the source region Dual Ion Funnel Removes the gas but captures the ions Removes neutral noise Extends turbo pump life 15

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17 Agilent QQQ and QTOF LC/MS/MS shared designs assist in moving work from untargeted profiling to targeted quantitative metabolomics: 6400 Series QQQ Octopole 1 Quad Mass Filter (Q1) Quad Mass Filter (Q3) Lens 1 and 2 Collision Cell 10KV Detector Rough Pump Turbo 1 Turbo 1 Turbo Series Q-TOF Quad Mass Filter (Q1) Octopole 1 Octopole 2 Lens 1 and 2 Collision Cell DC Quad Ion Pulser Rough Pump Turbo 1 Turbo 1 Turbo 1 Turbo 2 17

18 LC-MS based Metabolomic Profiling Separate & Detect Feature Finding Alignment & Statistics Identify Pathways Global LC-TOF/QTOF MassHunter Qual Molecular Feature Extraction Mass Profiler Professional ID Browser Pathway Architect Targeted LC-TOF/QTOF LC-QQQ** MassHunter Qual Find by Formula Mass Profiler Professional Pathway Architect 18

19 Agilent Software Solutions for Metabolomics Data acquisition software MassHunter Data processing software MassHunter Qual: Feature/Compound finding algorithms Molecular Feature Extractor (MFE) Find by Formula (FbF) Find by Auto MS/MS or Targeted MS/MS Compound Identification using Database/Library search DA Reprocessor: Batch Analysis for High Throughput workflows 19

20 Data Mining Process Monoisotopic mass (varies in ppm) Isotope distribution (varies in %) Isotope spacing (varies in ppm) 20

21 Agilent Software Solutions for Metabolomics Data analysis software Mass Profiler Professional (MPP): Uni-variate and multi-variate statistics IDBrowser for compound identification using Database searches Sample Class Prediction Modeling PathwayArchitect for biological pathway analysis Multi-omics Data Overlay and Interpretation Mass Profiler (MP): Uni-variate statistics, for profiling two experimental groups IDBrowser for compound identification using Database searches 21

22 Agilent Software Solutions for Metabolomics Data analysis software Personal Compound Database and Library Manager (PCDL): Add/Review/Edit compounds to database Add retention time and/or MS/MS spectral data to create MS/MS library Pathways to PCDL: Convert pathway metabolite information into Agilent personal compound database Database source - Wikipathways, BioCyc and KEGG Select one to many pathways Removes redundant metabolites Adds compound information Formula, CAS ID, IUPAC Name, Structure 22

23 Agilent Software Solutions for Metabolomics Data analysis software Molecular Structure Correlator (MSC): Aid in the structural elucidation of true unknown compounds, which cannot be found in accurate mass databases or MS/MS libraries Calculates Molecular Formulas from accurate mass precursor, fragment ions and neutral losses from an unknown MS/MS spectrum Match experimental fragments in the MS/MS spectra into proposed structure via a novel systematic bond breaking approach Calculates overall correlation score from individual scores and displays possible substructures overlaid over the parent structure Retrieves hundreds of structures for a molecular formula from web or PCDL MassHunter Quant: Quantitation curves for analytes profiled in LC-MS mode on Q-TOF 23

24 SimLipid** Lipidomics Software Solution Comprehensive lipidome MS and MS/MS database/analysis Lipids with MS level information Theoretical MS/MS fragmentation information/matching SimLipid 3.4 supports four other MS vendors data but Agilent data is the only one that: Allows import/searching of compounds (MassHunter Qual cef files) rather than raw spectra Does MS/MS pattern matching of multiple adducts (e.g. ammonium, sodium) Can annotate the cef files with the identified lipids for further processing Has a complete workflow with MassHunter Qual, MPP, and Pathway Architect for biological contextualization 24

25 Agilent Software Solutions for Multi-Omics Genomics Experiment Scaffold Pathway Architect MPP Metlin PCDL Molecular Structure Correlator Spectrum Mill Proteomics Experiment GeneSpring Metabolomics Experiment SimLipid Skyline Pathways to PCDL Literature and other sources 25

26 Books for Metabolomics and Statistics 26