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HOW MASS SPECTROMETRY CAN IMPROVE YOUR RESEARCH

HOW MASS SPECTROMETRY CAN IMPROVE YOUR RESEARCH. http://biosciences.exeter.ac.uk/facilities/spectrometry/. Hannah Florance H.V.Florance@exeter.ac.uk bs-mass-spec@exeter.ac.uk. The University of Exeter Science Strategy – Systems Biology. 13.30 Hannah Florance

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HOW MASS SPECTROMETRY CAN IMPROVE YOUR RESEARCH

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  1. HOW MASS SPECTROMETRY CAN IMPROVE YOUR RESEARCH http://biosciences.exeter.ac.uk/facilities/spectrometry/ Hannah Florance H.V.Florance@exeter.ac.uk bs-mass-spec@exeter.ac.uk The University of Exeter Science Strategy – Systems Biology

  2. 13.30 Hannah Florance “How Mass Spectrometry can improve your research - An overview of Biological Mass Spectrometry at Exeter” 13:50 Ashley Sage, Agilent Technologies "Improvements in Mass Spectrometry for Life Science Research - Does Agilent Have the Answer?“ 14:30 James Wakefield "Using Proteomics to Identify Microtubule Associated Proteins With Roles in Cell Division“ 14:45 George Taylor "Using LC-MS to Investigate Fatty Acid Oxidation in Cyanobacteria” 15:00 Nick Smirnoff “Current Examples of Research“ 15:30 Tea/Coffee in Geoffrey Pope Informal opportunity to discuss your research and how MS may help Tour of the facility 16:30 Finish

  3. What is Mass Spectrometry ? The determination of the mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion

  4. QQQ / Q-TOF Components of a Mass Spectrometer

  5. QQQ / Q-TOF Ions are formed by inducing a gain or loss of a charge Ions are directed into an analyser held at high vacuum by a series of electrostatic potentials Ions are separated by their m/z

  6. Analyte Introduction and Ionisation Electrospray Ionisation - ESI +ve ion mode = + H -ve ion mode = - H Analyser

  7. Mass Analyser: Quadrupole Time of Flight (Q-TOF) • Proteomics • Identification of purified proteins • Identifying protein from semi-complex and complex mixtures eg lysate • Intact protein analysis • PTM mapping • Metabolomics • Profiling • Comparative Quantitation

  8. Mass Analyser: Triple Quad (QQQ) • Proteomics & Metabolomics • PTM mapping • Targeted Identification • Comparative / Absolute Quantitation

  9. Data Interpretation - Mass Spectrum 523.2524 501.2693 [M+Na]+ [M+H]+ [13C M+Na]+ [13C M+H]+ Data courtesy of V.Perera

  10. Data Interpretation - MS/MS Extraction Tryptic Digest Untargeted MS/MS Tryptic Digest Protein Lysate Data courtesy of M. Grant

  11. Proteomics Exploratory Non-targetedAnalysis Identification Spectrum Mill Extract Data Molecular Feature Extraction (MFE) Sample Comparison Progenesis Extract Data Sample Comparison MAA Quantification Isotope Dilution Clustering MeV / GeneSpring [Identification] Targeted / Quantitative Analysis Metlin / PubChem Metabolomics

  12. Metabolomics • - Profiling • Sample Comparison • Alignment of extracted features (MAA) • Calculation of significant differences • Sample Clustering • Grouping of features across multiple samples (MeV / GeneSpring) • Global over-view of metabolic regulation MAA created and developed by Venura Perera, Grant Group, Biosciences Exp 2 Exp 4 Exp 5 Exp 6 Exp 7 Exp 8 Exp 9 Exp 10 Exp 3 Exp 1

  13. Metabolite Quantification Precursor CID Product • Metabolite • Extract 209 59, 151, 165 59 211 61, 151, 165 61 2H- labelled internal standards ●=2H2 ● ● Endogeneous JA Parent: 209; Product: 59 15.673 2H2-JA Standard Parent: 211; Product: 61 15.653 TIC: Total Ion Count Retention Time (mins) Data courtesy of N. Sultana

  14. Purified Protein; Immuno-precipitation; Pull-down assay; Whole cell lysate; Protein Identification Tryptic Digest Excise bands / spots from 1D or 2D gels Spectrum Mill Database Search Auto MS/MS Protein Solution Peptide Sequence Peptide Separation Targeted MS/MS Intact Protein Deconvolution Protein Mass Proteomics

  15. Protein Identification – Spectrum Mill • Customise databases • in silicodigests • Predict fragmentation of known peptides • de novo sequencing on unknown peptides • Clustal W alignments

  16. Protein Identification

  17. Protein Identification

  18. Protein Identification – Spectrum Mill y2 y9 y6 y4 y3 y1 y8 y7 y5 L A T S G A N F A R b2 b3 b5

  19. Sample Comparison - Progenesis Sample Alignment

  20. Sample Comparison - Progenesis Non-Labelled Quantification

  21. Current Methodologies • METABOLOMICS • Profiling sample analysis • Global over-view • Working on -Target identification, • Mapping back to pathways • System regulation • Targeted Analysis • Hormones • Flavonoids / Anthocyanins • Free Amino Acids • Sugars / Sugar Phosphates (on-going) • Acetyl CoA / Insecticides …………

  22. Current Methodologies • PROTEOMICS • Protein Identification • In-gel Digests • Complex Mixtures • Lysates (Soluble and Membrane Fractions) • Immuno-precipitations • Pull-down Assays • Working on • -Prefractionation to increase protein coverage, • Non-labelled Quantification

  23. METLIN Personal Point of Contact Hannah Florance H.V.Florance@exeter.ac.uk bs-mass-spec@exeter.ac.uk Geoffrey Pope Building Streatham Campus http://biosciences.exeter.ac.uk/facilities/spectrometry/ The University of Exeter Science Strategy – Systems Biology

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