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 Intro: The University of Exeter Science Strategy – Systems Biology

“How Mass Spectrometry can improve your research 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

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

Components of a Mass Spectrometer QQQ / Q-TOF Analyser dictates what type of mass spec you have. This in turn dictates the workflow Components of a Mass Spectrometer

Ions are formed by inducing a gain or loss of a charge QQQ / Q-TOF Ions are formed by inducing a gain or loss of a charge Analyser dictates what type of mass spec you have. This in turn dictates the workflow Ions are directed into an analyser held at high vacuum by a series of electrostatic potentials Ions are separated by their m/z

Analyte Introduction and Ionisation Electrospray Ionisation - ESI +ve ion mode = + H -ve ion mode = - H Need to get sample from the solution phase into the gas phase. Addition / subtraction of protons to basic / acidic amino acids denotes the charge. Dictated by pH of the solution Analyser

Quadrupole Time of Flight 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 Main uses: Trying to look at everything you have in the sample.

Mass Analyser: Triple Quad (QQQ) Proteomics & Metabolomics PTM mapping Targeted Identification Comparative / Absolute Quantitation For efficient use, need know what to look for.

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

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

Exploratory Non-targeted Analysis Proteomics 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

- Profiling Sample Comparison Sample Clustering 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 Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6 Exp 7 Exp 8 Exp 9 Exp 10 MAA created and developed by Venura Perera, Grant Group, Biosciences

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

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

Protein Identification – Spectrum Mill Customise databases in silico digests Predict fragmentation of known peptides de novo sequencing on unknown peptides Clustal W alignments

Protein Identification Same work path can be applied to protein analysis. In this instance the data is put into the search engine Spectrum Mill etc

Protein Identification

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

Sample Comparison - Progenesis Sample Alignment Not use for metabolites – Doesn’t deconvolute. Get too many features

Sample Comparison - Progenesis Non-Labelled Quantification

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 …………

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

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