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My contact details and information about submitting samples for MS

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1 My contact details and information about submitting samples for MS


3 Proteins are chains of amino acids each of which have slightly different masses The protein chain can be cut selectively by sequence specific proteases at particular amino acids Trypsin cuts after lysine or arginine Protein digestion

4 The peptides produced have distinct weights These are accurately measured by mass spectrometry A list of these weights is like a fingerprint (a PEPTIDE MASS FINGERPRINT) This is unique to the protein and can be used to identify it = = = 89.3

5 Laser energy Peptide ions enter the Time of Flight tube separated on basis of mass Peptides co- crystallised with matrix Ionise peptides Detector mass/charge of every peptide peptide mass fingerprint MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation)

6 peptide mass spectrum a trypsin digest of a single protein every peak corresponds to the mass (m/z) of a peptide ion m / z = mass / charge relative intensity

7 Peptide mass spectrum Data converted to text List of peptide masses a peak list (pkl) = fingerprint

8 run digested protein on MS Database searches with PeptideMassFingerprint data sequence databases theoretical trypsin digest of every predicted protein list of calculated peptide masses compare identification made if match is found list of measured peptide masses fingerprint

9 peptide mass fingerprinting rapid high throughput large scale identification of proteins from organisms whose genomes are completely sequenced good tool for a first look at a sample BUT……. peptide mass fingerprinting will not always give an identification genome is not completely sequenced the full length protein sequence is not in the database modifications are present more than one protein is present in the sample alternative method of analysis - tandem MS

10 Samples in solution Compatible with HPLC Complex protein mixtures Determine peptide masses Peptide fragmentation Peptide sequence ElectroSpray Ionisation (ESI) Mass Spectrometry on the Q-ToF2

11 MS2MS1 MS on the Q-ToF2

12 Tandem MS - peptide fragmentation series of peptide fragments each fragment is one amino acid longer than the next the series of fragments corresponds to the sequence of the peptide low energy collision fragments the peptide cleavage usually occurs at the amide bond i.e. between residues

13 peptide fragmentation the series of fragments corresponds to the sequence of the peptide

14 De novo sequencing Sequence reads in N to C direction - PSGASTGVHEAMR

15 Survey Mass Spectrum (MS) - intact peptides detected in a 1 second survey scan MSMS On-line LC-MSMS on the Q-ToF2 : peptides from a single protein Fragment Mass spectrum (MSMS) fragments from one peptide MSMS Many peptides are fragmented during a 60 minute run LC-MSMS generates much more data than fingerprinting mass of intact peptides & the fragment masses Search databases with much more data per protein

16 MSMS ions search data peak list (pkl) Peptide mass : charge state : intensity fragment mass : intensity : :

17 Examples of open access search tools MASCOT 3 types of searches Aldente PMF search tool / Phenyx an MS data analysis platform identification and characterization of proteins & peptides from mass spectrometry data

18 Mascot Search Overview Mascot is a search engine which uses mass spectrometry data to identify proteins from primary sequence databases

19 MASCOT provides 3 different search methods Peptide Mass Fingerprint peptide mass values Sequence Query peptide mass data plus amino acid sequence/composition MS/MS Ion Search uninterpreted MS/MS data from one or more peptides

20 Cut-off score for significance is different for every search Decoy database search

21 Peptide score Expect value Number of matching peptides Protein score Protein name Different species

22 Only the peptide masses and their fragment ion masses are matched – the peptides themselves have not actually been sequenced Predicted mass and predicted pI Sequence coverage

23 All these proteins are hit #1 All have the same score and the same peptide masses match The order of the list within each hit, is meaningless i.e. cow is top here, but the sample is mouse

24 Download the MSMS files 1 to 4 onto the desktop Click on the MS/MS Ions Search tool page

25 Standard search input your name & your use standard defaults swissprot trypsin, 1 missed cleavage variable on Carbamidomethyl C variable on Oxidation M peptide charge +2, +3, +4 Copy MSMS files to desktop Browse to add file to search page Micromass PKL ESI-QUAD-TOF

26 Vary some parameters in subsequent searches try NCBInr and swissprot databases for MSMS3 add in variable phosphorylations for MSMS4 semi-trypsin alter mass tolerances compare results with standard search

27 Selected references and reviews Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics Dec;4(12): Two-dimensional gel electrophoresis: an overview, Pages David E. Garfin Trends in Analytical Chemistry Volume 22, Issue 5, Pages (May 2003) The current state of two-dimensional electrophoresis with immobilized pH gradients. Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. Electrophoresis Apr;21(6): ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRY Matthias Mann, Ronald C. Hendrickson, and Akhilesh Pandey Annual Review of Biochemistry July 2001, Vol. 70, pp Challenges in mass spectrometry-based proteomics. Reinders J, Lewandrowski U, Moebius J, Wagner Y, Sickmann A. Proteomics Dec;4(12): Plant proteome analysis. Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M. Proteomics Feb;4(2): Subcellular proteomics. Dreger M. Mass Spectrom Rev Jan-Feb;22(1): Functional organization of the yeast proteome by systematic analysis of protein complexes. Gavin AC, Bosche M, et al Nature Jan 10;415(6868):141-7.

28 Development Feb;131(3): Drosophila ventral furrow morphogenesis: a proteomic analysis. Lei Gong, Mamta Puri, Mustafa Ünlü, Margaret Young, Katherine Robertson, Surya Viswanathan, Arun Krishnaswamy, Susan R. Dowd and Jonathan S. Minden State-of-the-art in phosphoproteomics Proteomics 2005 Early View i.e. find it on the journals early view section of the web site Joerg Reinders, Albert Sickmann Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF. Proteomics Jul;3(7): Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol Sep;5(9): Review.

29 links Swiss Proteomics Society. The digest provides a consolidated selection of articles published in all scientific publications that are pertinent to proteomics – finds all the interesting and relevant papers for you! proteomics special interest group at NIH, includes archived videocasts of research seminars proteome informatics tools e.g. peptidemass predicted digestion fragment tool British Society for Proteome Research British Mass Spectrometry society The Plasma Proteome Institute in Washington D.C. Unimod : protein modifications for mass spectrometry Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc

30 Example of good quality peptide match

31 Number of contiguous residues should be 5 or more Have 8 for this peptide – good quality match

32 Example of poor quality peptide

33 Longest stretch of contiguous reside calls is 2 – insufficient for good ID If this was the only peptide match it would be rejected by the user

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