We use 2 types of MS for PROTEIN IDENTIFICATION

1. My contact details and information about submitting samples for MShttp://www.nottingham.ac.uk/Biosciences/People/susan.liddell

2. We use 2 types of MS for PROTEIN IDENTIFICATION • PEPTIDE MASS FINGERPRINTING (PMF) • MALDI-ToF MS • TANDEM MS (aka MSMS) • ELECTROSPRAY Q-ToF2

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

4. 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 89.3 105.3 89.3 = 402.2 112.1 95.4 95.4 105.3 = 601 89.3 97.1 101.8 112.1 The peptides produced have distinct weights These are accurately measured by mass spectrometry

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

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

7. Peptide mass spectrum a peak list (pkl) . . . . 1051.54 1094.56 1244.64 1476.67 1542.84 1613.88 1664.97 1763.79 1952.89 2264.89 2238.23 . . . . Data converted to text List of peptide masses = fingerprint

8. Database searches with PeptideMassFingerprint data run digested protein on MS sequence databases theoretical trypsin digest of every predicted protein compare identification made if match is found list of calculated peptide masses . . . 1051.54 1094.56 1244.64 1476.67 1542.84 1613.88 1664.97 1763.79 1952.89 2264.89 2238.23 . . . 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. ElectroSpray Ionisation (ESI) Mass Spectrometry on the Q-ToF2 • Samples in solution • Compatible with HPLC • Complex protein mixtures • Determine peptide masses Peptide fragmentation • Peptide sequence

11. MS on the Q-ToF2 MS1 MS2

12. low energy collision fragments the peptide cleavage usually occurs at the amide bond i.e. between residues 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 Tandem MS - peptide fragmentation

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. On-line LC-MSMS on the Q-ToF2 : peptides from a single protein Fragment Mass spectrum (MSMS) fragments from one peptide MSMS MSMS MSMS MSMS SurveyMass Spectrum (MS) - intact peptides detected in a 1 second survey scan 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. 920.9598 241.0128 2 70.0629 15.5793 72.0767 22.1687 80.9474 6.1025 110.0635 8.3011 158.0875 11.9145 173.1226 71.9019 175.1129 9.3308 185.0797 7.5469 : 1769.7933 47.7946 1771.8080 43.3989 1839.8304 54.6593 1841.9095 44.5610 1843.8146 92.9938 1845.8208 58.4194 623.3281 243.3593 2 70.0612 91.0550 71.0651 3.5558 : MSMS ions search data peak list (pkl) Peptide mass : charge state : intensity fragment mass : intensity fragment mass : intensity fragment mass : intensity

17. Examples of open access search tools MASCOT 3 types of searches http://www.matrixscience.com/search_form_select.html Aldente PMF search tool http://ca.expasy.org/tools/aldente/ Phenyx an MS data analysis platform identification and characterization of proteins & peptides from mass spectrometry data http://ca.expasy.org/tools/aldente/

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

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

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

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 e-mail 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. 2004 Dec;4(12):3665-85. Two-dimensional gel electrophoresis: an overview, Pages 263-272 David E. Garfin Trends in Analytical Chemistry Volume 22, Issue 5, Pages 263-334 (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. 2000 Apr;21(6):1037-53. 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. 437-473 Challenges in mass spectrometry-based proteomics. Reinders J, Lewandrowski U, Moebius J, Wagner Y, Sickmann A. Proteomics. 2004 Dec;4(12):3686-703. Plant proteome analysis. Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M. Proteomics. 2004 Feb;4(2):285-98. Subcellular proteomics. Dreger M. Mass Spectrom Rev. 2003 Jan-Feb;22(1):27-56. Functional organization of the yeast proteome by systematic analysis of protein complexes. Gavin AC, Bosche M, et al Nature. 2002 Jan 10;415(6868):141-7.

28. Development. 2004 Feb;131(3):643-656. 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. 2003 Jul;3(7):1128-44. Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol. 2004 Sep;5(9):699-711. Review.

29. links http://www.swissproteomicsociety.org/digest 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! http://proteome.nih.gov proteomics special interest group at NIH, includes archived videocasts of research seminars http://ca.expasy.org/tools/ proteome informatics tools e.g. peptidemass predicted digestion fragment tool http://www.bspr.org/ British Society for Proteome Research http://www.bmss.org.uk/ British Mass Spectrometry society http://www.plasmaproteome.org/ The Plasma Proteome Institute in Washington D.C. http://www.unimod.org/ Unimod : protein modifications for mass spectrometry http://www.hupo.org/ http://www.spectroscopynow.com/coi/cda/home.cda?chId=0 http://www.ionsource.com/ Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm

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