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

2. 2 main types of MS are used 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 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 95.4 89.3 112.1 105.3 = 402.2 95.4 89.3 112.1 105.3 97.1 101.8 = 601 89.3 = 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. 1051.54 1094.56 1244.64 1476.67 1542.84 1613.88 1664.97 1763.79 1952.89 2264.89 2238.23. 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”. 1051.54 1094.56 1244.64 1476.67 1542.84 1613.88 1664.97 1763.79 1952.89 2264.89 2238.23.

9. peptide mass fingerprinting rapid high throughput large scale identification of proteins from organisms with completely sequenced genomes 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 – LC-MSMS

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 one fragmentation per peptide molecule 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. 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

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

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

17. 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

18. Cut-off score for significance is different for every search

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

20. A match is made by correlating the observed and predicted peptide masses and their fragment ion masses – the peptides themselves have not actually been sequenced Predicted mass and predicted pI Sequence coverage

21. 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

22. Click on the MS/MS Ions Search tool page INSTRUCTIONS FOR THE PRACTICAL COPY the 4 MSMS files onto the desktop

23. Standard search Input your name & your e-mail Use these standard defaults swissprot trypsin, 1 missed cleavage variable on Carbamidomethyl C variable on Oxidation M peptide charge +2, +3, +4 Browse to add an MSMS file to the search page Micromass PKL ESI-QUAD-TOF

24. Vary some parameters in subsequent searches try NCBInr and swissprot databases for MSMS3 add in variable phosphorylations for MSMS4 semi-trypsin for MSMS2 alter mass tolerances compare results with standard search

25. links http://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213 http://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213 MASCOT video tutorials http://www.ionsource.com/http://www.ionsource.com/ Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc http://proteome.nih.govhttp://proteome.nih.gov proteomics special interest group at NIH, includes archived videocasts of research seminars http://ca.expasy.org/tools/http://ca.expasy.org/tools/ informatics tools e.g. peptidemass predicted digestion fragment tool http://www.bspr.org/http://www.bspr.org/ British Society for Proteome Research http://www.bmss.org.uk/http://www.bmss.org.uk/ British Mass Spectrometry society http://www.plasmaproteome.org/http://www.plasmaproteome.org/ The Plasma Proteome Institute in Washington D.C. http://www.unimod.org/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.abrf.org/index.cfm/group.show/Proteomics.34.htm

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

27. Example of good quality peptide match

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

29. Example of poor quality peptide

30. 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