Lecture 10 Interpretation of Mass Spectra Peptide Mass Fingerprinting MS/MS sequencing

1. Lecture 10 • Interpretation of Mass Spectra • Peptide Mass Fingerprinting • MS/MS sequencing Oct 2010 SDMBT

2. Sample preparation Protein mixture Sample separationand visualisation Comparative analysis Digestion Peptides Mass spectrometry MS data Database search Protein identification General workflow for proteomic analysis Sample Oct 2010 SDMBT

3. Peptide Mass Fingerprinting (PMF) Protein separated on 2D-gel Tryptic digest Experimentally On MALDI-TOF match Virtual Tryptic Digest all known proteins Peptides Trypsin cuts at C-terminal side of lysine and arginine – size of peptides unique for each protein Oct 2010 SDMBT

4. Recall: Tryptic digest of β-casein Peptide Mass Fingerprinting (PMF) Major peaks at: 646 742 748 780 830 2186 King’s College London (Pierce) Oct 2010 SDMBT

5. Peptide Mass Fingerprinting (PMF) Virtual peptide digest Amino acid Sequence from GenBank http://www.ncbi.nlm.nih.gov/entrez/

6. Peptide Mass Fingerprinting (PMF) Virtual peptide digest Convert to FASTA format http://bioinformatics.org/sms2/genbank_fasta.html

7. Peptide Mass Fingerprinting (PMF) Virtual peptide digest http://www.expasy.org/

8. Peptide Mass Fingerprinting (PMF) Virtual peptide digest Results of virtual tryptic digest Compare with experimental peaks in MALDI-TOF of tryptic digest Major peaks at: 646 742 748 780 830 2186

9. Peptide mass fingerprinting (PMF) • Peptide masses are matched against theoretical digests of proteins in databases • Matches are ranked by the number of matching peptides • Confidence in the identity is given by • a large gap in the number of matching peptides between the 1st and 2nd ranked protein • good coverage of the 1st ranked protein with the experiment results Oct 2010 SDMBT

10. Peptide Mass Fingerprinting (PMF) Variables for database search • Choice of database (public or private) • Species of origin • Molecular weight and pI range • Enzyme used for digest • Modifications (reduction, alkylation, phosphorylation) • Tolerance Oct 2010 SDMBT

11. PMF using MS-FIT http://prospector.ucsf.edu/ Oct 2010 SDMBT

12. PMF using MS-FIT Choice of database Choice of enzyme Oct 2010 SDMBT

13. PMF using MS-FIT Tolerance Choice of modifications Peaks entered here Oct 2010 SDMBT

14. Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein • Same protein across 4 similar species Oct 2010 SDMBT

15. Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein Does this agree with position in 2D-gel? Note: do not need match all peaks or whole protein to identify protein! Oct 2010 SDMBT

16. Limitations of PMF • This method assumes that databases are complete • but the genomes of only some organisms are completely sequenced, high confidence matches might not be available • But homology between organisms allow for good results • No information about amino acid sequence, only identity of protein. The amino acid sequence in slide 15 is only the ‘predicted sequence’ based on virtual digest. Oct 2010 SDMBT

17. Peptide Mass Fingerprinting (PMF) Database search is only good as the database and the input data e.g. MALDI spectra often have peaks due to trypsin autolysis and keratin degradation (Promega) Oct 2010 SDMBT

18. Real world MS data Peptide Mass Fingerprinting (PMF) If the MS is too noisy….. Oct 2010 SDMBT (L&T Inc)

19. Peptide Mass Fingerprinting (PMF) Exercise: Identify this protein Oct 2010 SDMBT

20. MS/MSsequencing • Fragmentation of peptides causes cleavages along the peptide backbone • Comparison of MS-MS spectra allows in theory determination of possible amino acid sequences manually (slides 21-33) • Sequences matched to databases to determine identity and sequence of proteins (slides 34 onward) • Adds another layer of certainty in the identification of the peptide and hence to the protein Oct 2010 SDMBT

21. MS/MSsequencing TRYPTIC PEPTIDES IN MS/MS C-terminal always Arginine (R) or Lysine (K) By convention N-terminal on left N-terminal of peptide Trypsin cuts C terminal side of R/K Proteins digested into peptides by trypsin All tryptic peptides have similar structure – because digested by trypsin When peptides ionised usually– 2+ charge on either end of peptide MS/MS fragmentation of peptide in 6 ways leads to …..

22. By convention, ion fragments are called…. IMPORTANT Although 6 possible ways, generally b and y ions are most common It is in general not always to predict what sort of ions will be produced

23. Explain how does ionisation break up? In theory 8 y-ions and b-ions possible but not all may be observed Right-hand side C-terminus Left-hand side N-terminus

24. C-terminal Residue mass+19 Residue mass of amino acid N-terminal Residue mass+1 In practice, not all y and b ions observed (cannot be predicted)

25. MS/MSsequencing Difference betw y ions= Residue mass (see next page) Just looking at the y ions y7 y-ions contain the C-terminus y6 Gly (G) Ala (A) y4 Gly (G) Ala (A) y3 Cys (C) 57.1 70.9 102.8 57.3 71 y5 y2 246.2 303.3 374.2 477.0 534.3 605.3 therefore … AGCAG….CO2H

26. Residue masses of amino acids Residue mass = Molecular weight of amino acid –18 (2xH + 1xO) Note: some have very similar molecular weights Oct 2010 SDMBT (N.S. Weld)

27. MS/MSsequencing Just looking at the b ions b-ions contain the N-terminus b2 Ala (A) b5 Cys (C) Ala (A) Gly (G) b6 Ala (A) Gly (G) 71.1 57.2 102.8 b3 b4 b8 b7 70.3 57.5 70.5 170.9 242.0 299.2 402.0 472.5 530.2 600.7 therefore … NH2-…….AGCAGA

28. MS/MSsequencing Combine the results….. from y-ions… …….AGCAG….CO2H from b-ions … NH2-…….AGCAGA…. Partial sequence - NH2-….AGCAGA….CO2H Need to know how to interpret MS – which peaks are y- and b-? Which are y2, y3 etc? Difficult to tell the amino acids at the beginning and the end

29. MS/MSsequencing Useful numbers and Hints for MS-MS spectra ym ions - add all m residue masses + 19 bn ions – add all n residue masses + 1 cm ions – add all m residue masses +17 zn ions – add all n residue masses + 2 am ions – add all m residue masses - 27 xn ions – add all n residue masses + 45

30. b-ion (b1) MS/MSsequencing Where do these numbers come from? Definition of residue mass of amino acid = Molecular weight of amino acid –18 (2xH + 1xO) b ion has 1 extra hydrogen Compared to “residue mass of amino acid”

31. y-ion (y2) MS/MSsequencing Where do these numbers come from? Residue Mass of Gly Residue Mass of Lys Residue mass of Gly+Lys + 2xH + 1xH+1xO = sum of residue masses+19

32. MS/MSsequencing Draw the a,b,c and x,y,z ions from this dipeptide and Calculate the m/z ratios

33. MS/MSsequencing Draw the a,b,c and x,y,z ions from this tripeptide And calculate the m/z ratio

34. MS/MSsequencing Peptide after ionisation by MALDI or ESI match Virtual Fragmentation Fragmentation experimental Fragment peptides Oct 2010 SDMBT

35. eg peptide from human catalase LSQEDPDYGIR Protein Prospector – MS-Product http://prospector.ucsf.edu/ Paste amino acid sequence

36. All predicted a, b, y ions etc.

37. MS-MS data – amino acid sequence – protein identification e.g. if MS-MS of a A peptide of mass 1292.61 has the following peaks 1179.53 1092.50 964.44 835.39 720.37 623.31 508.29 345.22 288.20 175.12

38. First number - must be mass of peptide+1 i.e. [M+H]+

39. In ESI-MS tryptic peptide is usually 2+ – it is actually [M+2H]2+

40. MS/MSsequencing Output – protein identified

41. MS/MSsequencing Each of the fragments identified as y or b ions – the user does not have to assign the peaks or work out residual masses

42. MS/MSsequencing More complex example….. MS-MS of a peptide with mass 1217.58 with peaks at 1088.54 975.46 847.40 746.35 631.32 457.28 358.21 243.13 300.16 371.19 Yeast alcohol dehydrogenase – But deliberately missed out one y ion and all except 3 b ions

43. Still able to identify the protein. Even though info incomplete All peaks identified as y or b ions