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

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Presentation on theme: "Oct 2010 SDMBT Lecture 10 Interpretation of Mass Spectra A.Peptide Mass Fingerprinting B.MS/MS sequencing 1."— Presentation transcript:

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

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

3 Oct 2010 SDMBT3 Peptides Protein separated on 2D-gel Tryptic digestVirtual Tryptic Digest all known proteins Trypsin cuts at C-terminal side of lysine and arginine – size of peptides unique for each protein Peptide Mass Fingerprinting (PMF) Experimentally On MALDI-TOF match

4 Oct 2010 SDMBT King’s College London (Pierce) Major peaks at: Recall: Tryptic digest of β-casein 4 Peptide Mass Fingerprinting (PMF)

5 Amino acid Sequence from GenBank Virtual peptide digest Peptide Mass Fingerprinting (PMF)

6 Convert to FASTA format Peptide Mass Fingerprinting (PMF) Virtual peptide digest

7 Peptide Mass Fingerprinting (PMF) Virtual peptide digest

8 Results of virtual tryptic digest Peptide Mass Fingerprinting (PMF) Virtual peptide digest Major peaks at: Compare with experimental peaks in MALDI-TOF of tryptic digest

9 Oct 2010 SDMBT 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 1 st and 2 nd ranked protein good coverage of the 1 st ranked protein with the experiment results 9

10 Oct 2010 SDMBT 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 10 Peptide Mass Fingerprinting (PMF)

11 Oct 2010 SDMBT PMF using MS-FIT 11

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

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

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

15 Oct 2010 SDMBT15 Does this agree with position in 2D-gel? Peptide Mass Fingerprinting (PMF) results for tryptic digest of β-casein Note: do not need match all peaks or whole protein to identify protein!

16 Oct 2010 SDMBT 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. 16

17 Oct 2010 SDMBT Peptide Mass Fingerprinting (PMF) (Promega) 17 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

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

19 Oct 2010 SDMBT19 Peptide Mass Fingerprinting (PMF) Exercise: Identify this protein

20 Oct 2010 SDMBT  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 MS/MS sequencing 20

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

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

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

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

25 Just looking at the y ions y7 y6 y5 y4 y3 y Ala (A)Gly (G) Cys (C) Ala (A)Gly (G) y-ions contain the C-terminus therefore … AGCAG….CO 2 H Difference betw y ions= Residue mass (see next page) MS/MS sequencing

26 Oct 2010 SDMBT Residue masses of amino acids letternamemass, Da Gglycine57.02 Aalanine71.04 Sserine87.03 Pproline97.05 Vvaline99.07 Tthreonine Ccysteine Iisoleucine Lleucine Nasparagine letternamemass, Da Daspartic acid Qglutamine Klysine Eglutamic acid Mmethionine Hhistidine Fphenylalanine Rarginine Ytyrosine Wtryptophan (N.S. Weld) 26 Note: some have very similar molecular weights Residue mass = Molecular weight of amino acid –18 (2xH + 1xO)

27 Just looking at the b ions b7 b6 b5 b4b3 b Cys (C) Ala (A) Gly (G) b-ions contain the N-terminus therefore … NH 2 -…….AGCAGA b Ala (A) Gly (G) Ala (A) MS/MS sequencing

28 Combine the results….. from y-ions… …….AGCAG….CO2H from b-ions … NH2-…….AGCAGA…. Partial sequence - NH 2 -….AGCAGA….CO 2 H 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 MS/MS sequencing

29 a m ions – add all m residue masses - 27 x n ions – add all n residue masses + 45 c m ions – add all m residue masses +17 z n ions – add all n residue masses + 2 y m ions - add all m residue masses + 19 b n ions – add all n residue masses + 1 Useful numbers and Hints for MS-MS spectra MS/MS sequencing

30 Where do these numbers come from? b-ion (b 1 ) 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” MS/MS sequencing

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

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

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

34 Oct 2010 SDMBT34 Fragment peptides Peptide after ionisation by MALDI or ESI Fragmentation Virtual Fragmentation MS/MS sequencing experimental match

35 eg peptide from human catalase LSQEDPDYGIR Protein Prospector – MS-Product 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 has the following peaks

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 Output – protein identified MS/MS sequencing

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

42 More complex example….. MS-MS of a peptide with mass with peaks at Yeast alcohol dehydrogenase – But deliberately missed out one y ion and all except 3 b ions MS/MS sequencing

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


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