Interpretation of Mass Spectra

1. Interpretation of Mass Spectra Beatrix Ueberheide March 11th 2019

2. The Mass Spectrum

3. Charge State = number of H+ (H=1.008) +n M - molecular mass n - number of charges H – mass of a proton 4550.2805 [M+1H]+1 (910.8625 * 5)-(4*1.008) = 4550.2805

4. Charge State = number of H+ (H=1.008) M - molecular mass n - number of charges H – mass of a proton 1302.6759 [M+1H]+1

5. Charge State = number of H+ (H=1.008) M - molecular mass n - number of charges H – mass of a proton 1532.7666 [M+1H]+1

6. Biological Mass Spectrometry Proteolytic digestion Protein(s) Peptides Base Peak Chromatogram MS 500 1000 1500 m/z Time (min) Mass Spectrometer MS/MS Database Search Manual Interpretation 200 600 1000 m/z

7. Peptide Sequencing using Mass Spectrometry Fragments containing original N-terminus: b- or c- ion S G F L E E D E L K Fragments containing original C-terminus: y- or z- ion 100 % Relative Abundance 0 250 500 750 1000 m/z

8. Peptide Sequencing using Mass Spectrometry 88 145 292 405 534 663 778 907 1020 1166 b ions S G F L E E D E L K 1166 1080 1022 875 762 633 504 389 260 147 y ions 762 100 875 [M+2H]2+ % Relative Abundance 633 292 405 534 1022 260 389 504 907 1020 663 778 1080 0 250 500 750 1000 m/z

9. Searching Proteomics Data Protein GSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTP Digestion LFTFHADICTLPDTEK 1850.8993 RPCFSALTPDETYVPK 1823.8906 MPCTEDYLSLILNR 1667.8131 VPQVSTPTLVEVSR 1511.8427 DDPHACYSTVFDK 1497.6314 1850.8993 1850.8906 1850.8805 1850.8914 1850.8868 Peptide Mass Measurement MS 924.9537 500 1000 1500 m/z Peptide Fragmentation MS/MS 200 600 1000 m/z

10. Nomenclature of Fragment Ions http://www.ionsource.com/

11. Accurate Mass of Amino Acids

12. Common PTMs

13. Review from last class: • Major ions in a spectrum should be explained by the peptide sequence • Specific amino acids can show Neutral Losses (H20, NH3) • Water loss: S, T, E (must be at N-terminus) • Ammonia loss: R, K, Q and N • The y ion after P cleavage is often the dominant ion in a spectrum • P and to a lesser degree H can show internal cleavages • L and I have the exact same mass • K and Q have near identical mass (K = 128.09496; Q = 128.05858) • Two amino acids could near equal the mass of a single amino acid • The b2 ion is often observed along with the a2 ion

14. How to Sequence: CAD Residue Mass (RM) The very first N- and C-terminal fragment ions are not just their corresponding residue masses. The peptides N or C-terminus has to be taken into account. b ion y ion b1 = RM + 1 y1 = RM + 19

15. Example of how to calculate theoretical fragment ions 88 159 290 387 500 629 803 S A M P L E R 803 716 645 514 417 304 175 Residue Mass The first b ion The first y ion

16. How to calculate theoretical fragment ions RM+1 + RM + RM + RM + RM + RM +RM+18 88 159 290 387 500 629 803 S A M P L E R 803 716 645 514 417 304 175 + RM + RM + RM + RM + RM + RM RM+19 The first b ion The first y ion Residue Mass

17. Finding ‘pairs’ and ‘biggest’ ions: b ion If trypsin was used for digestion, one can assume that the peptide terminates in K or R. Therefore the biggest observable b ion should be: Mass of peptide [M+H] +1 -128 (K) -18 Mass of peptide [M+H] +1 -156 (R) -18

18. Finding ‘pairs’ and ‘biggest’ ions: y ion y ions are truncated peptides. Therefore subtract a residue mass from the parent ion [M+H] +1 . The highest possible ion could be at [M+H] +1 -57 (G) The lowest possible ion at [M+H] +1 -186 (W)

19. Finding ‘pairs’ and ‘biggest’ ions: pairs H+ H+ b and y ion pairs: Complementary b and y ions should add up and result in the mass of the intact peptide, but since both b and y ion carry 1H+ the peptide mass will be by 1H+ too high therefore: b (m/z) + y (m/z)-1H+ = [M+H] +1

20. How to start sequencing • Know the charge of the peptide • Calculate the [M+1H]+1 charge state of the peptide • Know the sample treatment (i.e. alkylation, other derivatizations that could change the mass of amino acids) • Know what enzyme was used for digestion • Find and exclude non sequence type ions (i.e. unreacted precursor, neutral loss from the parent ion, neutral loss from fragment ions) • Look for the biggest y or b ion in the spectrum. • Try to find sequence ions by finding b/y pairs • You usually can conclude you found the correct sequence if you can explain the major ions in a spectrum

21. Common observed neutral losses and mass additions: • Ammonia -17 • Water -18 • Carbon Monoxide from b ions -28 • Phosphoric acid from phosphorylated serine and threonine -98 • Carbamidomethyl modification on cysteines upon alkylation with iodoacetamide +57 • Oxidation of methionine+16 Calculate with nominal mass during sequencing, but use the monoisotopic masses to check if the sequence fits the parent mass fits. For high res. MS/MS check that the residue mass difference is correct.

22. Homework 1

27. Homework 2

30. H F V Q W C A L R

31. Example 1

36. Example 2

37. Example 2

38. Example 2

39. Example 2

40. Homework for next class

42. Ubiquitination - MS