Interpretation of Mass Spectra Beatrix Ueberheide March 11th 2019

The Mass Spectrum

Charge State = number of H+ (H=1.008) 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

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

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

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

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

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

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

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

Accurate Mass of Amino Acids

Common PTMs

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

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

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

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

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

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)

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

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

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.

Homework 1

Homework 2

H F V Q W C A L R

Example 1

Example 2

Example 2

Example 2

Example 2

Homework for next class

Ubiquitination - MS