Exercise MSMS6: Answers

1. How can you distinguish between peak picking choosing the ¹³C peak and deamidation?

   If you have low ppm accuracy, you can tell from the precursor mass. ¹³C is 1.0033 Da heavier than ¹²C while deamidation is 0.984 Da. For a peptide of mass 1800 Da, these differ by 11 ppm.

   If you don't have low ppm accuracy, it may be possible to tell from the fragment matches. If the peptide is deamidated, but you match it without this modification by allowing a precursor mass error of 1 Da, fragments containing the deamidated residue will not be matched. Exactly the same applies if the precursor is actually ¹³C, but a match is obtained by introducing a false deamidation. Only the "correct" match will give fragment matches spanning the deamidation site(s).

   You can see good illustrations by re-searching this data with a precursor tolerance of 15 ppm and adding Deamidated (NQ) as a variable modification.

2. Is there evidence of non-specific cleavage?

   Yes, an unusually high level of semi-specific peptides. Try using semi-trypsin as the enzyme or use model refinement / error tolerant search

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