Hi to everybody.
We are working in the field of marine proteomics and decided to use thermolysin instead of trypsin etc for digestion (bottom-up, using LC-MSMS; 4600 tripletof). Unfortunately the Mascot database does not support thermolysin and the support could not help us to solve the problem.
Of course we used the mascot configurator to include thermolysin into the enzyme list. Afterwards it work but comparing the results of mascot with peptide cutter resulted in significant differences. Especially the Prolin-problem (position 2 to cleavage side) is not handeled correctly.
Background cited from peptide cutter: Thermolysin preferentially cleaves sites with bulky and aromatic residues (Ile, Leu, Val, Ala, Met, Phe) in position P1' (Keil, 1992). Cleavage is favoured with aromatic sites in position P1 but hindered with acidic residues in position P1. Prolin blocks when located inposition P2' but not when found in position P1.
Our configuration in mascot:
title: thermolysin
Independent: 0
Semispecific: 0
Cleavage[0]:AFILMV
Restrict[0]:DE
Nterm[0]
Does anybody have similar problems or has a solution or hints for this issue? Thank you in advance
Hi Stefan,
I would suggest you to change restrict to P and allow semi-specific cleavages to account for DE. alternatively, restrict PDE and allow more missed cleavages. unfortunately mascot is not very flexible with regards of enzyme rules.
I would like to point out that there are many other free (and good!) search engine available that are more flexible.
Cheers,
David.
Hi Dave,
thanks for you suggestions. The major advantage of mascot in this case is the direct connection of Protein Pilot vom ABSciex to Mascot. Hence even though I aggree with you that Mascot insn't the best idea it fits best for future automization.
But regarding your suggestions we will give it a try
Cheers
Stefan
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