Attached the binding pocket of trypsin, citrulline should not bind, due to negative asp citrulline will not bind sufficiently in the enzyme, so no cleavage after citrulline

It is difficult to know for sure but I suspect it may be possible due to similar structures of Arginine (which trypsin happily cuts) and Citrulline. It is produced from arginine as a by-product of the reaction catalyzed by NOS family.

But these are mere speculations, you will need to confirm this by experimenting.

It shouldn't get cleaved by trypsin, at least most papers claim that (PMID:

19284558). Be extra careful as deamidation gives you the same mass increase in MS searches, you will have to look into the MSMS of your modified peptide(s) to ensure you pick up the correct fragments that cover the citrullination site.

Attached the binding pocket of trypsin, citrulline should not bind, due to negative asp citrulline will not bind sufficiently in the enzyme, so no cleavage after citrulline

Thank you all! And Thanks Bart for the illustrative chart. After all, citrulline shouldn't fit the pocket.

Hi Carlo,

While citrulline shouldn't fit the binding site and most papers report abrogation of cleavage, some groups do find C-terminal citrullinated peptides after trypsinisation (for instance Andrade et al, Arthritis Rheum 2010, 62, 1630), so I'd be careful using only trypsin miscleavage for identification of citrullination. We have had more success by using a different protease (such as chymotrypsin or GluC) and including citrullination as a variable modification in the database search.

Principally, Trypsin should not bind or cleave C-terminally of citrullinated residues. However, our experience with (porcine) Trypsin Gold (Promega) hints otherwise: Modified (i.e. alkylated) lysines or argines are accepted though less efficiently. Since this did not occur with previous preparations (bovine), we deduce this to a chymotrypsin contamination, which will readily accept such residues.

That's an interesting point, Knut, as I am using porcine trypsin from Promega thus far.

Arginine residues can be converted to citrulline by peptidylarginine deiminases, so if this enzyme is present and active after trypsin digestion, the citrullination can occur _after_ cleavage.

It has been investigated if citrullination could occur after tryptic digest, and while bacterial PAD preferentially acts on carboxyterminal arginines, mammalian PAD needs the amino acid context of internal arginines to work. There's some discussion of this in the article by Wegner (Arthritis Rheum 62, 9, 2662).

We did see cleavage at citrullinated arginine with trypsin gold from Promega.