I am analysing proteomic data, some of which was digested with the Thermo SMART Digest Kit and some by in-gel digest. I am not a laboratory scientist, and have noted that the samples processed with SMART Digest Kit have very low proportion containing cysteine residues (~0.5% compared to 30% with in-gel digestion). We now have a belated realisation that the SMART Digest Kit does not reduce or alkylate proteins, and that thus disulfide bonds will largely remain.
In an aim to recover as much data as possible, I tried removing the fixed carbamidomethyl mod, however only a few unmodified Cysteines are found. I performed an open search on a small subset of data obtained with high fragment ion resolution, and identified a number of peptides determined to containing cysteine with a -2.0157 Da modification, labelled as "2-amino-3-oxo-butanoic acid" or "didehydro [cysteine]" (shown is -2H on https://www.unimod.org/modifications_view.php?editid1=401). [ed - I appreciate this is not a recognised modification of cysteine!].
If I undertake a fixed search with this as a variable modification I find many more cysteine containing peptides (up to 5%) with a majority with this -2H modification.
However, this modification doesn't seem to be a recognised one for cysteine, and I am no chemist. I wonder if anyone has come across this modification before and can explain it? Ed - I presume it's more likely to represent a different amino acid with a modification which gives a near-identical mass to cysteine -2H.
Incidentally, despite the claims that the SMART Digest kit has immobilised trypsin which should not auto-digest, we have very strong signal from trypsin auto-digestion products!
