Good morning,
I have been working with a protein complex, after Orbitrap LC-MS/MS of trypsin digested sample only 2 pair of cysteines (peptide dimers) were detected. According to the protein structure, another cysteine is naturally exposed to the surface, but it was not detected by mass spectrometry involved in S-S bond. Could I still argue that this exposed cysteine may form disulphide bonds?
thank you for your comments,
daniel
Hey,
Depends if you had reduced and alkylated protein? There are few strategies to find it out like you first block free cysteine thiols with iodoacetamide (irreversible reaction) , then you reduce thiols with e.g., DTT or TCEP and block 'opened' bridges with MMTS. But still, only a fraction of protein might form disulphide bridges. Please have a look on a protein sequence, if your cysteine is within peptide, that can be visualised by MS.
Cheers,
Janusz
hi Janusz,
The protein sample had already S-S bonds when purified...it was digested with Trypsin Gold without any previous treatment with reducing or alkylating agents.... after Lc MS/MS analysis we found the molecular weight associated of 2 combinations of peptides each with 1 cysteine.... there are actually 4 theoretical peptides with 1 cistern after trypsin digestion in the monomers..... I just would like to know if any other of the 2 conbinantons founds are possible...
regards
daniel.
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