Hi,
it should be possible to enhance your confidence in PTM site localization by combining information from digests with different proteases, e.g. by considering where the peptides overlap and checking if fragment ions that are absent in one case might exist in the other. But that`s easier said than done.. I was wondering if anyone of you has tried this or is aware of any good literature?
Thanks.
Hi,
probably one of the earliest works and still an excellent reference is this publication from Yates and colleagues:
http://www.pnas.org/content/99/12/7900.long
If you are interested in phosphorylation, a recent paper that I co-authored could be useful for you:
http://jcb.rupress.org/content/206/4/541.long
It refers to several papers from the group of Eberhard Krause that contain more thorough descriptions of the employed workflow
1) I would split my sample in two aliquots
2) I would dephosphorylate or deglycosylate one and keep the other one as control
3) I would perform proteolysis compare hydrolysates between treated and untreated aliquots. (repeat for each protease you're willing to use)
for dephosphorylation this protocol was very effective (I used calf intestine alkaline phosphatase from SIGMA, cat P6674):
http://www.abcam.com/protocols/protein-dephosphorylation-protocol
and for deglycosylation I used the SIGMA Enzymatic protein deglycosylation kit (Cat. EDEGLY) following the product instructions.
Thanks a lot for the answers. As this is about protein methylation and not phosphorylation some of it won`t work unfortunately. I really like the idea of comparison with part of the sample where the PTM has been removed, but I don`t think it`ll work. The papers are a good starting point - thanks again.
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