mass spectrometry and tryptic digestion on membrane proteins - (Sep/24/2012 )
I have a band on my Coomassie blue stained SDS-PAGE and I want to cut it in order to identify the protein by mass spectrometry after tryptic digestion. If my purification is correct, this is a membrane protein.
For the tryptic digestion, do you use the protocol for soluble proteins?
why wouldn't you? anyhow it is denaturated from the SDS-PAGE gel. Just sayin' without any 100% sure (never did it). But how the MS people in my institute explained to me: you don't care too much about stuff from the SDS-PAGE, a peptide is a peptide.
I read somewhere that hydrophobic peptides can bind the surface of your tubes so you have to use specific tubes if you don't want to lose your peptides and that it was better to use another acid instead of TCA. Then somebody told me about heating as technique to obtain better results during the vaporisation or the HPLC (in LS-MS-MS).
But I'm not sure of that and I never found a real protocol.
The thing with the tubes is very true; even I heard about it from the MS people around The rest: I am not the expert.
You might find the following article discussing in-gel digestion of membrane proteins helpful;
"Comparative Study of Workflows Optimized for In-gel, In-solution, and On-filter Proteolysis in the Analysis of Plasma Membrane Proteins" found here
thank you KarenLL!