Generally, it is performed after Reduction and Alkylation steps.
But if trypsin digestion is performed prior to SDS PAGE, will it affect the peptide coverage? And how?
Since we performed that way and obtained three bands of BSA, MS analysis is yet to be done.
If the trypsin digestion goes to completion, you will end up with only small peptides and perhaps a few larger ones when you run the SDS-PAGE. If the digestion is incomplete, you will see larger fragments that may include the whole protein when combined, or may be missing some pieces because they run at the dye front.
If you are working with a pure protein, you can do the digestion in solution and skip the SDS-PAGE/in-gel digestion. You may want to do the digestion in 2 stages. After an initial digestion, you can boil the sample to denature the residual protein fragments, then add more trypsin to get complete digestion.
I agree with Adam! you could rather skip the SDS-PAGE step and analyze the sample by LC-MS. The objective of the in-gel digestion probably is to ease the peptides out of the gel matrix. I think you should have a better coverage without the SDS-PAGE since not all peptides may be extracted if you do an in-gel digestion.It may also be interesting to use a combination of enzymes if already not considered.
Hi Adam! How would the second digestion step help? I am just curious and havent come across this protocol. I think aggregation could also bury the sites of trypsin hydrolysis into the aggregates and hence may not give the same coverage! Although I may be wrong, hence the query!
The 2-step procedure was something I used a long time ago when trypsin digesting a large multisubunit protein, chloroplast coupling factor 1 (CF1), molecular weight 400,000. The idea was that the first digestion would leave some undigested pieces, but that boiling the partially digested sample would denature these pieces enough to allow them to be digested in the second step. Precipitation was not observed during the boiling because of the first digestion. It may not be needed for other proteins. One step may be sufficient. This can easily be checked by after adding serine protease inhibitor to kill any residual trypsin activity, then running the sample on a gel to see if any large pieces remain.
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