Enzymatic cleavage and HPLC peptide mapping of proteins

Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually > 10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reverse-phase HPLC. Since sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 "unknown" samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.