ADDITIONAL POSSIBLE TOOLS FOR IDENTIFICATION OF PROTEINS ON ONE-DIMENSIONAL OR 2-DIMENSIONAL ELECTROPHORESIS

Additional, essentially chemical, identification methods of proteins i n polyacrylamide gel electrophoresis are described. Two cleavages of p eptide bonds were used at the C-side of aspartic acid with a 0.2% pent afluoropropionic acid (PFPA) aqueous vapor at 90 degrees C for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioaceta te vapor at 50 degrees C for 6-24 h. The products were analyzed by mas s spectrometry - peptide mass fingerprinting. A new type of C-terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90 degrees C for 2-16 h provided cleavages at the C-sid e of aspartic acid and the N-side of serine/threonine and simultaneous successive truncation at the C-termini of the cleaved fragments. The product resulted in C-terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at -5 degrees C for 8 h deblocke d the N-formyl group,and the vapor at 20 degrees C for 4 h deblocked p yrrolidone carboxylate. N-acetylserine/threonine was deblocked by aque ous vapor of 75% PFPA at 50 degrees C for 1 h, followed by reaction wi th p-sulfophenylisothiocyanate at pH 6.0. These methods were applied t o a variety of protein spots on polyacrylamide gels. A new stepwise C- terminal sequencing of protein from polyacrylamide gels is also descri bed.