Additional possible tools for identification of proteins on one- or two-dimensional electrophoresis

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Abstract
Additional, essentially chemical, identification methods of proteins in polyacry‐lamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C‐side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90° for 4–16 h, and the N‐side of serine/threonine with an S‐ethyl trifluorothioacetate vapor at 50° for 6–24 h. The products were analyzed by mass 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° for 2–16 h provided cleavages at the C‐side 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° for 8 h deblocked the N‐formyl group, and the vapor at 20° for 4 h deblocked pyrrolidone carboxylate. N‐acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50° for 1 h, followed by reaction with p‐suifophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C‐terminal sequencing of protein from polyacrylamide gels is also described.