ABSTRACT
For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.
