Selective Chemical Cleavage Methods in Proteomics, Including C-Terminal Successive Degradation

The addition of trifluoroacetic acid (TFA) to HC1, in both liquid (1) and vapor phases (2), increases the hydrolysis efficiency of peptide bonds, suggesting a cleavage type other than conventional HC1 hydrolysis. Partial acid hydrolysis using high concentrations of perflooric acid have indicated a novel cleavage specificity of peptide bonds, different from those of either Sanger’s high-concentration acid hydrolysis (3) or the conventional dilute-acid hydrolysis (4). This chapter summarizes this high-concentration perfluoric acid, which essentially allows C-terminal protein sequencing as well as specific site cleavages at the C-side of Asp and the N-side of Ser/Thr. Akabori et al. (5) used anhydrous hydrazine for protein C-terminal determination. In this reaction, internal peptide bonds are hydrazinolyzed, yielding the hydrazides of the constituent amino acids and peptides and allowing the residual C-terminal amino acid residue to be identified (6). The similar reaction has been widely used for the deglycosylation of glycoprotein (7). Recently, milder conditions, including the use of hydrazine hydrate, were developed for the deblocking of N-blocked protein and for the specific cleavage of Asn residues (8,9). This chapter also includes a discussion of this Asn C-side peptide bond cleavage by hydrazine and hydrazine-hydrate.