Facile, effective means for determining the carboxyl-terminal amino acid sequence of proteins have long been sought. One promising approach utilizes mass spectrometric analysis of peptide fragments produced by enzymatic degradation of the polypeptide of interest with carboxypeptidases, wherein differences in the measured masses of the degradation products define the carboxyl-terminal sequence. However, the utility of this approach has been limited by large discrepancies in the rates of digestion of different terminal amino acid residues, leading to difficulties in producing uninterrupted sequence-defining peptide fragments. In order to ensure a mixture of uninterrupted sequence-defining peptide fragments (i.e., a continuous """"""""peptide ladder""""""""), it is necessary to either diminish differential rates of terminal amino acid removal or to render a fraction of each peptide product resistant to further degradation. Towards this objective, we have explored a new strategy in which hydrolysis and aminolysis are set up as competing reactions catalyzed by the same exopeptidase. The hydrolysis reaction removes the terminal amino acid residue, while the aminolysis (reverse proteolysis) reaction is designed to add a terminating group to the newly formed termini. We use an amino acid-amide (lysinamide) as a terminating reagent because it competes effectively as a nucleophile with water for the acyl-enzyme intermediate and because the resulting peptide amide is relatively resistant to hydrolysis. Our results demonstrate that kinetic effects resulting from the addition of a large molar excess of lysinamide can considerably improve the control of carboxypeptidase digestion for carboxyl-terminal sequencing by mass spectrometric readout of the resulting peptide ladders. Large discrepancies in enzyme digestion rates tend to be evened out because both hydrolysis and aminolysis are catalyzed by the enzyme. Although further optimization is desirable, the present strategy has the potential to provide an easy and reliable method for obtaining limited carboxyl-terminal sequences of peptides and proteins. To assist in the c-terminal sequencing of proteins by the method outlined above, we have also begun to develop a practical means for isolating the C-terminal peptide from a lys-C digest of a protein (see following subproject).
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