An Isotope Coding Strategy for Proteomics Involving Both Amine and Carboxyl Group Labeling

A stable isotope coding strategy is described for the analysis of all types of tryptic peptides, including those that are N-terminally blocked and from the C-terminus of proteins. The method exploits differential derivatization of amine and carboxyl groups generated during proteolysis as a means of coding. Carboxyl groups produced during proteolysis incorporate ¹⁸ O from H₂ ¹⁸ O. Peptides from the C-terminus of proteins were not labeled with ¹⁸ O unless they contained a basic C-terminal amino acid. Primary amines form controls, and experimental samples were differentially acylated after proteolysis with either ¹ H₃ - or ² H₃ -N -acetoxysuccinamide. When these two types of labeling were combined, unique coding patterns were achieved for peptides arising from the C-termini and blocked N-termini of proteins.