Approach for Identification and Quantification of C-Terminal Peptides: Incorporation of Isotopic Arginine Labeling Based on Oxazolone Chemistry

Abstract

C-termini of proteins often play an important role in various biological processes. The determination of the protein C-terminus is crucial because it provides not only distinct functional annotation but also a way to monitor the proteolysis-modified proteins. In this study, an isotopic labeling approach based on oxazolone chemistry was developed to achieve the identification and quantification of C-termini. Aminolysis reagents such as arginine selectively react with the α-carboxyl group at the peptide C-terminus via an oxazolone-like intermediate. Side chain carboxyl groups do not participate in this reaction. When an isotopic mixture consisting of 50% arginine ((0)Arg) and 50% C6-arginine ((6)Arg) was introduced to react with C-terminus of protein and followed by proteolysis, the C-terminal peptide could be directly recognized in the mass spectrum due to its unique isotopic paired peaks, and the sequence could be interpreted in MS2. Besides, the incorporation of an additional basic amino acid in the C-terminal peptide greatly enhanced the signal intensity for C-termini detection. Moreover, the isotopic arginine labeling strategy could be applied for relative C-termini quantitation. Our method showed an excellent correlation of the measured ratios to theoretical ratios and high reproducibility within 2 orders of magnitude of the dynamic range. The correlation coefficients (R(2)) were higher than 0.99, with the coefficients of variation (CVs) ranging from 1.16 to 10.91%. Finally, the approach was used to analyze the C-termini from Thermoanaerobacter tengcongensis , which was cultured under different temperatures. As a result, 68 C-termini have been identified, and 53 of them were quantified in total using our strategy. In addition, 24 neo-C-terminal peptides have also been discovered.