Microwave-assisted ¹⁸O-labeling of proteins catalyzed by formic acid

Oxygen exchange may occur at carboxyl groups catalyzed by acid. The reaction, however, takes at least several days at room temperature. The long-time exchanging reaction often prevents its application from protein analysis. In this study, an ¹⁸O-labeling method utilizing microwave-assisted acid hydrolysis was developed. After being dissolved in ¹⁶O/¹⁸O (1:1) water containing 2.5% formic acid, protein samples were exposed to microwave irradiation. LC-MS/MS analysis of the resulted peptide mixtures indicated that oxygen in the carboxyl groups from glutamic acid, aspartic acid, and the C-terminal residues could be efficiently exchanged with ¹⁸O within less than 15 min. The rate of back exchange was so slow that no detectable back exchange could be found during the HPLC run.