Proteolytic labeling in (H2O)-O-18 has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic O-16-to-O-18 exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger K-M for the Lys-peptide (4400 +/- 700 muM), when compared to that of the Arg-peptide (K-M 1300 +/- 300 muM), was mainly responsible for the slower reaction with YGGFMK (k(cat)/K-M 0.64 +/- 0.14 muM(-1)min(-1)) compared to YGGFMR (k(cat)/K-M 2.6 +/- 0.9 muM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous O-18(2)-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin O-18(2)-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic O-18 labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to O-18(2)-code peptides for comparative studies after proteolysis has taken place.
