A simple procedure for effective quenching of trypsin activity and prevention of 18O-labeling back-exchange.

Trypsin-catalyzed stable isotope 16O/18O-labeling of the C-terminal carboxyl groups of peptides is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by residual trypsin that can catalyze the back-exchange of 18O with 16O after labeling. Here, we demonstrate through a detailed evaluation that boiling the peptide sample for 10 min provides a simple means for completely quenching residual trypsin activity and preventing oxygen back-exchange in 18O-labeled samples. We also observed that the presence of organic solvents such as acetonitrile made boiling less efficient for inactivating trypsin. Finally, current 18O-labeling methods that typically employ immobilized trypsin result in significant sample losses due to nonspecific binding of peptides to the resin, making their application toward smaller biological samples increasingly impractical. We present here an improved 18O-labeling protocol that is more applicable to microscale biological samples by using solution-phase trypsin instead of immobilized trypsin. The ability to generate stably 18O-labeled samples without back-exchange should enable more effective applications of 18O-labeling toward large-scale biomarker discovery and validations where an 18O-labeled sample can be used as a common reference for quantitation.