PNNL

Abstract

Stable isotope 16O/18O-labeling of the C-terminal carboxyl groups of peptides is among the methods used in shotgun proteomics for relative protein quantitation. However, precise quantitative measurements can be difficult when 18O is replaced with the more common isotope 16O during a trypsin-catalyzed process called “back-exchange” that occurs after labeling. Attempts to improve this method by targeting trypsin activity and/or implementing immobilized trypsin because it can be easily separated from the sample ultimately lead to sample loss. Because of this, the 18O-labeling method cannot be applied toward small-abundant samples that are commonly found in clinical studies. Here, we show that sample loss is minimized by using solution-phase trypsin rather than immobilized trypsin during the labeling process and that trypsin can be effectively quenched by boiling the sample after labeling for 10 minutes.