Proteolytic Labeling With 18O for Comparative Proteomics Studies: Preparation of 18O-Labeled Peptides and the 18O/16O Peptide Mixture

The method reported here uses proteolytic catalysis to introduce two
18
O atoms into the carboxyl termini of peptides in mixtures, and is intended to be part of the work-flow in comparative proteomicsstrategies. Proteins are first cleaved with trypsin in water, and subsequently the peptide products are dried and labeledby incubation with trypsin in
18
O-enriched water. One important aspect of this two-step procedure is that peptides, and not proteins, are dried and redissolvedin H
218
O for the labeling reaction. Incorporation can exceed 95% if it is carried out in water that is sufficiently enriched withH
218
O. The byproduct of the reaction is water. The use of catalytic enzyme immobilized on beads facilitates its removal and terminationof the exchange. In differential proteomic studies, heavy isotope-labeled peptides are combined with peptides carrying
16
O for isotope ratio measurements by mass spectrometry.