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 18O atoms into the carboxyl termini of peptides in mixtures, and is intended to be part of the work-flow in comparative proteomics strategies. Proteins are first cleaved with trypsin in water, and subsequently the peptide products are dried and labeled by incubation with trypsin in 18O-enriched water. One important aspect of this two-step procedure is that peptides, and not proteins, are dried and redissolved in H2(18)O for the labeling reaction. Incorporation can exceed 95% if it is carried out in water that is sufficiently enriched with H2(18)O. The byproduct of the reaction is water. The use of catalytic enzyme immobilized on beads facilitates its removal and termination of the exchange. In differential proteomic studies, heavy isotope-labeled peptides are combined with peptides carrying 16O for isotope ratio measurements by mass spectrometry.