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 proteomics strategies. Proteins are first cleaved with trypsin in water, and subsequently the peptide products are dried and labeled by 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 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 ¹⁶ O for isotope ratio measurements by mass spectrometry.