Minimizing back exchange in 18O/16O quantitative proteomics experiments by incorporation of immobilized trypsin into the initial digestion step

Differential labeling of peptides via the use of the O-18-water proteolytic labeling method has been widely adopted for quantitative shotgun proteomics studies due to its simplicity and low reagent costs. In this report, the use of immobilized trypsin in the initial digestion step, in addition to the initial digestion step, is explored as a means to minimize postlabeling back exchange of O-18-labeled peptides into the O-16 form when multidimensional peptide separation methods (here, isoelectric focusing of peptides) are incorporated into the sample workflow. Examples are shown with a mixture of standard proteins and a sample from an ongoing clinical proteomics study.