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

Abstract

Differential labeling of peptides via the use of the 18O-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 18O-labeled peptides into the 16O 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.

Figure 1

MALDI mass spectra of the peptide LVNELTEFAK derived from bovine serum albumin after tryptic digestion, differential labeling with ¹⁶O/¹⁸O at a ratio of 1:1 and isoelectric focusing. The sample in Panel A was digested with soluble trypsin at a protease: substrate ratio of 1:50, followed by addition of PMSF and centrifugal ultrafiltration post-labeling. The sample in Panel B was subjected to the same procedures as a, save initial proteolytic digestion with immobilized trypsin,