title: 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.
authors.affiliation: Mass Spectrometry Research Program, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Sevinsky
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Brown
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Cargile
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Bundy
authors.forename: James L
authors.initials: JL
authors.lastname: Stephenson
mesh_terms: D001798:Blood Proteins / Q000032:analysis; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D001798:Blood Proteins; D004800:Enzymes, Immobilized; D010103:Oxygen Isotopes; D010455:Peptides; D014357:Trypsin