PICquant: A Quantitative Platform To Measure Differential Peptide Abundance Using Dual-Isotopic Labeling with 12C6- and 13C6-Phenyl Isocyanate

摘要

ABSTRACT: We have developed a complete system for thenisotopic labeling, fractionation, and automated quantificationnof differentially expressed peptides that significantly facilitatesncandidate biomarker discovery.We describe a new stablemassntagging reagent pair,n12nC6- and 13nC6-phenyl isocyanate (PIC),nthat offers significant advantages over currently available tags.nPeptides are labeled predominantly at their amino termini andnexhibit elution profiles that are independent of label isotope.nImportantly, PIC-labeled peptides have unique neutral-massnlosses upon CID fragmentation that enable charge state andnlabel isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. Tonexploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS2ndata acquisition strategynand a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach,ndubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwisenconfound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the systemnallows rapid identification of peptides that are differentially expressed between control and patient groups.