title: FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides.
abstract: Measurements of protein abundance and quantitative assessment of multiple post-translational modifications (PTMs) within a single protein are increasingly used to understand the control of protein activity, particularly in metazoan cells. General methods of wide applicability and precision/accuracy for quantitative estimation of protein post-translational regulation are lacking. Protein mass spectrometry has evolved from a high-throughput qualitative technique to a potentially general quantitative tool, but there are still serious limitations in dynamic range and coverage. To address some of these limitations, we introduce a novel MS-based quantitative strategy, FLEXIQuant, (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification), which can track changes in relative peptide abundances as a function of PTM, and determine absolute quantity of a protein from its lysate. We examined two subunits of the anaphase-promoting complex, CDC27 and APC5, as a test of our ability to monitor quantitatively, the PTM status of several peptides over time. We find evidence of differential regulation at different sites, a phenomenon we believe will be very widespread. FLEXIQuant proved itself to be capable of serving as a general quantitative tool.
authors.affiliation: Department of Pathology, Harvard Medical School and Children's Hospital Boston, Boston, Massachusetts 02115, USA.
authors.forename: Sasha
authors.initials: S
authors.lastname: Singh
authors.forename: Michael
authors.initials: M
authors.lastname: Springer
authors.forename: Judith
authors.initials: J
authors.lastname: Steen
authors.forename: Marc W
authors.initials: MW
authors.lastname: Kirschner
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen