FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides

J Proteome Res. 2009 May;8(5):2201-10. doi: 10.1021/pr800654s.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anaphase-Promoting Complex-Cyclosome
  • Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Apc5 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Cell Cycle Proteins / analysis
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cloning, Molecular
  • Gene Expression
  • HeLa Cells
  • Humans
  • Isotope Labeling / methods
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Oligopeptides / chemistry
  • Proteins / analysis*
  • Proteins / chemistry
  • Proteins / genetics
  • Proteomics / methods*
  • Reproducibility of Results
  • Ubiquitin-Protein Ligase Complexes / analysis
  • Ubiquitin-Protein Ligase Complexes / chemistry
  • Ubiquitin-Protein Ligase Complexes / genetics

Substances

  • ANAPC5 protein, human
  • Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Apc5 Subunit, Anaphase-Promoting Complex-Cyclosome
  • CDC27 protein, human
  • Cell Cycle Proteins
  • Oligopeptides
  • Proteins
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome