Absolute Quantification of Specific Proteins in Complex Mixtures Using Visible Isotope-Coded Affinity Tags

There is intense interest in determining the absolute abundance of specific proteins in complex mixtures, for example in thearea of disease biomarker discovery. We have developed a set of protein-tagging reagents called visible isotope-coded affinitytags (VICAT reagents) that contain a protein-tagging reagent for reaction with cysteine SH groups, a visible probe for monitoringthe chromatographic behavior of the target peptides, a photo-releasable biotin affinity tag for selective capture and releaseof tagged peptides, and a heavy isotope tag for differentiating analyte from internal standards. These reagents are used togetherwith isoelectric focusing and reverse-phase microbore chromatography/electrospray ionization/tandem mass spectrometry to determinethe absolute abundance of a set of target proteins in a complex mixture, such as a cell lysate. VICAT reagents should alsobe useful for detecting low abundance proteins in biological fluids such as serum, and for the detection of posttranslationalprotein modifications and different splice variants.