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

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Abstract
The identification of proteins in complex mixtures is most useful when quantitative information is also obtained. We describe a new type of protein tagging reagent called the visible isotope-coded affinity tag (VICAT) which allows the absolute amount of a target protein or proteins to be quantified in a complex biological sample such as a eukaryotic cell lysate. VICAT reagents tag thiol groups of cysteines or thioacetylated amino groups and introduce into the tryptic peptide a biotin affinity handle, a visible moiety for tracking the chromatographic location of the target peptide by a method other than mass spectrometry, a photocleavable linker for removing a portion of the tag, and an isotope tag for distinguishing sample and internal standard peptides. We demonstrate the use of VICAT reagents together with isoelectric focusing of peptides on an immobilized gel strip followed by combined micro-liquid chromatography/electrospray ionization mass spectrometry operating in selected reaction monitoring mode to determine the absolute abundance of a specific protein, human group V phospholipase A2, in eukaryotic cell lysates. It is found that human lung macrophages contain 66 fmol of this protein per 100 μg of cell protein. Western blot analysis of human group V phospholipase A2 in macrophages gave inconclusive data. VICAT reagents should be useful for numerous applications including the analysis of candidate disease markers in complex mixtures such as serum.