Analytical reagents and
mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or
protein function in mixtures of proteins. The methods employ affinity labeled
protein reactive reagents having three portions: an
affinity label (A) covalently linked to a
protein reactive group (PRG) through a
linker group (L). The
linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the
linker with a stable
isotope thereof. These reagents allow for the
selective isolation of
peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated
peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a
protein function in those mixtures. Isolated peptides or reaction products are characterized by
mass spectrometric (MS) techniques. The reagents also provide for differential
isotopic labeling of the isolated peptides or reaction products which facilitates
quantitative determination by
mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global
protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or
biological fluids is affected by a stimulus or by a change in condition or state of the
cell, tissue or
organism from which the sample originated.