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.
