Mapping Tyrosine Phosphorylation Networks by Chemical Proteomics

Abstract

Chemical Proteomics is an unbiased approach to cellular signaling, but it relies on the ability to chemically react a specific post‐translational modification for analysis. Previously, a Beta‐elimination/Michael's addition reaction has been developed to chemically probe phosphorylated Serine and Threonine residues. The stability provided to the phosphate in by attachment to the ring structure of Tyrosine makes this process impossible. Herein, I present a procedure for chemically labeling phospho‐Tyrosine (pY) through a modified Suzuki coupling on tryptic peptides. This is followed by a biotin‐streptavidin affinity based enrichment and mass spectrometric (MS) analysis. This procedure solves the two main problems of pY analysis. First, the “flyability” of the peptide is increased drastically by removing the negative charge that is present on the phosphate (pKa=1.76) at the standard pH (~2.0) used in MS. Second, and as importantly, the problem of rarity of phosphorylated peptides in a cellular system is also solved. With the addition of SILAC, ICAT, or other isotopic labeling techniques it is straightforward to map the propagation of signaling along pathways and possibly uncover novel effects by that signaling that can not be determined via conventional methods.Support or Funding InformationW.E.R is a recipient of an Arne N. Wick pre‐doctoral research fellowship from the California Metabolic Research Foundation.