Quantitative Proteomic Analysis of Phosphotyrosine-Mediated Cellular Signaling Networks

Receptor tyrosine kinases receive extracellular cues, such as ligand binding, and transmit this information to the cell throughboth autophosphorylation and phosphorylation of tyrosine residues on selected substrates, stimulating a variety of signaltransduction pathways. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues,may play a role in determining cellular response, but experimental data required for analysis of these features have not previouslybeen available. We have recently developed a methodology enabling the simultaneous quantification of tyrosine phosphorylationof specific residues on dozens of key proteins in a time-resolved manner, downstream of receptor tyrosine kinase activation. In this chapter, we present a detailed description of this mass spectrometry-based method, including conditions for cell cultureand stimulation, sample preparation for stable isotope labeling and peptide immunoprecipitation, immobilized metal affinitychromatography-liquid chromatography -tandem mass spectrometry analysis of affinity-enriched tyrosine phosphorylated peptides,and analysis of the resulting MS data.