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 through both autophosphorylation and phosphorylation of tyrosine residues on selected substrates, stimulating a variety of signal transduction 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 previously been available. We have recently developed a methodology enabling the simultaneous quantification of tyrosine phosphorylation of 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 culture and stimulation, sample preparation for stable isotope labeling and peptide immunoprecipitation, immobilized metal affinity chromatography-liquid chromatography-tandem mass spectrometry analysis of affinity-enriched tyrosine phosphorylated peptides, and analysis of the resulting MS data.