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.
CITATION STYLE
Zhang, Y., Wolf-Yadlin, A., & White, F. M. (2007). Quantitative Proteomic Analysis of Phosphotyrosine-Mediated Cellular Signaling Networks (pp. 203–212). https://doi.org/10.1007/978-1-59745-255-7_14
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