Humana Press, Methods in Molecular Biology, p. 203-212, 2007
DOI: 10.1007/978-1-59745-255-7_14
Quantitative Proteomics by Mass Spectrometry, p. 203-212
DOI: 10.1385/1-59745-255-6:203
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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.