title: Quantitative proteomic analysis of phosphotyrosine-mediated cellular signaling networks.
abstract: 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.
authors.affiliation: Biological Engineering Division, MIT, Cambridge, MA, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Alejandro
authors.initials: A
authors.lastname: Wolf-Yadlin
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D019000:Phosphotyrosine