Quantitative Proteomics in the Study of Phosphotyrosine-Mediated Signal Transduction Pathway

Author(s): Xiquan Liang, Mahbod Hajivandi, Paul Predki, Marshall R. Pope

Journal Name: Current Proteomics

Volume 5 , Issue 3 , 2008

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Many biological functions are regulated by tyrosine phosphorylation: cell proliferation, migration, differentiation, and tumorgenesis among them. Hence, aspects of cellular behavior may be illustrated by monitoring the global dynamics of cellular tyrosine phosphorylation in response to stimuli. In this review, we describe an approach that combines phosphoprotein/peptide enrichment with stable isotope labeling which can quantify unambiguously those changes in phosphorylation status that occur specifically in response to stimulation by growth factor, autocrines, drug treatment or cellular physiology. Current technologies for enrichment of phosphoproteins and peptides including phosphotyrosine or phosphospecific antibodies, SH2 domains, metal oxide and IMAC chemistries coupled to magnetic particles are discussed. Phosphopeptides from cultures supplemented with either light or heavy lysine and arginine appear in mass spectra as sequence-matched isotopomers, but are chemically identical and co-migrate in any other separation. Hence, the quantification of stimulated phosphorylation (dephosphorylation) at specific sites of a protein may be accomplished with MScoupled chromatographic separation by comparing the relative peak area response of sequence matched phosphopeptides arising from two (or more) cell states. Furthermore, we illustrate a crossover methodology that may be used to disentangle true protein binding partners from nonspecific interactions, which is a common problem in affinity enrichment endeavors.

Keywords: SILAC, tyrosine phosphorylation, quantitation, mass spectrometry, IMAC, SH2 domain, anti-phosphotyrosine antibody

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Article Details

Year: 2008
Page: [146 - 156]
Pages: 11
DOI: 10.2174/157016408785909631
Price: $25

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