Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Journal of the American Society for Mass Spectrometry
Zhengping YiSusan T Weintraub

Abstract

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using...Continue Reading

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Citations

Mar 25, 2008·Journal of the American Society for Mass Spectrometry·Pegah R Jalili, Haydn L Ball
Jul 3, 2010·Journal of the American Society for Mass Spectrometry·Paul LanglaisZhengping Yi
Feb 18, 2011·Endocrinology·Paul LanglaisLawrence J Mandarino
Dec 2, 2008·Biochimica Et Biophysica Acta·Sara FröjdöLuciano Pirola
Jan 3, 2007·Journal of Mass Spectrometry : JMS

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