Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Zhengping Yi, Moulun Luo, Christopher A. Carroll, Susan T. Weintraub, Lawrence J. Mandarino

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Serine phosphorylation of insulin receptor substrate-1 (IRS-I) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandein mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutatliione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-tenninal regions of 1RS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.

Original languageEnglish (US)
Pages (from-to)5693-5699
Number of pages7
JournalAnalytical Chemistry
Issue number17
StatePublished - Sep 1 2005

ASJC Scopus subject areas

  • Analytical Chemistry

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