Identification of Phosphorylation Sites in Insulin Receptor Substrate-1 by Hypothesis-Driven High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry
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- 作者：Zhengping Yi ; Moulun Luo ; Christopher A. Carroll ; Susan T. Weintraub ; and Lawrence J. Mandarino
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：September 1, 2005
- 年：2005
- 卷：77
- 期：17
- 页码：5693 - 5699
- 全文大小：298K
- 年卷期：v.77,no.17(September 1, 2005)
- ISSN：1520-6882
文摘
Serine phosphorylation of insulin receptor substrate-1(IRS-1) can regulate tyrosine phosphorylation of IRS-1and subsequent insulin signaling. The 182 serine and 60threonine residues in IRS-1 make position-by-positionanalysis of potential phosphorylation sites by mutagenesisdifficult. Tandem mass spectrometry provides a moreefficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated themin vitro with various kinases followed by identification ofphosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in thetryptic digests of middle and C-terminal regions of IRS-1treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previouslybeen detected by any method and provide novel candidates for identification in cells or in vivo.