TY - JOUR
T1 - Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry
AU - Yi, Zhengping
AU - Luo, Moulun
AU - Carroll, Christopher A.
AU - Weintraub, Susan T.
AU - Mandarino, Lawrence J.
PY - 2005/9/1
Y1 - 2005/9/1
N2 - 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.
AB - 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.
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U2 - 10.1021/ac050760y
DO - 10.1021/ac050760y
M3 - Article
C2 - 16131083
AN - SCOPUS:24644509696
SN - 0003-2700
VL - 77
SP - 5693
EP - 5699
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 17
ER -