TY - JOUR
T1 - Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS
AU - Zhang, Xiangmin
AU - Højlund, Kurt
AU - Luo, Moulun
AU - Meyer, Christian
AU - Geetha, Thangiah
AU - Yi, Zhengping
N1 - Funding Information:
This work was supported by Clinical/translational Research Award from the American Diabetes Association 7‐09‐CT‐56 (ZY), a Clinical Research Grant from the American Diabetes Association 1‐09‐CR‐39 (CM) and NIH grants R01DK081750 (ZY) and R21DK082820 (CM).
PY - 2012/7/16
Y1 - 2012/7/16
N2 - Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca 2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.
AB - Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca 2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.
KW - HPLC-ESI-MS/MS
KW - Rat muscle
KW - Tyrosine immunoprecipitation
KW - Tyrosine phosphorylation
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U2 - 10.1016/j.jprot.2012.05.009
DO - 10.1016/j.jprot.2012.05.009
M3 - Article
C2 - 22609512
AN - SCOPUS:84862644519
SN - 1874-3919
VL - 75
SP - 4017
EP - 4026
JO - Journal of Proteomics
JF - Journal of Proteomics
IS - 13
ER -