Journal
JOURNAL OF PROTEOME RESEARCH
Volume 8, Issue 11, Pages 4954-4965Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr9007267
Keywords
Human skeletal muscle; Phosphoproteome; HPLC-ESI-MS/MS; Phosphopeptide enrichment; SCX; TiO2; in vivo
Categories
Funding
- American Diabetes Association [7-09-CT-56, 1-09-CR-39]
- NIH [R01DK081750, R01DK47936, R01DK66483]
- Novo Nordisk Foundation
- Danish Medical Research Council
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Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin-resistant muscle using phospho-epitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines, and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis, and glycogen metabolism Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disk proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo
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