In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

Kurt Højlund, Benjamin P. Bowen, Hyonson Hwang, Charles R. Flynn, Lohith Madireddy, Thangiah Geetha, Paul Langlais, Christian Meyer, Lawrence J. Mandarino, Zhengping Yi

Research output: Contribution to journalArticlepeer-review

76 Scopus citations


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 3-5mg 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.

Original languageEnglish (US)
Pages (from-to)4954-4965
Number of pages12
JournalJournal of Proteome Research
Issue number11
StatePublished - Nov 6 2009


  • Human skeletal muscle
  • In vivo
  • Phosphopeptide enrichment
  • Phosphoproteome
  • SCX
  • Tio

ASJC Scopus subject areas

  • Biochemistry
  • General Chemistry


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