Abstract
Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.
Highlights
Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance
We identified 954 proteins, including all enzymes participating in the major pathways of glucose and lipid metabolism, a large number of proteins involved in mitochondrial oxidative phosphorylation and calcium homeostasis, and most isoforms of the proteins that constitute the myofibrillar apparatus
Characteristics of Human Skeletal Muscle Proteome—To obtain a comprehensive proteomic characterization of the human vastus lateralis muscle, we carried out HPLC-ESI-MS/ MS-based analysis of lysates of whole muscle from which proteins were first fractionated by one-dimensional gel electrophoresis
Summary
Subjects—The skeletal muscle samples used for the proteomics analyses in this study were obtained from three healthy, nonobese volunteers (I-III; age, 30 – 47 years; body mass index, 24 –28 kg/m2; percent body fat, 23–34%) with normal glucose tolerance and no family history of type 2 diabetes. The abbreviations used are: FA, formic acid; ACN, acetonitrile; CID, collision-induced dissociation, OXPHOS, oxidative phosphorylation; MW, molecular weight; MS/MS, tandem mass spectrometry; HPLC, high performance liquid chromatography; ESI electrospray ionization. Experiment 1: subject I, 75 g of muscle lysate, proteins separated on a 10% SDS-polyacrylamide gel, and HPLC-MS/MS run in duplicate (Experiments 1–1 and 1–2). Experiments 2–1 and 2–2: 60 g of muscle lysate proteins from subjects II and III, respectively, were separated on 4 –20% gradient SDS-polyacrylamide gels; proteins were visualized with Coomassie blue (Sigma Chemical Co., St. Louis, MO). Charge states and monoisotopic peak assignments were verified using DTA-SuperCharge, part of the MSQuant suite of software [32], before all “DTA” files from each gel lane in an experiment were combined into a single Mascot Generic format file. Probability assessment of peptide assignments and protein identifications were made through use of Scaffold (version Scaffold-01_06_17, Proteome Software Inc., Portland, OR). IPI accession numbers for identified proteins were input into the UniProt database (www.pir.uniprot.org) to obtain Gene Ontology annotation
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