In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC−ESI−MS/MS

Abstract

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 phosphoepitope-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 TiO 2 . 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 Ca 2+ -cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc 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.

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

“…Moreover, significant positive correlations were also observed for summative spot volumes (sum of volumes of all spots with the same identified protein) relating to cytoplasmic malate dehydrogenase (MDH1), an enzyme controlling TCA cycle pool size and providing contractile function [27], and the mitochondrial enzymes ACO2, ATP5A1, ATP5B and GBAS. These results, suggesting reduced TCA cycle and mitochondrial protein content in insulin resistance, are consistent with previous findings of reduced enzyme activities [10,28,29], protein expression [16,17,30], altered phosphorylation [16,31,32], altered transcript levels [10,33,34] or altered flux through mitochondrial ATP synthase [8].…”

“…In any case, regulation of PYGM by PTMs might be quite complex. For example, three other phosphorylation sites of PYGM have recently been detected [31]. Activating and deactivating modifications may therefore counterbalance and thus ensure a normal glycogenolysis in insulin-resistant muscle under basal conditions.…”

“…Stronger changes were found in sarcomeric proteins, including insulin-stimulated upregulation of ACTA1, MYL1 and MYL3, and downregulation of MYH2 spots. These changes may largely be due to phosphorylations [31,46] and/or other PTMs. Insulin elicits rapid dynamic remodelling of actin filaments, and this is necessary for GLUT4 translocation [47,48].…”

Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis

“…Given the relatively high expression levels of RALDHs in the brain (4,30), it is likely that the enzymatic activity of RALDHs is acutely regulated by posttranslational modifications, such as phosphorylation. Indeed, phosphorylation sites on RALDHs have been reported by recent quantitative phosphoproteomics studies (32)(33)(34)(35)(36)(37)(38). Further work is needed to elucidate how CaN may directly or indirectly modulate RALDH activity in neurons through regulation of various phosphorylation sites.…”

Mefloquine interferes with glycolysis in schistosomula of Schistosoma mansoni via inhibition of enolase

“…For example, in mammalian cells such as human muscle fibres, enolases may be phosphorylated (Hojlund et al 2009). The functional implication of this phosphorylation is unknown so far.…”