Dataset Information

The exercise-regulated skeletal muscle phosphoproteome

ABSTRACT: Four healthy male volunteers (age: 24 - 27; BMI: 24.2 - 25.9 kg/m2) underwent a single bout of high-intensity bicycle exercise reaching a VO2 max approximately 95% with blood samples and muscle biopsies taken immediately pre- and post-exercise. Subjects reached 95% VO2 max in approximately 9 to 12 min and, significant increases in blood glucose and lactate were observed. Quantitative phosphoproteomic analysis of muscle biopsies pre- and post-exercise was performed with tandem mass tags, phosphopeptide enrichment using titanium dioxide chromatography, sequential elution from immobilized metal ion affinity chromatography and hydrophilic interaction liquid chromatography (TiSH) (PMID: 22906719) followed by nano-ultra high pressure liquid chromatography coupled to tandem mass spectrometry (nanoUHPLC-MS/MS). The analysis included the quantification of non-phosphorylated peptides to investigate changes in protein abundance. A total of 11,903 unique phosphopeptides (8,511 phosphosites with >90% localization probability) and 4,317 protein groups were quantified in all four subjects. The phosphorylation profile in each subject was highly reproducible with an average Pearson’s correlation coefficient r = 0.72. We identified 1,322 phosphopeptides (1,004 phosphosites with >90% localization probability) that were significantly regulated with acute exercise and only 5 protein groups quantified with altered abundance (Fig. 1D; Table S3; t-test P < 0.05 and false discovery rate (FDR) < 0.01). Of the significantly regulated phosphosites, a total of 592 were annotated in the PhosphoSitePlus database (PMID: 22135298) with 412 not previously annotated, representing potentially novel phosphosphorylation sites.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Benjamin Parker

PROVIDER: MSV000080777 | MassIVE | Wed Mar 29 13:38:00 BST 2017

SECONDARY ACCESSION(S): PXD001543

REPOSITORIES: MassIVE

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Publications

Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

Hoffman Nolan J NJ Parker Benjamin L BL Chaudhuri Rima R Fisher-Wellman Kelsey H KH Kleinert Maximilian M Humphrey Sean J SJ Yang Pengyi P Holliday Mira M Trefely Sophie S Fazakerley Daniel J DJ Stöckli Jacqueline J Burchfield James G JG Jensen Thomas E TE Jothi Raja R Kiens Bente B Wojtaszewski Jørgen F P JF Richter Erik A EA James David E DE

Cell metabolism 20151001 5

Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinas ...[more]

PMID: 26437602

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Project description:Purpose. Insulin resistant muscle is resistant to gene expression changes induced by acute exercise. This study was undertaken to identify transcription factors that differentially respond to exercise in insulin resistance. Candidate transcription factors were identified from analysis of 5’-untranslated regions (5’-UTRs) of exercise responsive genes and from analysis of the 5’-UTRs of genes coding for proteins that differ in abundance in insulin resistance. Research design and methods. Muscle biopsies were obtained from lean and obese subjects before and after a single exercise bout. Euglycemic glucose clamps assessed insulin sensitivity. Global proteomics analysis identified differentially abundant proteins. The 5’-UTRs of genes coding for significant proteins were subjected to transcription factor enrichment analysis to identify candidate transcription factors. Q-rt-PCR to determine expression of candidate transcription factors was performed on RNA from resting and post-exercise muscle biopsies; immunoblots quantified protein abundance. Results. Obese subjects were insulin resistant compared to lean but performed exercise at the same intensity. Proteins involved in mitochondrial function, protein targeting and translation, and metabolism were among those significantly different between the groups. Transcription factor enrichment analysis of genes coding for these proteins revealed new candidate transcription factors. Q-rt-PCR analysis of RNA and immunoblot analysis from pre- and post-exercise muscle biopsies revealed several transcription and growth factors that had altered responses to exercise in insulin resistant subjects. Conclusions. These results confirm findings of an association between insulin sensitivity and transcription factor mRNA response to exercise and extend these results to show that obesity also may be a sufficient prerequisite for exercise resistance. Analysis of the muscle proteome together with determination of effects of exercise on expression of transcription factors suggests that abnormal responses of transcription factors to exercise may be responsible for differences in protein abundances in insulin resistant muscle.

Project description:Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is an widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346 ± 18 g) were assigned to 3 independent groups (n= 6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0 h) after or 3 h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (v1.3.0.5). The VO2peak of rats in the 0 h and 3 h groups was 66 ± 5 ml•kg-1•min-1 and 69.8 ± 5 ml•kg-1•min-1, respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10 %) differences. Almost all (97 %) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β1-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation.