The exercise-regulated skeletal muscle phosphoproteome

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.