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Phosphoproteomics and proteomics of insulin signaling in aged mouse bone

ABSTRACT: We compared the in vivo insulin signaling response in bone tissue of 10- verses 73-week old C57BL/6J male mice (Figure 1A). Saline or insulin (2.5 mU/kg) was interperitoneally injected and mice sacrificed after 20 min. Tibia bone was rapidly dissected and flushed with ice-cold PBS within 30 sec to remove marrow and quench phosphorylation before being snap frozen in liquid nitrogen. Protein was extracted from powdered bone tissue and digested with trypsin and LysC followed by multiplexed stable isotope labelling with Tandem Mass Tags (TMT). Enriched phosphopeptides were analyzed by multi-dimensional ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (2DLC-MS/MS). In parallel, we also quantified non-enriched peptides to analyze relative proteome abundance differences. All mice were kept on a standard chow diet.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Tibia

SUBMITTER: Benjamin Parker

LAB HEAD: Benjmain Parker

PROVIDER: PXD054205 | Pride | 2025-12-16

REPOSITORIES: Pride

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			Action	DRS
	200221_Eclipse_BenP_YvO_total_1.raw	Raw
	200221_Eclipse_BenP_YvO_total_10.raw	Raw
	200221_Eclipse_BenP_YvO_total_11.raw	Raw
	200221_Eclipse_BenP_YvO_total_12.raw	Raw
	200221_Eclipse_BenP_YvO_total_2.raw	Raw

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Project description:Ten healthy male participants were recruited and successfully completed all preliminary testing and the two exercise bouts (Figure 1A). This randomized crossover design permitted signaling responses to workload- and duration-matched HIIT and MICT exercise to be mapped and interrogated within the same participant. Baseline whole-body anthropometric and blood glucose and lipid measurements confirmed these participants (age 25.4 ± 3.2 y; BMI 23.5 ± 1.6 kg/m2) were metabolically healthy, and maximal exercise capacity testing confirmed they were recreationally active but relatively untrained (relative V̇O2 peak 37.9 ± 5.2) in line with our recruitment strategy to maximize detection of skeletal muscle signaling responses to exercise (Figure 1B). Peak power output (Wpeak; 207.5 ± 40.3 W) was used to prescribe the relative work-matched intensities for HIIT and MICT. Lean mass from the DXA scan and resting metabolic rate (Figure 1B) were used to prescribe a standardized meal for each participant to consume prior to each exercise trial day. Following consumption of a standardized dinner meal the night before the HIIT or MICT exercise trial, blood and skeletal muscle biopsy samples were collected in the fasted state at baseline and at 5 min and 10 min of each respective exercise intensity. Participants completing the acute bout of HIIT, which consisted of 10 min total of 1-min ‘on’ intervals at 85 ± 0.1% of individual Wpeak (176 ± 34 W) and 1-min ‘off’ intervals at 50 W, displayed increased plasma lactate concentrations at 5 and 10 min of exercise relative to pre-exercise baseline (Figure 1C; P < 0.0001 and P < 0.001, respectively; main effect of time P < 0.0001) and no changes in plasma glucose (Figure 1D). In response to total work- and duration-matched acute bout of MICT at 55 ± 2% individual Wpeak (113 ± 17 W), participants displayed increased plasma lactate concentrations at 5 and 10 min of exercise versus baseline (Figure 1C; P < 0.001; main effect of time P < 0.0001) and no changes in plasma glucose (Figure 1D), with a main effect of higher plasma lactate levels in response to HIIT (Figure 1C; P < 0.05). To map the signaling networks regulated by HIIT and MICT, proteins from each skeletal muscle biopsy sample were extracted, digested to peptides with trypsin, isobarically labelled prior to phosphopeptide enrichment, fractionation and LC-MS/MS phosphoproteomic analysis (Figure 2A).

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Phosphoproteomics and proteomics of insulin signaling in aged mouse bone

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