DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

Abstract

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.

Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera)

“…ATP synthase expression also increased with advancing age in the trophocytes and fat cells of workers. Previous studies have reported similar findings, showing that ATP synthase expression increases with advancing age in the brains and muscle of rats (Nicoletti et al 1995;Chang et al 2007;Doran et al 2008;Donoghue et al 2010), and in muscle of humans (Gelfi et al 2006). Although ND1 and ATP synthase expression measured in absolute terms increased with advancing age in the trophocytes and fat cells of workers, the relative expression of ND1 and ATP synthase normalized to mitochondrial density and VDAC1 was not significantly different.…”

Proteomic profiling of bovine M. longissimus lumborum from Crossbred Aberdeen Angus and Belgian Blue sired steers varying in genetic merit for carcass weight1

“…As differences exist in fat accumulation between BB-and AA-sired steers, the authors took care during tissue collection to harvest lean muscle tissue only, avoiding intramuscular fat. In addition the protocol used in this study was optimized for protein extraction from muscle tissue (Mullen et al, 2011;Donoghue et al, 2010). There is also no evidence of differential expression of markers of fat metabolism between the AA-and BB-sired steers in the current study.…”

“…Progenesis SameSpots analysis (SSA) was used to identify proteins of interest as described by Donoghue et al (2010) and Mullen et al (2011). The gel images were placed into groups (AA, BB, AA-High, and AALow), and analysis was carried as follows: AA vs. BB, AA-High vs. AA-Low, and BB-High vs. BB-Low.…”

“…Progenesis Samespots V3.2.3 software (NonLinear Dynamics, Newcastle upon Tyne, UK) was used for the detection of protein spots, background subtraction and detection of proteins with statistically signifi cant differences across groups. Before analysis of individual gels, a single reference gel, representing electrophoretically separated proteins from muscle tissue from all animals in the study, was analyzed and further matched and normalized with non-gel spots manually fi ltered out on the basis of spot volume (Donoghue et al, 2010;Mullen et al, 2011). Differentially expressed protein spots (P < 0.05) between AA and BB were classifi ed as biologically signifi cant with a fold change greater than 1.9 and were selected for tryptic digestion from gels stained with Coomassie Brilliant Blue G-250.…”

Use of Quantitative Membrane Proteomics Identifies a Novel Role of Mitochondria in Healing Injured Muscles

“…Thus, we used this approach to enrich plasma membrane proteins from cultured myoblasts. Cell surface proteins were biotinylated using Sulfo-NHS-LC-Biotin (biotin) and solubilized using RIPA buffer (35,36), and the biotinylated proteins were isolated using streptavidin-conjugated agarose or magnetic beads, as described under "Experimental Procedures." Magnetic beads allowed greater recovery of proteins as compared with agarose beads (data not shown) and were used for all subsequent experiments.…”