iTRAQ-coupled 2D LC–MS/MS analysis on differentially expressed proteins in denervated tibialis anterior muscle of Rattus norvegicus

Abstract

To understand the molecular aspects of denervation-induced atrophy of skeletal muscles, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry were performed to detect a total of 260 proteins that were differentially expressed in the rat tibialis anterior muscle at different times (1, 4, and 8 weeks) after rat sciatic nerve transection. Western blot, gene ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted for protein validation, functional annotation, and pathway identification, respectively. Among 260 dysregulated proteins, metabolic enzymes represented the largest class of proteins differentially expressed; a down-regulation of β-enolase might be associated with a decreased expression of fast-twitch myosin-4; the 14-3-3 proteins displayed an up-regulation, which might facilitate the inhibition of mTOR signaling; an up-regulation of α-crystallin B chain might be related to the later onset and the slower progress of atrophy; an up-regulation of phosphatidylethanolamine-binding protein-1 perhaps progressively abrogated the cell survival and antiapoptotic properties during muscle atrophy. These results might contribute to the understanding of molecular mechanisms regulating denervation-induced muscle atrophy.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

“…More recent proteomic approaches have quantified changes in the abundances of specific proteins (58)(59)(60). Our quantitative profile of FSR changes of >100 specific proteins in response to denervation extend these findings and are consistent with gene expression changes in denervated muscle reported for structural proteins, enzymes involved in glycolysis, and mitochondrial ATP synthesis (61).…”

Proteomic and bioinformatic analysis of differentially expressed proteins in denervated skeletal muscle

“…Animal handling procedures followed the Institutional Animal Care Guidelines of Nantong University and were approved by the Administration Committee of Experimental Animals, Jiangsu Province, China. The rats were subjected to sciatic nerve transection (operated groups) or sham operation (control group), as previously described (12). At 1 and 4 weeks following the operation, the TA muscles were rapidly dissected from the operated side of the animals and immediately immersed in liquid nitrogen prior to use.…”

“…Protein samples were extracted from the harvested muscles and quantified using the Protein Assay kit (Bio-Rad, Hercules, CA, USA). iTRAQ labeling of the protein samples was performed as previously described (12). Briefly, 100 µg of each protein sample, obtained by acetone precipitation, was dissolved in 20 µl of dissolution buffer and sequentially reduced, alkylated and digested, followed by labeling with the iTRAQ tags 114, 115 and 116, and pooling for further analysis.…”

“…Online 2D nano LC-MS/MS analysis was performed as previously described (12). Briefly, the peptides in each fraction were resuspended in 20 µl solvent A (water with 0.1% formic acid), separated by nano LC, and analyzed by on-line electrospray tandem mass spectrometry using the LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany).…”

“…The MS raw data were analyzed as previously described (12). Briefly, MS/MS spectra were compared to rat data from the Swiss-Prot database (Release 2010_04) using the SEQUEST software v.28 (revision 12; Thermo Electron Corp.).…”