Molecular and Cellular Biochemistry

, Volume 364, Issue 1–2, pp 193–207 | Cite as

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

  • Hualin Sun
  • Meiyuan Li
  • Leilei Gong
  • Mei Liu
  • Fei Ding
  • Xiaosong Gu


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.


Skeletal muscle atrophy Denervation iTRAQ 2D LC–MS/MS 



This study was supported by Hi-Tech Research and Development Program of China (863 Program, Grant No. 2006AA02A128), National Natural Science Foundation of China (Grant Nos. 81130080, 81171180, 30870811 and 30670667), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Research Program of Education Ministry (Grant No. 208044), and Nantong Science and Technology Innovation Program (Grant No. BK2011045). We thank Professor Jie Liu for the assistance in manuscript preparation.


  1. 1.
    Tews DS, Behrhof W, Schindler S (2005) Expression patterns of initiator and effector caspases in denervated human skeletal muscle. Muscle Nerve 31:175–181PubMedCrossRefGoogle Scholar
  2. 2.
    Baumann AP, Ibebunjo C, Grasser WA, Paralkar VM (2003) Myostatin expression in age and denervation-induced skeletal muscle atrophy. J Musculoskelet Neuronal Interact 3:8–16PubMedGoogle Scholar
  3. 3.
    Donoghue P, Ribaric S, Moran B, Cebasek V, Erzen I, Ohlendieck K (2004) Early effects of denervation on Ca(2+)-handling proteins in skeletal muscle. Int J Mol Med 13:767–772PubMedGoogle Scholar
  4. 4.
    Isfort RJ, Wang F, Greis KD, Sun Y, Keough TW, Farrar RP, Bodine SC, Anderson NL (2002) Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy. Proteomics 2:543–550PubMedCrossRefGoogle Scholar
  5. 5.
    Sun H, Liu J, Ding F, Wang X, Liu M, Gu X (2006) Investigation of differentially expressed proteins in rat gastrocnemius muscle during denervation-reinnervation. J Muscle Res Cell Motil 27:241–250PubMedCrossRefGoogle Scholar
  6. 6.
    Jia L, Xu L, Jiang M, Gu Y, Zhang Z (2005) Protein abnormality in denervated skeletal muscles from patients with brachial injury. Microsurgery 25:316–321PubMedCrossRefGoogle Scholar
  7. 7.
    Finamore F, Pieroni L, Ronci M, Marzano V, Mortera SL, Romano M, Cortese C, Federici G, Urbani A (2010) Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study. Blood Transfus 8(3):s140–s148PubMedGoogle Scholar
  8. 8.
    Saito H, Dahlin LB (2008) Expression of ATF3 and axonal outgrowth are impaired after delayed nerve repair. BMC Neurosci 9:88PubMedCrossRefGoogle Scholar
  9. 9.
    Lu H, Yang Y, Allister EM, Wijesekara N, Wheeler MB (2008) The identification of potential factors associated with the development of type 2 diabetes: a quantitative proteomics approach. Mol Cell Proteomics 7:1434–1451PubMedCrossRefGoogle Scholar
  10. 10.
    Sun H, Zhu T, Ding F, Hu N, Gu X (2009) Proteomic studies of rat tibialis anterior muscle during postnatal growth and development. Mol Cell Biochem 332:161–171PubMedCrossRefGoogle Scholar
  11. 11.
    Sato Y, Shimizu M, Mizunoya W, Wariishi H, Tatsumi R, Buchman VL, Ikeuchi Y (2009) Differential expression of sarcoplasmic and myofibrillar proteins of rat soleus muscle during denervation atrophy. Biosci Biotechnol Biochem 73:1748–1756PubMedCrossRefGoogle Scholar
  12. 12.
    Ong SE, Pandey A (2001) An evaluation of the use of two-dimensional gel electrophoresis in proteomics. Biomol Eng 18:195–205PubMedCrossRefGoogle Scholar
  13. 13.
    Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R (2000) Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci USA 97:9390–9395PubMedCrossRefGoogle Scholar
  14. 14.
    Elliott MH, Smith DS, Parker CE, Borchers C (2009) Current trends in quantitative proteomics. J Mass Spectrom 44:1637–1660PubMedGoogle Scholar
  15. 15.
    Hakimov HA, Walters S, Wright TC, Meidinger RG, Verschoor CP, Gadish M, Chiu DK, Stromvik MV, Forsberg CW, Golovan SP (2009) Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization. Proteomics 9:4000–4016PubMedCrossRefGoogle Scholar
  16. 16.
    Schuchardt S, Borlak J (2008) Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics. Mass Spectrom Rev 27:51–65PubMedCrossRefGoogle Scholar
  17. 17.
    DeSouza LV, Grigull J, Ghanny S, Dube V, Romaschin AD, Colgan TJ, Siu KW (2007) Endometrial carcinoma biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry. Mol Cell Proteomics 6:1170–1182PubMedCrossRefGoogle Scholar
  18. 18.
    DeSouza LV, Romaschin AD, Colgan TJ, Siu KW (2009) Absolute quantification of potential cancer markers in clinical tissue homogenates using multiple reaction monitoring on a hybrid triple quadrupole/linear ion trap tandem mass spectrometer. Anal Chem 81:3462–3470PubMedCrossRefGoogle Scholar
  19. 19.
    O’Leary MF, Hood DA (2008) Effect of prior chronic contractile activity on mitochondrial function and apoptotic protein expression in denervated muscle. J Appl Physiol 105:114–120PubMedCrossRefGoogle Scholar
  20. 20.
    Adhihetty PJ, O’Leary MF, Chabi B, Wicks KL, Hood DA (2007) Effect of denervation on mitochondrially mediated apoptosis in skeletal muscle. J Appl Physiol 102:1143–1151PubMedCrossRefGoogle Scholar
  21. 21.
    Batt J, Bain J, Goncalves J, Michalski B, Plant P, Fahnestock M, Woodgett J (2006) Differential gene expression profiling of short and long term denervated muscle. FASEB J 20:115–117PubMedGoogle Scholar
  22. 22.
    Kato K, Shimizu A, Semba R, Satoh T (1985) Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles. Biochim Biophys Acta 841:50–58PubMedCrossRefGoogle Scholar
  23. 23.
    Merkulova T, Dehaupas M, Nevers MC, Creminon C, Alameddine H, Keller A (2000) Differential modulation of alpha, beta and gamma enolase isoforms in regenerating mouse skeletal muscle. Eur J Biochem 267:3735–3743PubMedCrossRefGoogle Scholar
  24. 24.
    Shinozaki S, Chiba T, Kokame K, Miyata T, Ai M, Kawakami A, Kaneko E, Yoshida M, Shimokado K (2007) Site-specific effect of estradiol on gene expression in the adipose tissue of ob/ob mice. Horm Metab Res 39:192–196PubMedCrossRefGoogle Scholar
  25. 25.
    Patterson MF, Stephenson GM, Stephenson DG (2006) Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat. Am J Physiol Cell Physiol 291:C518–C528PubMedCrossRefGoogle Scholar
  26. 26.
    Zanin M, Germinario E, Dalla Libera L, Sandona D, Sabbadini RA, Betto R, Danieli-Betto D (2008) Trophic action of sphingosine 1-phosphate in denervated rat soleus muscle. Am J Physiol Cell Physiol 294:C36–C46PubMedCrossRefGoogle Scholar
  27. 27.
    Miyazaki M, Esser KA (2009) REDD2 is enriched in skeletal muscle and inhibits mTOR signaling in response to leucine and stretch. Am J Physiol Cell Physiol 296:C583–C592PubMedCrossRefGoogle Scholar
  28. 28.
    Sakurai T, Fujita Y, Ohto E, Oguro A, Atomi Y (2005) The decrease of the cytoskeleton tubulin follows the decrease of the associating molecular chaperone alphaB-crystallin in unloaded soleus muscle atrophy without stretch. FASEB J 19:1199–1201PubMedGoogle Scholar
  29. 29.
    Schulte L, Peters D, Taylor J, Navarro J, Kandarian S (1994) Sarcoplasmic reticulum Ca2+ pump expression in denervated skeletal muscle. Am J Physiol 267:C617–C622PubMedGoogle Scholar
  30. 30.
    Atomi Y, Yamada S, Nishida T (1991) Early changes of α B-crystallin mRNA in rat skeletal muscle to mechanical tension and denervation. Biochem Biophys Res Commun 181:1323–1330PubMedCrossRefGoogle Scholar
  31. 31.
    Smith SC, Theodorescu D (2009) Learning therapeutic lessons from metastasis suppressor proteins. Nat Rev Cancer 9:253–264PubMedCrossRefGoogle Scholar
  32. 32.
    Zeng L, Imamoto A, Rosner MR (2008) Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target. Expert Opin Ther Targets 12:1275–1287PubMedCrossRefGoogle Scholar
  33. 33.
    Park S, Rath O, Beach S, Xiang X, Kelly SM, Luo Z, Kolch W, Yeung KC (2006) Regulation of RKIP binding to the N-region of the Raf-1 kinase. FEBS Lett 580:6405–6412PubMedCrossRefGoogle Scholar
  34. 34.
    Beshir AB, Ren G, Magpusao AN, Barone LM, Yeung KC, Fenteany G (2010) Raf kinase inhibitor protein suppresses nuclear factor-kappaB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression. Cancer Lett 299:137–149PubMedCrossRefGoogle Scholar
  35. 35.
    Raffaello A, Laveder P, Romualdi C, Bean C, Toniolo L, Germinario E, Megighian A, Danieli-Betto D, Reggiani C, Lanfranchi G (2006) Denervation in murine fast-twitch muscle: short-term physiological changes and temporal expression profiling. Physiol Genomics 25:60–74PubMedCrossRefGoogle Scholar
  36. 36.
    Tang H, Cheung WM, Ip FC, Ip NY (2000) Identification and characterization of differentially expressed genes in denervated muscle. Mol Cell Neurosci 16:127–140PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Hualin Sun
    • 1
  • Meiyuan Li
    • 1
  • Leilei Gong
    • 1
  • Mei Liu
    • 1
  • Fei Ding
    • 1
  • Xiaosong Gu
    • 1
  1. 1.Jiangsu Key Laboratory of NeuroregenerationNantong UniversityNantongChina

Personalised recommendations