Skip to main content
SpringerLink
Log in

Proteomic Profiling of the Dystrophin-Deficient Brain

  • Protocol
  • First Online:
Duchenne Muscular Dystrophy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1687))

Abstract

Duchenne muscular dystrophy is a highly progressive neuromuscular disorder caused by primary abnormalities in the Dmd gene encoding the membrane cytoskeletal protein dystrophin. Dystrophinopathies are multi-systems disorders that are characterized by severe skeletal muscle wasting, with loss of independent ambulation in the early teenage years, followed by cardio-respiratory complications and premature death. Nonprogressive cognitive impairments are estimated to affect approximately one-third of dystrophic children. To identify the molecular mechanisms behind the impaired brain function in dystrophinopathy, liquid chromatography-based mass spectrometry offers an unbiased and technology-driven approach. In this chapter, we give a detailed description of a label-free mass spectrometric method to investigate proteome-wide changes in the dystrophin-deficient brain from a genetic mouse model of Duchenne muscular dystrophy.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509–517

    Article  CAS  PubMed  Google Scholar 

  2. Koenig M, Monaco AP, Kunkel LM (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53:219–228

    Article  CAS  PubMed  Google Scholar 

  3. Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes AH, Belfall B, Klamut HJ, Talbot J, Hodges RS, Ray PN, Worton RG (1988) The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333:466–469

    Article  CAS  PubMed  Google Scholar 

  4. Murphy S, Ohlendieck K (2015) The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle. Comput Struct Biotechnol J 14:20–27

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, Campbell KP (1990) Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 345:315–319

    Article  CAS  PubMed  Google Scholar 

  6. Allen DG, Whitehead NP, Froehner SC (2016) Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy. Physiol Rev 96:253–305

    Article  CAS  PubMed  Google Scholar 

  7. Deconinck N, Dan B (2007) Pathophysiology of duchenne muscular dystrophy: current hypotheses. Pediatr Neurol 36:1–7

    Article  PubMed  Google Scholar 

  8. Shin J, Tajrishi MM, Ogura Y, Kumar A (2013) Wasting mechanisms in muscular dystrophy. Int J Biochem Cell Biol 45:2266–2279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Holland A, Murphy S, Dowling P, Ohlendieck K (2016) Pathoproteomic profiling of the skeletal muscle matrisome in dystrophinopathy associated myofibrosis. Proteomics 16:345–366

    Article  CAS  PubMed  Google Scholar 

  10. Muntoni F, Torelli S, Ferlini A (2003) Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2:731–740

    Article  CAS  PubMed  Google Scholar 

  11. Perronnet C, Vaillend C (2010) Dystrophins, utrophins, and associated scaffolding complexes: role in mammalian brain and implications for therapeutic strategies. J Biomed Biotechnol 2010:849426

    PubMed  PubMed Central  Google Scholar 

  12. Culligan K, Ohlendieck K (2002) Diversity of the brain dystrophin-glycoprotein complex. J Biomed Biotechnol 2:31–36

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Snow WM, Fry M, Anderson JE (2013) Increased density of dystrophin protein in the lateral versus the vermal mouse cerebellum. Cell Mol Neurobiol 33:513–520

    Article  CAS  PubMed  Google Scholar 

  14. Lidov HG, Selig S, Kunkel LM (1995) Dp140: a novel 140 kDa CNS transcript from the dystrophin locus. Hum Mol Genet 4:329–335

    Article  CAS  PubMed  Google Scholar 

  15. Culligan K, Glover L, Dowling P, Ohlendieck K (2001) Brain dystrophin-glycoprotein complex: persistent expression of beta-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy. BMC Cell Biol 2:2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Doran P, Martin G, Dowling P, Jockusch H, Ohlendieck K (2006) Proteome analysis of the dystrophin-deficient MDX diaphragm reveals a drastic increase in the heat shock protein cvHSP. Proteomics 6:4610–4621

    Article  CAS  PubMed  Google Scholar 

  17. Guevel L, Lavoie JR, Perez-Iratxeta C, Rouger K, Dubreil L, Feron M, Talon S, Brand M, Megeney LA (2011) Quantitative proteomic analysis of dystrophic dog muscle. J Proteome Res 10:2465–7248

    Article  CAS  PubMed  Google Scholar 

  18. Rayavarapu S, Coley W, Cakir E, Jahnke V, Takeda S, Aoki Y, Grodish-Dressman H, Jaiswal JK, Hoffman EP, Brown KJ, Hathout Y, Nagaraju K (2013) Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse. Mol Cell Proteomics 12:1061–1073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Holland A, Dowling P, Meleady P, Henry M, Zweyer M, Mundegar RR, Swandulla D, Ohlendieck K (2015) Label-free mass spectrometric analysis of the mdx-4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy-related fibrosis. Proteomics 15:2318–2331

    Article  CAS  PubMed  Google Scholar 

  20. Murphy S, Dowling P, Zweyer M, Mundegar RR, Henry M, Meleady P, Swandulla D, Ohlendieck K (2016) Proteomic analysis of dystrophin deficiency and associated changes in the aged mdx-4cv heart model of dystrophinopathy-related cardiomyopathy. J Proteomics 145:24–36

    Article  CAS  PubMed  Google Scholar 

  21. Anderson JL, Head SI, Rae C, Morley JW (2002) Brain function in Duchenne muscular dystrophy. Brain 125:4–13

    Article  CAS  PubMed  Google Scholar 

  22. Cyrulnik SE, Hinton VJ (2008) Duchenne muscular dystrophy: a cerebellar disorder? Neurosci Biobehav Rev 32:486–496

    Article  CAS  PubMed  Google Scholar 

  23. Waite A, Brown SC, Blake DJ (2012) The dystrophin-glycoprotein complex in brain development and disease. Trends Neurosci 35:487–496

    Article  CAS  PubMed  Google Scholar 

  24. Duchenne G (1867) The pathology of paralysis with muscular degeneration (paralysie myosclerotique), or paralysis with apparent hypertrophy. Br Med J 2:541–542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cotton S, Voudouris NJ, Greenwood KM (2001) Intelligence and Duchenne muscular dystrophy: full-scale, verbal, and performance intelligence quotients. Dev Med Child Neurol 43:497–501

    Article  CAS  PubMed  Google Scholar 

  26. Moizard MP, Billard C, Toutain A, Berret F, Marmin N, Moraine C (1989) Are Dp71 and Dp140 brain dystrophin isoforms related to cognitive impairment in Duchenne muscular dystrophy? Am J Med Genet 80:32–41

    Article  Google Scholar 

  27. Pane M, Lombardo ME, Alfieri P, D’Amico A, Bianco F, Vasco G, Piccini G, Mallardi M, Romeo DM, Ricotti V, Ferlini A, Gualandi F, Vicari S, Bertini E, Berardinelli A, Mercuri E (2012) Attention deficit hyperactivity disorder and cognitive function in Duchenne muscular dystrophy: phenotype-genotype correlation. J Pediatr 161:705–709

    Article  PubMed  Google Scholar 

  28. Banihani R, Smile S, Yoon G, Dupuis A, Mosleh M, Snider A, McAdam L (2015) Cognitive and neurobehavioral profile in boys with duchenne muscular dystrophy. J Child Neurol 30:1472–1482

    Article  PubMed  Google Scholar 

  29. McGreevy JW, Hakim CH, McIntosh MA, Duan D (2015) Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis Model Mech 8:195–213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Partridge TA (2013) The mdx mouse model as a surrogate for Duchenne muscular dystrophy. FEBS J 280:4177–4186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vaillend C, Billard JM, Laroche S (2004) Impaired long-term spatial and recognition memory and enhanced CA1 hippocampal LTP in the dystrophin-deficient Dmd(mdx) mouse. Neurobiol Dis 17:10–20

    Article  CAS  PubMed  Google Scholar 

  32. Vaillend C, Rendon A, Misslin R, Ungerer A (1995) Influence of dystrophin-gene mutation on mdx mouse behavior. I. Retention deficits at long delays in spontaneous alternation and bar-pressing tasks. Behav Genet 25:569–579

    Article  CAS  PubMed  Google Scholar 

  33. Muntoni F, Mateddu A, Serra G (1991) Passive avoidance behaviour deficit in the mdx mouse. Neuromuscul Disord 1:121–123

    Article  CAS  PubMed  Google Scholar 

  34. Holland A, Carberry S, Ohlendieck K (2013) Proteomics of the dystrophin-glycoprotein complex and dystrophinopathy. Curr Protein Pept Sci 14:680–697

    Article  CAS  PubMed  Google Scholar 

  35. Dowling P, Holland A, Ohlendieck K (2014) Mass spectrometry-based identification of muscle-associated and muscle-derived proteomic biomarkers of dystrophinopathies. J Neuromus Dis 1:15–40

    Google Scholar 

  36. Fuller HR, Graham LC, Llavero Hurtado M, Wishart TM (2016) Understanding the molecular consequences of inherited muscular dystrophies: advancements through proteomic experimentation. Expert Rev Proteomics 13:659–671

    Article  CAS  PubMed  Google Scholar 

  37. Hathout Y, Seol H, Han MH, Zhang A, Brown KJ, Hoffman EP (2016) Clinical utility of serum biomarkers in Duchenne muscular dystrophy. Clin Proteomics 13:9

    Article  PubMed  PubMed Central  Google Scholar 

  38. Murphy S, Zweyer M, Henry M, Meleady P, Mundegar RR, Swandulla D, Ohlendieck K (2015) Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy. Clin Proteomics 12:27

    Article  PubMed  PubMed Central  Google Scholar 

  39. Craft GE, Chen A, Nairn AC (2013) Recent advances in quantitative neuroproteomics. Methods 61:186–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rabilloud T, Chevallet M, Luche S, Lelong C (2010) Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteomics 73:2064–2077

    Article  CAS  PubMed  Google Scholar 

  41. Chen EI, Cociorva D, Norris JL, Yates JR III (2007) Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J Proteome Res 6:2529–2253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2009) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol Chapter 10:Unit10.25

    Google Scholar 

  43. Naldrett MJ, Zeidler R, Wilson KE, Kocourek A (2005) Concentration and desalting of peptide and protein samples with a newly developed C18 membrane in a microspin column format. J Biomol Tech 16:423–428

    PubMed  PubMed Central  Google Scholar 

  44. Gorr TA, Vogel J (2015) Western blotting revisited: critical perusal of underappreciated technical issues. Proteomics Clin Appl 9:396–405

    Article  CAS  PubMed  Google Scholar 

  45. Bradd SJ, Dunn MJ (1993) Analysis of membrane proteins by western blotting and enhanced chemiluminescence. Methods Mol Biol 19:211–218

    CAS  PubMed  Google Scholar 

  46. Methogo RM, Dufresne-Martin G, Leclerc P, Leduc R, Klarskov K (2005) Mass spectrometric peptide fingerprinting of proteins after Western blotting on polyvinylidene fluoride and enhanced chemiluminescence detection. J Proteome Res 4:2216–2224

    Article  CAS  PubMed  Google Scholar 

  47. Haan C, Behrmann I (2007) A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background. J Immunol Methods 318:11–19

    Article  CAS  PubMed  Google Scholar 

  48. Mruk DD, Cheng CY (2011) Enhanced chemiluminescence (ECL) for routine immunoblotting. Spermatogenesis 1:121–122

    Article  PubMed  PubMed Central  Google Scholar 

  49. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  50. Kruger NJ (1994) The Bradford method for protein quantitation. Methods Mol Biol 32:9–15

    CAS  PubMed  Google Scholar 

  51. Noble JE, Bailey MJ (2009) Quantitation of protein. Methods Enzymol 463:73–95

    Article  CAS  PubMed  Google Scholar 

  52. Olsen JV, Ong SE, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614

    Article  CAS  PubMed  Google Scholar 

  53. Smith BJ (1994) SDS polyacrylamide gel electrophoresis of proteins. Methods Mol Biol 32:23–34

    CAS  PubMed  Google Scholar 

  54. Walker JM (1994) Gradient SDS polyacrylamide gel electrophoresis of proteins. Methods Mol Biol 32:35–38

    CAS  PubMed  Google Scholar 

  55. Gallagher SR (2006) One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Mol Biol. Chapter 10:Unit 10.2A

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Maynooth University, National University of Ireland Maynooth, Callan Building, North Campus, Maynooth, Co. Kildare, Ireland

    Sandra Murphy & Kay Ohlendieck

Authors
  1. Sandra Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kay Ohlendieck
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kay Ohlendieck .

Editor information

Editors and Affiliations

  1. Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, Roma, Italy

    Camilla Bernardini

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Murphy, S., Ohlendieck, K. (2018). Proteomic Profiling of the Dystrophin-Deficient Brain. In: Bernardini, C. (eds) Duchenne Muscular Dystrophy. Methods in Molecular Biology, vol 1687. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7374-3_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7374-3_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7373-6

  • Online ISBN: 978-1-4939-7374-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation