Abstract
Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation and this has been successfully achieved using gel-based techniques, particularly that of two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often under-represented, but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative DIGE analysis.
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Lander ES, Linton LM, Birren B et al (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921
C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018
Adams MD, Celniker SE, Holt RA et al (2000) The genome sequence of Drosophila melanogaster. Science 287:2185–2195
Bensmail H, Haoudi A (2003) Postgenomics: proteomics and bioinformatics in cancer research. J Biomed Biotechnol 2003:217–230
Gregorich ZR, Ge Y (2014) Top-down proteomics in health and disease: challenges and opportunities. Proteomics 14:1195–1210
Rifai N, Gillette MA, Carr SA (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24:971–983
Deshmukh AS, Murgia M, Nagaraj N et al (2015) Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors. Mol Cell Proteomics 14:841–853
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243
Lilley KS, Friedman DB (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomic. Expert Rev Proteomics 1:401–409
Unlü M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077
Oliva K, Barker G, Rice GE et al (2013) 2D-DIGE to identify proteins associated with gestational diabetes in omental adipose tissue. J Endocrinol 218:165–178
Doran P, O'Connell K, Gannon J et al (2008) Opposite pathobiochemical fate of pyruvate kinase and adenylate kinase in aged rat skeletal muscle as revealed by proteomic DIGE analysis. Proteomics 8:364–377
Rong Y, Jin D, Hou C et al (2010) Proteomics analysis of serum protein profiling in pancreatic cancer patients by DIGE: up-regulation of mannose-binding lectin 2 and myosin light chain kinase 2. BMC Gastroenterol 10:68
Ummanni R, Mundt F, Pospisil H et al (2011) Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform. PLoS One e16833:6
Lewis C, Ohlendieck K (2010) Mass spectrometric identification of dystrophin isoform Dp427 by on-membrane digestion of sarcolemma from skeletal muscle. Anal Biochem 404:197–203
Anderson NL, Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 1:845–867
Coenen-Stass AM, McClorey G, Manzano R et al (2015) Identification of novel, therapy-responsive protein biomarkers in a mouse model of Duchenne muscular dystrophy by aptamer-based serum proteomics. Sci Rep 5:17014
Hortin GL, Jortani SA, Ritchie JC (2006) Proteomics: a new diagnostic frontier. Clin Chem 52:1218–1222
Pieper R, Gatlin CL, Makusky AJ et al (2003) The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3:1345–1364
Schiaffino S, Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol Rev 91:1447–1531
Ohlendieck K (2011) Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques. Skelet Muscle 1:6
Gygi SP, Corthals GL, Zhang Y et al (2000) Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 97:9390–9395
Fonslow BR, Carvalho PC, Academia K et al (2011) Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT. J Proteome Res 10:3690–3700
Rabilloud T, Lelong C (2011) Two-dimensional gel electrophoresis in proteomics: a tutorial. J Proteome 74:1829–1841
Tu C, Rudnick PA, Martinez MY et al (2010) Depletion of abundant plasma proteins and limitations of plasma proteomics. J Proteome Res 9:4982–4991
O'Connell K, Ohlendieck K (2009) Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal muscle. Proteomics 9:5509–5524
Wallace DC (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488
Wallace DC (2000) Mitochondrial defects in cardiomyopathy and neuromuscular disease. Am Heart J 139:S70–S85
Carberry S, Zweyer M, Swandulla D, Ohlendieck K (2014) Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle. Anal Biochem 446:108–115
Maughan DW, Henkin JA, Vigoreaux JO (2005) Concentrations of glycolytic enzymes and other cytosolic proteins in the diffusible fraction of a vertebrate muscle proteome. Mol Cell Proteomics 4:1541–1549
Alban A, David SO, Bjorkesten L et al (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3:36–44
Malm C, Hadrevi J, Bergström SA et al (2008) Evaluation of 2-D DIGE for skeletal muscle: protocol and repeatability. Scand J Clin Lab Invest 68:793–800
Lilley KS, Razzaq A, Dupree P (2002) Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr Opin Chem Biol 6:46–50
Acknowledgments
Research in the author’s laboratory has been supported by project grants from the Irish Health Research Board and Muscular Dystrophy Ireland, as well as a Hume Scholarship from Maynooth University.
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Murphy, S. (2018). Subcellular Fractionation for DIGE-Based Proteomics. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 1664. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7268-5_18
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DOI: https://doi.org/10.1007/978-1-4939-7268-5_18
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