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Evaluation of 2-D DIGE for skeletal muscle: Protocol and repeatability
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
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2008 (English)In: The Scandinavian Journal of Clinical & Laboratory Investigation, Vol. 68, no 8, 793-800 p.Article in journal (Refereed) Published
Abstract [en]

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 1824 cm gels, scanned with a Typhoon™ 9410 laser scanner and analysed in the DeCyder™ software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.

Place, publisher, year, edition, pages
Informa Healthcare , 2008. Vol. 68, no 8, 793-800 p.
Keyword [en]
DIGE, disease, exercise, human, mass spectrometry, skeletal muscle
National Category
Ophthalmology Cell and Molecular Biology
Research subject
Human Anatomy; Ophtalmology
URN: urn:nbn:se:umu:diva-10396DOI: 10.1080/00365510802277464Local ID: 744OAI: diva2:150067
Available from: 2008-12-09 Created: 2008-12-09 Last updated: 2014-08-29Bibliographically approved
In thesis
1. Applying proteomics and metabolomics for studying human skeletal muscle with a focus on chronic trapezius myalgia
Open this publication in new window or tab >>Applying proteomics and metabolomics for studying human skeletal muscle with a focus on chronic trapezius myalgia
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[en]
Tillämpning av proteomiska och metabolomiska metoder på human skelettmuskel med inriktning mot kronisk trapezius myalgi
Abstract [en]

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle.

Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.

 The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle.

Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2012. 60 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1533
Trapezius myalgia, proteomics, 2D-DIGE, metabolomics, microdialysate, biopsies
National Category
Other Basic Medicine
Research subject
Human Anatomy
urn:nbn:se:umu:diva-61399 (URN)978-91-7459-515-4 (ISBN)
Public defence
2012-12-06, BiA 201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2012-11-15 Created: 2012-11-12 Last updated: 2012-11-15Bibliographically approved

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Malm, ChristerHadrevi, JennyBergström, Sven-AndersPedrosa-Domellof, FatimaAntti, HenrikSvensson, MichaelFrängsmyr, Lars
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