Application of fluorescence two-dimensional difference in-gel electrophoresis as a proteomic biomarker discovery tool in muscular dystrophy research

Abstract

In this article, we illustrate the application of difference in-gel electrophoresis for the proteomic analysis of dystrophic skeletal muscle. The mdx diaphragm was used as a tissue model of dystrophinopathy. Two-dimensional gel electrophoresis is a widely employed protein separation method in proteomic investigations. Although two-dimensional gels usually underestimate the cellular presence of very high molecular mass proteins, integral membrane proteins and low copy number proteins, this method is extremely powerful in the comprehensive analysis of contractile proteins, metabolic enzymes, structural proteins and molecular chaperones. This gives rise to two-dimensional gel electrophoretic separation as the method of choice for studying contractile tissues in health and disease. For comparative studies, fluorescence difference in-gel electrophoresis has been shown to provide an excellent biomarker discovery tool. Since aged diaphragm fibres from the mdx mouse model of Duchenne muscular dystrophy closely resemble the human pathology, we have carried out a mass spectrometry-based comparison of the naturally aged diaphragm versus the senescent dystrophic diaphragm. The proteomic comparison of wild type versus mdx diaphragm resulted in the identification of 84 altered protein species. Novel molecular insights into dystrophic changes suggest increased cellular stress, impaired calcium buffering, cytostructural alterations and disturbances of mitochondrial metabolism in dystrophin-deficient muscle tissue.

Figure 1

Overview of the fluorescence difference in-gel electrophoresis method: Shown are the main approaches used in fluorescence gel-based proteomics, whereby 2-dye or 3-dye difference in-gel electrophoresis (DIGE) is most commonly applied for studying global changes in complex protein populations. In the case of muscular dystrophy research, normal and dystrophic specimens are differentially labeled with CyDyes prior to electrophoresis, then proteins separated based on the unique combination of their isoeletric point and molecular masses and finally image analysis used to evaluate differential expression pattern of fluorescently labeled proteins.

Figure 2

Mass spectrometry-based proteomic profiling of the mdx mouse model of Duchenne muscular dystrophy: The upper panel of the figure summarizes the types of muscle and age groups of mdx mice that have been used over the last few years to identify global changes in the dystrophic muscle proteome. All listed proteomic studies have been recently discussed in comprehensive reviews of the proteomics of the dystrophin-glycoprotein complex and dystrophinopathy [50,69]. The lower panel gives an overview of the difference in-gel electrophoretic (DIGE) analysis of the aged mdx diaphragm muscle presented in this report.

Figure 3

Image analysis of fluorescent DIGE gels representing normal versus dystrophic diaphragm muscle: Shown are a 2D-DIGE master gel used for the analysis of aged mdx diaphragm (A), the visualization of the altered expression levels of the cytosolic Ca²⁺-binding protein parvalbumin (PVA; B, D, E) and the small molecular chaperone cvHsp (C, F, G), the variability of detected alterations between normal and dystrophic samples (H, I) and the graphical presentation of the drastic decrease in parvalbumin (J, K) and the up-regulation of cvHsp (L, M) in dystrophic muscle. In this study, a Typhoon Trio variable mode imager was employed for the visualization of electrophoretically separated and CyDye-labelled diaphragm proteins. Expression changes of proteins between wild type samples and dystrophic mdx samples were determined with the help of Progenesis SameSpots analysis software.

Figure 4

2D-DIGE master gel of the aged mdx diaphragm muscle: Shown is a 2D master gel that is representative of the findings of the fluorescence two-dimensional difference in-gel electrophoretic analysis of the aged mdx diaphragm. Protein spots with a significant change in expression levels between normal and dystrophic specimens are marked by circles and are numbered 1 to 84. See Table 1 for the mass spectrometric identification of individual diaphragm-associated proteins. The pH-values of the first dimension gel system and molecular mass standards of the second dimension are indicated on the top and on the left of the panels, respectively.

Figure 5

Molecular function of altered mdx diaphragm-associated proteins: The bioinformatics software programme PANTHER (database; version 8.1; [64,65]) was applied to identify the clustering of molecular functions of the mass spectrometrically identified proteins with a changed abundance in aged mdx diaphragm as compared to normal muscle (Table 1).

Figure 6

Interaction map of altered mdx diaphragm-associated proteins: The bioinformatics STRING database (version 9.1; [66,67]) was used to generate a protein interaction map with known and predicted protein associations that include direct physical and indirect functional protein linkages of the mass spectrometrically identified proteins with a changed abundance in aged mdx diaphragm as compared to normal muscle (Table 1).

Figure 7

Gel electrophoretic and immunoblot analysis of the aged mdx diaphragm muscle: Shown is a silver-stained gel (A) and representative immunoblots (B–D) with expanded views of antibody-decorated bands. Lanes 1 and 2 represent preparations from non-dystrophic wild type (WT) versus dystrophic (MDX) diaphragm muscle, respectively. Immuno-decoration was carried out with primary antibodies to laminin (B), the dystrophin-associated glycoprotein β-dystroglycan (C) and collagen (D). Below the individual immunoblots are shown panels, which give a graphical representation of the immuno-decoration levels in normal versus mdx preparations (Student’s t-test, unpaired; n = 4; * p < 0.05; ** p < 0.01).

Figure 8

Immunoblot analysis of the aged mdx diaphragm muscle: Shown are representative immunoblots with expanded views of antibody-decorated bands. Lanes 1 and 2 represent preparations from non-dystrophic wild type (WT) versus dystrophic (MDX) diaphragm muscle, respectively. Immuno-decoration was carried out with primary antibodies to heat shock protein cvHsp (A), heat shock protein Hsp70 (B), prohibitin (C), parvalbumin (D), the luminal Ca²⁺-binding protein calsequestrin (E) and the mitochondrial enzyme ATP synthase (F). Besides the individual immunoblots are shown panels, which give a graphical representation of the immuno-decoration levels in normal versus mdx preparations (Student’s t-test, unpaired; n = 4; * p < 0.05; ** p < 0.01, *** p < 0.001).

Figure 9

Proteomic profile of the aged mdx diaphragm muscle: The diagram summarizes the main classes of diaphragm proteins identified by the proteomic profiling of the aged and dystrophin-deficient mdx mouse model of X-linked muscular dystrophy. The severely perturbed protein expression pattern of dystrophic muscle tissue includes extracellular matrix proteins, contractile proteins, structural proteins, molecular chaperones, mitochondrial enzymes, glycolytic enzymes, calcium-binding proteins and metabolite transporters. Thus, the deficiency in dystrophin isoform Dp427 and concomitant reduction in dystrophin-associated glycoproteins in the dystrophic sarcolemma appears to trigger a large range of secondary abnormalities in muscular dystrophy.