Abstract
The skeletal muscle proteome consists of a large number of diverse protein species with a broad and dynamic concentration range. Since mature skeletal muscles are characterized by a distinctive combination of contractile cells with differing physiological and biochemical properties, it is essential to determine specific differences in the protein composition of fast, slow, and hybrid fibers. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a powerful comparative tool to analyze fiber type-specific differences between predominantly fast contracting versus slower twitching muscles. In this chapter, the application of the 2D-DIGE method for the comparative analysis of different subtypes of skeletal muscles is outlined in detail. A standardized proteomic workflow is described, involving sample preparation, protein extraction, differential fluorescence labeling using a 3-CyDye system, first-dimension isoelectric focusing, second-dimension slab gel electrophoresis, 2D-DIGE image analysis, protein digestion, and mass spectrometry.
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