Ohlendieck Kay
Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland.
Methods Mol Biol. 2018;1664:93-108. doi: 10.1007/978-1-4939-7268-5_9.
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 specific 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 gel electrophoresis (DIGE) is a powerful comparative tool to analyze fiber type-specific differences between fast and slow muscles. In this chapter, the application of the 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-dye system, first-dimension isoelectric focusing, second-dimension slab gel electrophoresis, DIGE image analysis, protein digestion, and mass spectrometry.
骨骼肌蛋白质组由大量不同的蛋白质种类组成，其浓度范围广泛且动态变化。由于成熟的骨骼肌具有不同生理和生化特性的收缩细胞的特定组合，因此确定快肌、慢肌和混合纤维蛋白质组成的特定差异至关重要。荧光二维凝胶电泳（DIGE）是分析快肌和慢肌之间纤维类型特异性差异的强大比较工具。在本章中，将详细概述DIGE方法在骨骼肌不同亚型比较分析中的应用。描述了一种标准化的蛋白质组学工作流程，包括样品制备、蛋白质提取、使用三染料系统进行差异荧光标记、一维等电聚焦、二维平板凝胶电泳、DIGE图像分析、蛋白质消化和质谱分析。
