Charles River Laboratories, Saffron Walden, UK.
Methods Mol Biol. 2023;2596:351-362. doi: 10.1007/978-1-0716-2831-7_24.
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 fluorescence 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 underrepresented 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 the skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative 2D-DIGE analysis.
基于质谱的蛋白质分析方法彻底改变了分析生物化学领域，使人们能够鉴定生物体液、细胞系和组织中的数百到数千种蛋白质。该方法需要对蛋白质进行初始分离，这已经成功地使用基于凝胶的技术实现，特别是荧光二维差异凝胶电泳（2D-DIGE）。然而，鉴于蛋白质组的复杂性，可能需要采用分馏技术来优化低丰度蛋白质的检测，这些低丰度蛋白质往往代表性不足，但它们可能是健康和疾病中的重要参与者。这种亚细胞分馏方案通常利用密度梯度离心，并已能够富集粗微体、细胞质、质膜、核和线粒体。在本章中，我们描述了使用差速离心从骨骼肌中富集粗微体的实验步骤，以及随后通过凝胶电泳和免疫印迹进行的富集验证，然后进行比较 2D-DIGE 分析。
