Carberry Steven, Ohlendieck Kay
Department of Biology, National University of Ireland Maynooth, Maynooth, Kildare, Ireland.
Methods Mol Biol. 2013;1048:229-46. doi: 10.1007/978-1-62703-556-9_17.
Cellular aging is a fundamental biological process, and mass spectrometry-based proteomics has been widely used for the global identification of age-related changes in a variety of tissues. The proteomic profiling of senescent skeletal muscles has revealed a variety of alterations in proteins associated with the contractile apparatus, cell signaling, ion homeostasis, metabolism, and the cellular stress response. Here, we outline the two-dimensional gel electrophoretic separation and fluorescent labeling of the urea-soluble protein complement from aged diaphragm muscle. This chapter describes the various experimental steps involved in gel electrophoresis-based proteomics, including protein extraction, isoelectric focusing, slab gel electrophoresis, fluorescence labeling, image analysis, protein digestion, mass spectrometric identification of proteins and immunoblotting.
细胞衰老乃是一个基本的生物学过程，基于质谱的蛋白质组学已被广泛应用于全面鉴定多种组织中与衰老相关的变化。衰老骨骼肌的蛋白质组学分析揭示了与收缩装置、细胞信号传导、离子稳态、新陈代谢以及细胞应激反应相关的蛋白质存在多种改变。在此，我们概述了从老龄膈肌中提取尿素可溶性蛋白质组分的二维凝胶电泳分离及荧光标记方法。本章描述了基于凝胶电泳的蛋白质组学所涉及的各种实验步骤，包括蛋白质提取、等电聚焦、平板凝胶电泳、荧光标记、图像分析、蛋白质消化、蛋白质的质谱鉴定以及免疫印迹。
