Piechura Heike, Oeljeklaus Silke, Warscheid Bettina
Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2012;893:201-21. doi: 10.1007/978-1-61779-885-6_14.
Through crucial advancements in quantitative mass spectrometry (MS), proteomics has evolved from taking mere "snapshots" of proteomes to thoroughly studying dynamic changes in entire proteomes and characterizing intricate protein-protein interaction or signaling networks. Thus, quantitative MS-based proteomics offers the unique potential to place proteins into their functional context and, moreover, to improve our understanding of the molecular processes involved in the development, survival, or pathology of cells and organisms. Among the vast variety of techniques developed for the accurate quantification of proteins via MS, stable isotope labeling by amino acids in cell culture (SILAC) arguably represents the most elegant method. In this chapter, we provide a detailed protocol for the establishment of SILAC for mammalian cell culture systems. In addition, to exemplify the high versatility of SILAC for addressing different biological questions, we describe the successful "pairing" of SILAC with conventional affinity purification (AP)-MS approaches allowing for accurately characterizing protein complexes.
通过定量质谱（MS）技术的关键进展，蛋白质组学已从仅仅对蛋白质组进行“快照”发展到全面研究整个蛋白质组的动态变化，并对复杂的蛋白质-蛋白质相互作用或信号网络进行表征。因此，基于定量质谱的蛋白质组学具有独特的潜力，能够将蛋白质置于其功能背景中，进而增进我们对细胞和生物体发育、存活或病理过程中所涉及分子过程的理解。在通过质谱对蛋白质进行准确定量所开发的众多技术中，细胞培养中氨基酸稳定同位素标记（SILAC）可谓是最为精妙的方法。在本章中，我们提供了在哺乳动物细胞培养系统中建立SILAC的详细方案。此外，为了例证SILAC在解决不同生物学问题方面的高度通用性，我们描述了SILAC与传统亲和纯化（AP）-MS方法的成功“配对”，从而能够准确表征蛋白质复合物。
