Blagoev Blagoy, Mann Matthias
Center for Experimental Bioinformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Methods. 2006 Nov;40(3):243-50. doi: 10.1016/j.ymeth.2006.08.001.
In the last several years, the impact of mass spectrometry (MS)-based proteomics on cell signaling research has increased dramatically. This development has been driven both by better instrumentation and by the progression of proteomics from mainly qualitative measurements towards quantitative analyses. In this regard, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has established itself as one of the most popular and useful quantitative proteomic methodologies to study signaling networks. SILAC relies on the metabolic incorporation of non-radioactive heavy isotopes in the whole proteome of desired cell line, making all proteins from these cells easily distinguishable in the mass spectrometers from the proteins originating from control cells. The procedure does not involve any chemical derivatization steps and, importantly, allows mixing of the two cell populations for combined additional sample manipulation, thus leading to highly reliable results with minimal errors. In this chapter, we describe in detail the SILAC labeling procedure and explain how to design SILAC experiments to examine the level and duration of phosphorylation of endogenous MAP kinases and their substrates in cell culture systems.
在过去几年中,基于质谱(MS)的蛋白质组学对细胞信号研究的影响急剧增加。这一发展既得益于更好的仪器设备,也得益于蛋白质组学从主要的定性测量向定量分析的进展。在这方面,细胞培养中氨基酸稳定同位素标记(SILAC)已成为研究信号网络最流行和最有用的定量蛋白质组学方法之一。SILAC依赖于在所需细胞系的整个蛋白质组中代谢掺入非放射性重同位素,使得这些细胞中的所有蛋白质在质谱仪中很容易与来自对照细胞的蛋白质区分开来。该过程不涉及任何化学衍生步骤,重要的是,允许将两个细胞群体混合以进行额外的联合样品处理,从而以最小的误差获得高度可靠的结果。在本章中,我们将详细描述SILAC标记过程,并解释如何设计SILAC实验以检测细胞培养系统中内源性丝裂原活化蛋白激酶(MAP激酶)及其底物的磷酸化水平和持续时间。