A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)
A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)
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DOI：
10.1038/nprot.2006.427
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Ong, Shao-En;Mann, Matthias
细胞培养中氨基酸稳定同位素标记(SILAC)是基于质谱仪(MS)的定量蛋白质组学中一种简单、可靠但功能强大的方法。SILAC通过正常的代谢过程标记细胞蛋白质组，在新合成的蛋白质中加入非放射性的、稳定的含有同位素的氨基酸。用“重”的SILAC氨基酸代替天然的(“轻的”)氨基酸，制备生长介质。在该培养基中生长的细胞经5次倍增后含有较重的氨基酸，而SILAC氨基酸对细胞形态和生长速度没有影响。当轻细胞和重细胞混合在一起时，它们仍然可以通过MS区分开来，蛋白质丰度是由相对的MS信号强度确定的。SILAC提供准确的相对定量，无需任何化学衍生化或操作，并使蛋白质组学中优雅的功能分析得以发展。在这个方案中，我们描述了如何应用SILAC和纳米级液相色谱与电喷雾电离质谱联用来鉴定和定量蛋白质。这一过程可以在8天内完成。
Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural ("light") amino acids are replaced by "heavy" SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.