Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics
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DOI：
10.1074/mcp.m200025-mcp200
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发表时间：
2002-05-01
影响因子：
7
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Ong, SE;Blagoev, B;Mann, M
定量蛋白质组学传统上是通过二维凝胶电泳进行的，但最近，基于稳定同位素定量的质谱方法在复杂蛋白质混合物的同时和自动鉴定和定量方面显示出很大的前景。在这里，我们描述了一种称为SILAC的方法，用于在细胞培养中通过氨基酸进行稳定同位素标记，用于将特定氨基酸结合到所有哺乳动物蛋白质中。哺乳动物细胞系在缺乏标准必需氨基酸的培养基中生长，但补充了该氨基酸的非放射性同位素标记形式，在这种情况下是氘化亮氨酸（Leu-d3）。我们发现在这些培养基中维持的细胞的生长与在正常培养基中的生长没有什么不同，这可以通过细胞形态、倍增时间和分化能力来证明。在研究的细胞系和蛋白质中，经过五次加倍后，Leu-d3完全结合。来自实验和对照样品的蛋白质群体在收获后直接混合，质谱鉴定很简单，因为每个含亮氨酸的肽都含有所有正常亮氨酸或所有亮氨酸-d3。我们将该技术应用于肌肉细胞分化过程中蛋白表达变化的相对定量。在这一过程中被发现上调的蛋白包括甘油醛-3-磷酸脱氢酶、纤维连接蛋白和丙酮酸激酶M2。SILAC是一种简单、廉价、准确的方法，可以在任何细胞培养系统中用作定量蛋白质组学方法。
Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.