Quantitative cancer proteomics: Stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research
Quantitative cancer proteomics: Stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research
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DOI：
10.1074/mcp.m400021-mcp200
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发表时间：
2004-07-01
影响因子：
7
通讯作者：
Gygi, SP
中科院分区：
文献类型：
--
作者：
Everley, PA;Krijgsveld, J;Gygi, SP
微阵列一直是大规模分析癌症进展相关基因的主要手段。然而，最近人们认识到需要直接在蛋白质水平上研究癌症的发展。在本报告中，我们将比较蛋白质组学技术应用于转移性前列腺癌的研究。这项技术被称为细胞培养物中氨基酸稳定同位素标记 (SILAC)，最近因其能够在单个实验中比较数百种蛋白质的表达水平而受到欢迎。 SILAC 利用添加到单独培养的细胞系的生长培养基中的 C-12 和 C-13 标记氨基酸，产生分别含有“轻”或“重”蛋白质的细胞。混合从这些细胞收集的裂解物后，可以通过串联质谱法鉴定蛋白质。稳定同位素的掺入还可以对两个样品进行定量比较。使用这种方法，我们比较了具有不同转移潜力的前列腺癌细胞微粒体部分中 440 多种蛋白质的表达水平。其中，60 个在高度转移的细胞中升高超过 3 倍，而 22 个则降低了等量。蛋白质印迹进一步证实了基于质谱的定量。我们的结果证明了这种新方法对于使用确定的细胞系研究癌症进展的适用性。
Microarrays have been the primary means for large-scale analyses of genes implicated in cancer progression. However, more recently a need has been recognized for investigating cancer development directly at the protein level. In this report, we have applied a comparative proteomic technique to the study of metastatic prostate cancer. This technology, termed stable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC makes use of C-12- and C-13-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either "light" or "heavy" proteins, respectively. Upon mixing lysates collected from these cells, proteins can be identified by tandem mass spectrometry. The incorporation of stable isotopes also allows for a quantitative comparison between the two samples. Using this method, we compared the expression levels for more than 440 proteins in the microsomal fractions of prostate cancer cells with varying metastatic potential. Of these, 60 were found elevated greater than 3-fold in the highly metastatic cells, whereas 22 were reduced by equivalent amounts. Western blotting provided further confirmation of the mass spectrometry-based quantification. Our results demonstrate the applicability of this novel approach toward the study of cancer progression using defined cell lines.