通过生物素连接酶标记分析K-Ras相互作用
Analysis of K-Ras Interactions by Biotin Ligase Tagging.
作者信息
Ritchie Christopher, Mack Andrew, Harper Logan, Alfadhli Ayna, Stork Philip J S, Nan Xiaolin, Barklis Eric
机构信息
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, U.S.A.
Department of Vollum Institute, Oregon Health & Science University, Portland, OR, U.S.A.
出版信息
Cancer Genomics Proteomics. 2017 Jul-Aug;14(4):225-239. doi: 10.21873/cgp.20034.
BACKGROUND
Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network.
MATERIALS AND METHODS
We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing.
RESULTS
In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers.
CONCLUSION
Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.
背景
人类K-Ras 4B（K-Ras）G蛋白的突变与相当一部分人类癌症相关。尽管如此，目前仍缺乏对K-Ras相互作用的全面分析。我们的研究重点是K-Ras相互作用网络的表征。
材料与方法
我们采用了生物素连接酶标记方法，其中标记的K-Ras蛋白以邻近依赖的方式将生物素连接到相邻蛋白上，并通过质谱（MS）测序鉴定蛋白。
结果
在转染细胞中，从表达生物素连接酶标记的K-Ras变体的细胞中总共鉴定出748种生物素化蛋白。在膜相关变体和法尼基化缺陷突变体之间观察到显著差异。在胰腺癌细胞中，鉴定出56个K-Ras相互作用伙伴。其中大多数是细胞骨架或质膜蛋白，许多先前已被鉴定为潜在的癌症生物标志物。
结论
生物素连接酶标记为K-Ras相互作用网络的表征提供了一种快速便捷的方法。
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