含炔基类异戊二烯探针代谢标记的优化
Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes.
作者信息
Ahmadi Mina, Suazo Kiall Francis, Distefano Mark D
机构信息
Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
出版信息
Methods Mol Biol. 2019;2009:35-43. doi: 10.1007/978-1-4939-9532-5_3.
Abstract
Protein prenylation, found in eukaryotes, is a posttranslational modification in which one or two isoprenoid groups are added to the C terminus of selected proteins using either a farnesyl group or a geranylgeranyl group. Prenylation facilitates protein localization mainly to the plasma membrane where the prenylated proteins, including small GTPases, mediate signal transduction pathways. Changes in the level of prenylated proteins may serve a critical function in a variety of diseases. Metabolic labeling using modified isoprenoid probes followed by enrichment and proteomic analysis allows the identities and levels of prenylated proteins to be investigated. In this protocol, we illustrate how the conditions for metabolic labeling are optimized to maximize probe incorporation in HeLa cells through a combination of in-gel fluorescence and densitometric analysis.
摘要
蛋白质异戊二烯化存在于真核生物中，是一种翻译后修饰，其中一个或两个类异戊二烯基团通过法尼基基团或香叶基香叶基基团添加到选定蛋白质的C末端。异戊二烯化主要促进蛋白质定位于质膜，在质膜上，包括小GTP酶在内的异戊二烯化蛋白质介导信号转导途径。异戊二烯化蛋白质水平的变化可能在多种疾病中起关键作用。使用修饰的类异戊二烯探针进行代谢标记，然后进行富集和蛋白质组分析，可以研究异戊二烯化蛋白质的身份和水平。在本方案中，我们说明了如何通过凝胶内荧光和光密度分析相结合的方法优化代谢标记条件，以最大限度地提高HeLa细胞中探针的掺入率。
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