Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)
复制标题
DOI：
10.1073/pnas.0601637103
复制
发表时间：
2006-06-20
影响因子：
11.1
通讯作者：
Schuman, Erin M.
中科院分区：
文献类型：
--
作者：
Dieterich, Daniela C.;Link, A. James;Schuman, Erin M.
在正常和病理状态下，细胞通过合成新的蛋白质对环境信号做出快速反应。新合成的蛋白质组的选择性鉴定受到一个基本事实的阻碍，即所有蛋白质，无论新旧，都共享相同的氨基酸库，因此在化学上是不可区分的。我们在这里描述了一种技术，该技术基于将叠氮基共翻译引入蛋白质，并使用炔亲和标签对叠氮标记的蛋白质进行化学选择性标记，以专门分离和鉴定哺乳动物细胞中新合成的蛋白质。含叠氮化物的氨基酸叠氮基高丙氨酸的掺入是无偏的，无毒的，并且不增加蛋白质降解。作为该方法的第一个演示，我们报告了195代谢标记的蛋白质的选择性纯化和鉴定与多维液相色谱串联MS。此外，结合基于亮氨酸的质量标记，候选人立即验证为新合成的蛋白质。所确定的蛋白质，在2小时的窗口合成，具有广泛的生化特性和跨越大多数功能基因本体论类别。这项技术使得解决任何细胞类型中新合成蛋白质组的时间和空间特征成为可能。
In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.