Smits Arne H, Borrmann Annika, Roosjen Mark, van Hest Jan C M, Vermeulen Michiel
Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen, The Netherlands.
Department of Bio-organic Chemistry, Faculty of Science, Institute for Molecules and Materials, Radboud University Nijmegen , Nijmegen, The Netherlands.
ACS Chem Biol. 2016 Dec 16;11(12):3245-3250. doi: 10.1021/acschembio.6b00520. Epub 2016 Oct 17.
Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.
表位标签是一种从粗细胞提取物中促进蛋白质富集的有效工具。传统上，使用N端或C端融合标签，然而，这可能会干扰蛋白质功能。带有小反应基团的非天然氨基酸（UAA）可以位点特异性地掺入蛋白质中，因此可作为传统蛋白质标签的潜在替代物。在这里，我们介绍Click-MS，它结合了位点特异性UAA掺入、生物正交化学和基于定量质谱的蛋白质组学的力量，以从哺乳动物粗细胞提取物中特异性富集单一目标蛋白质。通过对对叠氮基-L-苯丙氨酸进行遗传编码，可使用无铜点击化学选择性捕获目标蛋白质。我们使用Click-MS富集在不同细胞区室中发挥作用的蛋白质，并鉴定蛋白质-蛋白质相互作用，显示出Click-MS在相互作用蛋白质组学工作流程中的巨大潜力。
