College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Science, Beijing National Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, China.
Angew Chem Int Ed Engl. 2021 Feb 19;60(8):4028-4033. doi: 10.1002/anie.202010981. Epub 2020 Dec 21.
Protein lipoylation is a post-translational modification of emerging importance in both prokaryotes and eukaryotes. However, labeling and large-scale profiling of protein lipoylation remain challenging. Here, we report the development of iLCL (iodoacetamide-assisted lipoate-cyclooctyne ligation), a chemoselective reaction that enables chemical tagging of protein lipoylation. We demonstrate that the cyclic disulfide of lipoamide but not linear disulfides can selectively react with iodoacetamide to produce sulfenic acid, which can be conjugated with cyclooctyne probes. iLCL enables tagging of lipoylated proteins for gel-based detection and cellular imaging. Furthermore, we apply iLCL for proteomic profiling of lipoylated proteins in both bacteria and mammalian cells. In addition to all of the eight known lipoylated proteins, we identified seven candidates for novel lipoylated proteins. The iLCL strategy should facilitate uncovering the biological function of protein lipoylation.
蛋白质脂酰化是原核生物和真核生物中一种新兴的翻译后修饰方式。然而，蛋白质脂酰化的标记和大规模分析仍然具有挑战性。在这里，我们报告了 iLCL（碘乙酰胺辅助脂酰环辛炔连接）的发展，这是一种化学选择性反应，可实现蛋白质脂酰化的化学标记。我们证明脂酰基辅酶 A 的环二硫键而不是线性二硫键可以选择性地与碘乙酰胺反应生成亚磺酸，然后可以与环辛炔探针结合。iLCL 可用于凝胶检测和细胞成像中脂酰化蛋白的标记。此外，我们还将 iLCL 应用于细菌和哺乳动物细胞中脂酰化蛋白的蛋白质组学分析。除了所有已知的 8 种脂酰化蛋白外，我们还鉴定了 7 种新型脂酰化蛋白的候选蛋白。iLCL 策略应有助于揭示蛋白质脂酰化的生物学功能。
