Chemical Tagging of Protein Lipoylation

Protein lipoylation is a post-translational modification of emerging importance in both prokaryotes and eukaryotes. However, effective methods for labeling and analyzing, particularly in large scale, protein lipoylation remain limited. 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. Using iLCL, lipoylation on recombinant bacterial lipoylated proteins and in cell and tissue lysates is tagged with fluorophores for analysis by in-gel fluorescence scanning 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, seven new ones are identified. The iLCL-based tagging method should facilitate the studies and understanding of biological function of protein lipoylation.蛋白质脂酰化（Protein lipoylation）是一类在原核生物与真核生物中愈发受到关注的翻译后修饰。然而，当前针对蛋白质脂酰化的高效标记与分析方法仍十分有限，尤其是大规模分析场景下的技术手段。本研究报道了iLCL（碘乙酰胺辅助脂酸-环炔连接反应，iodoacetamide-assisted lipoate-cyclooctyne ligation）的开发，这是一种可实现蛋白质脂酰化化学标记的化学选择性反应。我们证实，仅脂酰胺的环状二硫键（而非线性二硫键）可与碘乙酰胺发生选择性反应，生成亚磺酸中间体，该中间体可与环炔探针完成共轭结合。借助iLCL技术，我们可对重组细菌脂酰化蛋白，以及细胞与组织裂解物中的脂酰化位点进行荧光基团标记，进而通过凝胶内荧光扫描与细胞成像开展分析。此外，我们将iLCL应用于细菌与哺乳动物细胞内脂酰化蛋白的蛋白质组学表征。除已报道的8种脂酰化蛋白外，本研究还新鉴定出7种脂酰化蛋白。该基于iLCL的标记方法将有助于推动蛋白质脂酰化的生物学功能研究与认知。