Yang Yu-Ying, Grammel Markus, Raghavan Anuradha S, Charron Guillaume, Hang Howard C
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.
Chem Biol. 2010 Nov 24;17(11):1212-22. doi: 10.1016/j.chembiol.2010.09.012.
The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na₂S₂O₄-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.
生物正交连接方法的进展为使用叠氮化物/炔烃功能化化学报告分子和基于活性的探针进行新合成蛋白质、翻译后修饰以及特定酶家族的蛋白质组学分析提供了新机会。利用具有选择性可切割亲和标签高效富集和洗脱叠氮化物/炔烃标记的蛋白质对于蛋白质鉴定和定量应用至关重要。在此，我们报告了用于生物正交化学蛋白质组学的Na₂S₂O₄可切割偶氮苯基亲和标签的合成及比较分析。我们证明了邻羟基取代基是偶氮苯键有效切割所必需的，并表明这些可切割亲和标签可用于鉴定氨基酸化学报告分子靶向的细菌中新合成的蛋白质及其在内源表达蛋白质上的修饰位点。基于偶氮苯的亲和标签与凝胶内、溶液内和磁珠富集策略兼容，应为各种生物正交蛋白质组学应用提供有用的工具。
