Biochemistry III-Regensburg Center for Biochemistry-Institute for Biochemistry, Genetics and Microbiology, University of Regensburg, Regensburg, Germany.
Methods Mol Biol. 2022;2533:229-246. doi: 10.1007/978-1-0716-2501-9_14.
The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea .
以定量和时间依赖的方式研究蛋白质的产生和降解，是更好地了解细胞生理反应的主要挑战。在现有的技术中，生物正交非天然氨基酸标记（BONCAT）是一种有效的方法，通过引入化学反应性的非天然氨基酸，如 L-叠氮高丙氨酸（L-AHA），可以实现蛋白质的时间依赖性标记。含叠氮基的氨基酸衍生物能够进行高效和特异性的反应，称为点击化学，其中 L-AHA 的叠氮基团与反应性炔烃衍生物（如二苯并环辛炔（DBCO）衍生物）反应，使用应变促进的炔烃-叠氮环加成（SPAAC）。此外，现有的含 DBCO 的试剂具有多功能性，可以与荧光团（例如 Cy7）或亲和标签（例如生物素）衍生物偶联，分别用于易于可视化和亲和纯化。在这里，我们描述了针对模式古菌 Haloferax volcanii 优化的分步 BONCAT 方案，但也适用于利用其他生物系统。最后，我们还描述了下游可视化、L-AHA 标记蛋白的亲和纯化和差异表达分析的示例。总之，以下 BONCAT 方案扩展了可用工具包，以使用时间分辨的半定量蛋白质组学分析在古菌中探索蛋白质稳态。
