<br>用于 ATP 结合蛋白全局分析的可点击光反应 ATP 亲和探针,Analytical Chemistry

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用于 ATP 结合蛋白全局分析的可点击光反应 ATP 亲和探针
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-11-22 , DOI: 10.1021/acs.analchem.3c02694
Zhiming Wang _{1,

2} , Jing Tan _{1,

2} , Mengxuan Li _{1,

3} , Can Gao ₁ , Wenwen Li _{1,

3} , Jing Xu _{1,

3} , Changchuan Guo ₄ , Zhenzhen Chen ₅ , Rong Cai _{1,

2,

3}

Affiliation

三磷酸腺苷（ATP）是生物体内主要的能量载体，有许多细胞蛋白可以与ATP结合。在这些蛋白质中，激酶是多种细胞信号传导过程的关键调节因子，异常的激酶信号传导导致许多人类疾病（包括癌症）的发生。因此，小分子激酶抑制剂已成功用于治疗多种疾病。由于许多激酶的 ATP 结合袋相似，因此评估不同激酶抑制剂的选择性非常重要。我们在这里报告了一种可点击的 ATP 光亲和探针，用于 ATP 结合蛋白的全局分析。将蛋白质裂解物与 ATP 探针一起孵育，然后进行紫外线 (UV) 照射，然后用炔烃手柄标记 ATP 结合蛋白，以便随后通过点击化学进行生物素缀合。标记的蛋白质用链霉亲和素珠富集，用胰蛋白酶消化，并使用液相色谱-串联质谱 (LC-MS/MS) 进行分析。超过 400 种 ATP 结合蛋白，包括大约 200 种激酶，可以在数据依赖采集模式下的单次 LC-MS/MS 运行中进行鉴定。然后，我们应用这种方法来分析三种选定的 ATP 竞争性激酶抑制剂的靶标。我们能够从无标记定量结果中成功识别出他们报告的一些靶蛋白，并使用蛋白质印迹分析验证结果。我们共同开发了一种可点击的 ATP 光亲和探针，用于 ATP 结合蛋白的蛋白质组范围分析，并证明这种化学蛋白质组方法适合激酶抑制剂的高通量靶标鉴定。

"点击查看英文标题和摘要"

Clickable Photoreactive ATP-Affinity Probe for Global Profiling of ATP-Binding Proteins

Adenosine triphosphate (ATP) is the major energy carrier in organisms, and there are many cellular proteins that can bind to ATP. Among these proteins, kinases are key regulators in several cell signaling processes, and aberrant kinase signaling contributes to the development of many human diseases, including cancer. Hence, small-molecule kinase inhibitors have been successfully used for the treatment of various diseases. Since the ATP-binding pockets are similar for many kinases, it is very important to evaluate the selectivity of different kinase inhibitors. We report here a clickable ATP photoaffinity probe for the global profiling of ATP-binding proteins. After incubating the protein lysate with the ATP probe followed by ultraviolet (UV) irradiation, ATP-binding proteins were labeled with an alkyne handle for subsequent biotin conjugation through click chemistry. Labeled proteins were enriched with streptavidin beads, digested with trypsin, and analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS). More than 400 ATP-binding proteins, including approximately 200 kinases, could be identified in a single LC-MS/MS run in the data-dependent acquisition mode. We then applied this method to the analysis of targets of three selected ATP-competitive kinase inhibitors. We were able to successfully identify some of their reported target proteins from label-free quantification results and validated the results using Western blot analyses. Together, we developed a clickable ATP photoaffinity probe for proteome-wide profiling of ATP-binding proteins and demonstrated that this chemoproteomic method is amenable to high-throughput target identification of kinase inhibitors.

更新日期：2023-11-22

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