CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian, 116023, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Anal Chim Acta. 2021 Jul 11;1168:338612. doi: 10.1016/j.aca.2021.338612. Epub 2021 May 6.
The process of protein precipitation can be used to decipher the interaction of ligand and protein. For example, the classic Thermal Proteome Profiling (TPP) method uses heating as the driving force for protein precipitation, to discover the drug target protein. Under heating or other denature forces, the target protein that binds with the drug compound will be more resistant to precipitation than the free protein. Similar to thermal stress, mechanical stress can also induce protein precipitation. Upon mechanical stress, protein will gradually precipitate along with protein conformational changes, which can be exploited for the study of the ligand-protein interaction. Herein, we proposed a Mechanical Stress Induced Protein Precipitation (MSIPP) method for drug target deconvolution. Its streamlined workflow allows in situ sample preparation on the surface of microparticles, from protein precipitation to digestion. The mechanical stress was generated by vortexing the slurry of protein solution and microparticle materials. The mechanical stress induced protein precipitate was captured by the microparticles, which guarantees the MSIPP method to be scalable and user-friendly. The MSIPP method was successfully applied to four drug compounds, Methotrexate, Raltitrexed, SHP099, Geldanamycin and a pan-inhibitor of protein kinases, Staurosporine. Besides, DHFR was demonstrated to be a target of Raltitrexed, which has not been revealed by any other modification-free drug target discovery method yet. Thus, MSIPP is a complementary method to other drug target screening methods.
蛋白质沉淀过程可用于解析配体与蛋白质的相互作用。例如，经典的热蛋白质组分析（TPP）方法利用加热作为蛋白质沉淀的驱动力，以发现药物靶标蛋白。在加热或其他变性力的作用下，与药物化合物结合的靶标蛋白比游离蛋白更能抵抗沉淀。与热应激类似，机械应激也能诱导蛋白质沉淀。在机械应力下，蛋白质会随着蛋白质构象变化逐渐沉淀，这可用于研究配体-蛋白质相互作用。在此，我们提出了一种用于药物靶标剖析的机械应力诱导蛋白质沉淀（MSIPP）方法。其简化的工作流程允许在微孔颗粒表面上进行原位样品制备，从蛋白质沉淀到消化。通过搅拌蛋白质溶液和微孔材料的浆料来产生机械应力。机械应力诱导的蛋白质沉淀物被微孔颗粒捕获，这保证了 MSIPP 方法的可扩展性和易用性。MSIPP 方法成功应用于四种药物化合物，即甲氨蝶呤、雷替曲塞、SHP099、格尔德霉素和一种蛋白激酶的泛抑制剂，司他夫定。此外，还证明 DHFR 是雷替曲塞的靶标，这是其他任何无修饰药物靶标发现方法都没有揭示的。因此，MSIPP 是其他药物靶标筛选方法的补充方法。
