Thermostability-assisted limited proteolysis-coupled mass spectrometry for capturing drug target proteins and sites.

BACKGROUND: Identifying drug-binding targets and their corresponding sites is crucial for drug discovery and mechanism studies. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a sophisticated method used for the detection of compound and protein interactions. However, in some cases, LiP-MS cannot identify the target proteins due to the small structure changes or the lack of enrichment of low-abundant protein. To overcome this drawback, we developed a thermostability-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) approach for efficient drug target discovery. RESULTS: We proved that the novel strategy, TALiP-MS, could efficiently identify target proteins of various ligands, including cyclosporin A (a calcineurin inhibitor), geldanamycin (an HSP90 inhibitor), and staurosporine (a kinase inhibitor), with accurately recognizing drug-binding domains. The TALiP protocol increased the number of target peptides detected in LiP-MS experiments by 2- to 8-fold. Meanwhile, the TALiP-MS approach can not only identify both ligand-binding stability and destabilization proteins but also shows high complementarity with the thermal proteome profiling (TPP) and machine learning-based limited proteolysis (LiP-Quant) methods. The developed TALiP-MS approach was applied to identify the target proteins of celastrol (CEL), a natural product known for its strong antioxidant and anti-cancer angiogenesis effect. Among them, four proteins, MTHFD1, UBA1, ACLY, and SND1 were further validated for their strong affinity to CEL by using cellular thermal shift assay. Additionally, the destabilized proteins induced by CEL such as TAGLN2 and CFL1 were also validated. SIGNIFICANCE: Collectively, these findings underscore the efficacy of the TALiP-MS method for identifying drug targets, elucidating binding sites, and even detecting drug-induced conformational changes in target proteins in complex proteomes.

Advanced Development of Supercritical Fluid Chromatography in Herbal Medicine Analysis.

The greatest challenge in the analysis of herbal components lies in their variety and complexity. Therefore, efficient analytical tools for the separation and qualitative and quantitative analysis of multi-components are essential. In recent years, various emerging analytical techniques have offered significant support for complicated component analysis, with breakthroughs in selectivity, sensitivity, and rapid analysis. Among these techniques, supercritical fluid chromatography (SFC) has attracted much attention because of its high column efficiency and environmental protection. SFC can be used to analyze a wide range of compounds, including non-polar and polar compounds, making it a prominent analytical platform. The applicability of SFC for the separation and determination of natural products in herbal medicines is overviewed in this article. The range of applications was expanded through the selection and optimization of stationary phases and mobile phases. We also focus on the two-dimensional SFC analysis. This paper provides new insight into SFC method development for herbal medicine analysis.