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.

A molecular networking-assisted automatic database screening strategy for comprehensive annotation of small molecules in complex matrices.

Liquid chromatography-tandem with high-resolution mass spectrometry (LCHRMS) has proven challenging for annotating multiple small molecules within complex matrices due to the complexities of chemical structure and raw LCHRMS data, as well as limitations in previous literatures and reference spectra related to those molecules. In this study, we developed a molecular networking assisted automatic database screening (MN/auto-DBS) strategy to examine the combined effect of MS1 exact mass screening and MS2 similarity analysis. We compiled all previously reported compounds from the relevant literatures. With the development of a Python software, the in-house database (DB) was created by automatically calculating the m/z and data from experimental MS1 hits were rapid screened with DB. We then performed a feature-based molecular network analysis on the auto-MS2 data for supplementary identification of unreported compounds, including clustered FBMN and annotated GNPS compounds. Finally, the results from both strategies were merged and manually curated for correct structural assignment. To demonstrate the applicability of MN/auto-DBS, we selected the Huangqi-Danshen herb pair (HD), commonly used in prescriptions or patent medicines to treat diabetic nephropathy and cerebrovascular disease. A total of 223 compounds were annotated, including 65 molecules not previously reported in HD, such as aromatic polyketides, coumarins, and diarylheptanoids. Using MN/auto-DBS, we can profile and mine a wide range of complex matrices for potentially new compounds.

Discovery of effective combination from Renshen-Fuzi herbal pair against heart failure by spectrum-effect relationship analysis and zebrafish models.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal pair Ginseng Radix et Rhizoma (roots and rhizomes of Panax ginseng C.A. Mey, Renshen in Chinese) and Aconiti Lateralis Radix Praeparata (lateral roots of Aconitum carmichaelii Debeaux, Fuzi in Chinese), composition of two traditional Chinese medicinal herbs, has been widely used in traditional Chinese medicine formula, in which Shenfu decoction has been used clinically in China for the treatment of heart failure at present. AIM OF THE STUDY: Although the ginsenosides and aconite alkaloids have been proven as the essential bioactive components in Renshen-Fuzi herbal pair, the exact composition of effective components to combat heart failure are still unclear. Therefore, spectrum-effect relationship analysis was performed to reveal its effective combination for anti-heart failure effect. MATERIALS AND METHODS: Firstly, the chemical constituents of Renshen-Fuzi herbal pair were identified using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). The 39 major compounds in Renshen-Fuzi with five different compatibility ratios were simultaneously quantified using ultra high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ MS/MS). Subsequently, zebrafish models induced by verapamil hydrochloride were constructed and four heart failure-related indexes were selected for pharmacodynamic evaluation of Renshen-Fuzi. To analyze the spectrum-effect relationships, partial least squares regression (PLSR) models were established among the contents of 39 compounds in Renshen-Fuzi with each pharmacodynamic index. According to the contribution of each compound to the whole efficacy, 12 compounds were finally screened out as the effective combination. RESULTS: A total of 157 chemical compounds of Renshen-Fuzi herbal pair were identified, in which 39 components were simultaneously determined. The pharmacological effects indicated that Renshen-Fuzi with 1:2 ratio exhibited the best effect based on zebrafish model, which could improve cardiac output and blood flow velocity and inhibit pericardial enlargement and venous blood stasis significantly. A combination of 9 ginsenosides and 3 aconite alkaloids based on a component-efficacy modeling by PLSR was screened, and exerted approximately equivalent pharmacological effects compared with Renshen-Fuzi herbal pair. CONCLUSIONS: Our findings elucidated the effective combination of Renshen-Fuzi herbal pair that has been used in clinic for the treatment of heart failure, which could also promote the pharmacological research and quality control of their formula such as Shenfu decoction.

Comprehensive chemical profiling of volatile constituents of Angong Niuhuang Pill in vitro and in vivo based on gas chromatography coupled with mass spectrometry.

BACKGROUND: Angong Niuhuang Pill (ANP), a renowned precious traditional Chinese medicine prescription, is extensively utilized for the clinical treatment of stroke, meningitis and encephalorrhagia in China. As a classic resuscitation-inducing aromatic prescription, ANP has been investigated for its pharmacological effects in recent years, while the volatile composition in ANP still lacks comprehensive elucidation. METHOD: To better explore the volatile constituents in ANP, a qualitative analysis method was developed based on gas chromatography coupled with mass spectrometry. Furthermore, a validated quantitative method was established to determine 21 main compounds in 8 batches of commercially available ANP samples by gas chromatography-tandem mass spectrometry. The quantitative data were successively subjected to Pearson correlation coefficient analysis. Additionally, the absorbed volatile constituents in rat plasma after single oral administration of ANP have also been characterized. RESULTS: A total of 93 volatile constituents including 29 sesquiterpenoids, 28 monoterpenoids, 13 fatty acids and their esters, 7 alkanes, 6 ketones, 3 phenols, 3 aldehydes, 2 benzoate esters, and 2 other types, were preliminarily characterized, which primarily originated from Borneolum, Moschus, Curcumae Radix, and Gardeniae Fructus. D-Borneol, isoborneol and muscone were the top three abundant ingredients (> 600 µg/g) in 8 batches of ANP samples. Subsequently, the average Pearson correlation coefficient of the contents of 21 analytes was 0.993, inferring the high batch-to-batch similarity among 8 batches. After oral administration of ANP, D-borneol, isoborneol, muscone and camphor were the main volatile constituents absorbed in the rat plasma. CONCLUSION: This research may be helpful for the comprehensive quality control study of ANP, and provide for guarantee the clinical efficacy of ANP.