Integrated analysis of liquid chromatography-mass spectrometry and network pharmacology identified active compounds of Paeoniae Radix Alba for myasthenia gravis treatment

A number of reports are available in the literature that describe liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) analysis of amanitins, very toxic mushroom toxins, in biological samples. However, the extractive pretreatment methods and LC separation column materials vary remarkably according to the different reports. This communication presents a very simple and suffi ciently sensitive method for LC-MS analysis of amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC-MS analysis with a conventional octadecyl LC separation column. The selected ion monitoring of α-amanitin and β-amanitin at m/z 919–921 and m/z 920–922, respectively, gave symmetrical peaks and good separation of both amanitin peaks. Using an external calibration method, linearity, detection limits, recovery rates, and precision were tested; they were all satisfactory. To our knowledge, the present method gives the simplest LC-MS analysis for amanitins among those so far reported. We recommend the method for use in actual forensic and clinical toxicological analysis of amanitins in biological samples.

Evaluating the interplay among stationary phases/ion-pairing reagents/sequences for liquid chromatography mass spectrometry analysis of oligonucleotides

Analysis of liquid chromatography-mass spectrometry (LC-MS) data requires the differentiation between a small number of relevant chemical signals and a larger amount of noise. This is often done based, at least partially, on a threshold which assumes that low intensity m/z signals arise from the noise. This eliminates low-intensity fragments, isotopes, and adducts and may exclude relevant low-intensity compounds all together. This work describes the use of multivariate curve resolution-alternating least-squares with an additional sparse regression step using elastic net (MCR-ENALS) to distinguish relevant m/z signals without the use of a harsh thresholding step, thus allowing for discovery of low-intensity m/z signals corresponding to the analytes. This strategy is demonstrated first on a unit mass analysis of amphetamines in which relevant m/z signals are found at as low as a 0.1% intensity relative to the molecular m/z peak. The incorporation of MCR-ENALS into our previously reported data reduction strategy for analysis of high-resolution LC-MS is also demonstrated. Analysis based on only 0.3% of the original data set, while retaining low-intensity isotope peaks, was accomplished without the use of thresholding, allowing for the application of MCR-ENALS to the high-resolution LC-MS data.

Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis

Malaria is a deadly disease that continues to pose a threat to children and maternal well-being. This study was designed to identify the chemical constituents in the ethanolic fruit extract of Azadirachta indica, elucidate the pharmacological potentials of identified phytochemicals through the density functional theory method and carry out the antimalarial activity of extract using chemosuppression and curative models. The liquid chromatography-mass spectrometry (LC-MS) analysis of the ethanolic extract was carried out, followed by the density functional theory studies of the identified phytochemicals using B3LYP and 6-31G (d, p) basis set. The antimalarial assays were performed using the chemosuppression (4 days) and curative models. The LC-MS fingerprint of the extract led to the identification of desacetylnimbinolide, nimbidiol, O-methylazadironolide, nimbidic acid, and desfurano-6α-hydroxyazadiradione. Also, the frontier molecular orbital properties, molecular electrostatic potential, and dipole moment studies revealed the identified phytochemicals as possible antimalarial agents. The ethanolic extract of A indica fruit gave 83% suppression at 800 mg/kg, while 84% parasitaemia clearance was obtained in the curative study. The study provided information about the phytochemicals and background pharmacological evidences of the antimalarial ethnomedicinal claim of A indica fruit. Thus, isolation and structure elucidation of the identified phytochemicals from the active ethanolic extract and extensive antimalarial studies towards the discovery of new therapeutic agents is recommended for further studies.

Multidimensional LC-MS with 1D multi-method option and parallel middle-up and bottom-up MS acquisition for in-depth characterization of antibodies

Tyrosinase (TYR) is a rate-limiting enzyme in the synthesis of melanin, while direct TYR inhibitors are a class of important clinical antimelanoma drugs. This study established a spectrum-effect relationship analysis method and high-performance liquid chromatography-mass spectrometry (LC-MS) analysis method to screen and identify the active ingredients that inhibited TYR in Salvia miltiorrhiza–Carthamus tinctorius (Danshen–Honghua, DH) herbal pair. Seventeen potential active compounds (peaks) in the extract of DH herbal pair were predicted, and thirteen of them were tentatively identified by LC-MS analysis. Furthermore, TYR inhibitory activities of five pure compounds obtained from the DH herbal pair were validated in the test in which kojic acid served as a positive control drug. Among them, three compounds including protocatechuic aldehyde, hydroxysafflor yellow A, and tanshinone IIA were verified to have high TYR inhibitory activity (IC50 value of 455, 498, and 1214 μM, resp.) and bind to the same amino acid residues in TYR catalytic pocket according to the results of the molecular docking test. However, the other two compounds lithospermic acid and salvianolic acid A had a weak effect on TYR, as they do not combine with the active amino acid residues or act on the active center of TYR. Therefore, the developed methods (spectrum-effect relationship analysis and molecular docking) could be used to effectively screen TYR inhibitors in complex mixtures such as natural products.

A novel cholesterol-reducing mechanism of polygonati rhizoma: Dual action via Bacteroides-mediated cholesterol sulfonation and feedback inhibition of ACAT2 by sulfated metabolite.

Protective effect of Tecoma stans (L.) Juss.ex Kunth in CFA-induced arthritic rats

Pulveroboletus ravenelii (Beck. et Curt.) Murr. (Boletaceae), commonly known as Ravenel's bolete, is an edible and medicinal mushroom, and is also used for preparing mushroom-based dyes. As part of a continuing project to discover the bioactive natural products from wild mushrooms, we analyzed the methanol (MeOH) extract of P. ravenelii to identify metabolites with the anticancer activity. Chemical analysis of the MeOH extract combined with liquid chromatography-mass spectrometry (LC-MS) analysis led to the isolation of a phenolic compound, pulveraven A (PA), whose chemical structure was determined using a combination of 1D and 2D NMR and LC-MS analysis. In the present study, we investigated the cytotoxicity and anticancer mechanisms of pulveraven A using human breast cancer (MCF-7) cells, and demonstrated that it reduced cell viability of MCF-7 cells below 50% (71.74 ± 3.61 μM). Annexin V Alexa Fluor 488 binding assay and western blot results revealed that pulveraven A induced apoptotic cell death via the extrinsic apoptosis pathway, as indicated by the activation of initiator caspase-8 and executioner caspase-7. Furthermore, it was accompanied by an increase in the Bax/Bcl-2 ratio. These results suggest that pulveraven A induces apoptosis in breast cancer cells via the extrinsic apoptotic signaling pathway.

Protocol to pinpoint oxidized and reduced cysteine residues in brown fat using differential alkylation labeling techniques

Introduction: The development, physiology function, and pathogenesis of the tissues are intricately orchestrated by the spatial organization of diverse cell populations. Proteins are the fundamental components within individual cells that control every function. Therefore, by integrating both information on cellular location and molecular profiling, spatial proteomics has emerged as an indispensable field for understanding mechanisms governing tissue homeostasis, disease progression, and therapeutic responses. Here, I introduce a spatial proteomics method designed to target selective cell phenotypes within definite tissue niches for in-depth spatial biology analysis. Method: In this approach, we developed a sequential pipeline highlighted with immunofluorescence staining, microscopic photobleaching, fluorescence cell sorting, and liquid chromatography-mass spectrometry (LC-MS) analysis. The process begins with staining 400-μm-thick tissue macrosections with fluorescence-conjugated antibodies to visualize specific cell types of interest. We employed two different fluorescence-conjugated antibodies of the same clone (e.g., Alexa488-anti-CD11c and Alexa647-anti-CD11c antibodies) for the staining on every targeted cell (e.g., CD11c+ immune cells). We then introduced “photobleaching barcodes” using a confocal microscope to photobleach/exhaust either one of the fluorescence signals stained on cells by exposing it to the matched laser. This allows us to create differentiable fluorescence barcodes that label cells at distinct regions, incorporating cell spatial information into our fluorescence detection. After tissue dissociation, barcoded cells were sorted into different groups which were also classified by their original locations in the macrosection. Finally, protein extraction and LC-MS analysis were conducted on the collected cells to enable comprehensive spatial proteomic analysis. Results: The initial application in investigating dendritic cell (DC) subsets in mouse spleen during lipopolysaccharide (LPS)-induced inflammation reveals significant proteome differences among three splenic DC subsets, categorized by their locations in or outside spleen T-cell zones as well as control DCs. Our result aligns with previously published proteome data in splenic DCs, underscoring the feasibility and reliability of our technology. Currently, we are in the process of evaluating breast cancer cell heterogeneity in human biopsy specimens using our approach to profile the tumor-immune microenvironment. Conclusion: Spatial proteomic findings from both applications are poised to discover potential drug targets that benefit treatment efficacy for inflammatory diseases and breast cancer. This technology features deep protein profiling for cell-type-specific spatial proteomics and is designed for broad applications across diverse tissue specimens in various diseases. Citation Format: Yi-Chien Wu, Elie Abi Khalil, Samuel Weng, Steve Lee. Tissue-niche-based and cell-type-selective in-depth proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3772.

Chemical labeling (CL) in combination with liquid chromatography-mass spectrometry (LC-MS) analysis has been demonstrated to be a promising technology in metabolomic analysis. However, identification of chemically labeled metabolites remains to be challenging. Retention time (RT) is one of the most important parameters for the identification of metabolites, but it could vary greatly in LC-MS analysis. In this work, we developed a chemical labeling-based HPLC retention index (CL-HPLC RI) strategy to facilitate the identification of metabolites. In this CL-HPLC RI strategy, a series of 2-dimethylaminoethylamine (DMED)-labeled fatty acids were used as calibrants to establish RIs for DMED-labeled carboxylated compounds and a series of 4-( N, N-dimethylamino)phenyl isothiocyanate (DMAP)-labeled fatty amines were used as calibrants for DMAP-labeled amine compunds. To calculate the RIs, the whole LC chromatogram was divided into 24 time intervals by 23 DMED-labeled fatty acid standards or 15 time intervals by 14 DMAP-labeled fatty amine standards. Then, we established the RIs of 854 detected DMED-labeled carboxylated metabolites and 1057 DMAP-labeled amine metabolites in fecal samples and demonstrated that RIs were highly reproducible under different elution gradients, columns, and instrument systems. Finally, we applied this strategy to the identification of metabolites in human serum. Using RIs, 267 DMED-labeled carboxylated metabolites and 273 DMAP-labeled amine metabolites in human serum matched well with the fecal metabolome database. Taken together, the developed CL-HPLC RI strategy was demonstrated to be a promising method to facilitate the identification of metabolites in metabolomic analysis.

Honey from stingless bee is highly regarded as a medicine in many global communities due to its nutritional and therapeutic value. There is a comparatively restricted amount of research on the biological characteristics of stingless bee honey (SBH) in contrast to Indian bee honey (IBH). Therefore, the objective of this research is to explore the anti-cancer properties of Tetragonula travancorica honey. In the present investigation, antioxidant activity of honey was evaluated using ABTS, DPPH and SOD radical scavenging assays. Anti-proliferative effects was examined on various cell lines using the MTT assay. Liquid chromatography-mass spectrometry (LCMS) analysis was employed to characterize the honey. Binding capabilities of compounds identified in SBH on estrogen receptors (ER) α and ER β were explored through Schrödinger Maestro software. Results indicated that SBH effectively scavenged ABTS, DPPH and SOD free radicals with IC50 values of 22, 36.15, and 79.85 v/v, respectively. SBH exhibited anti-proliferative activity against MCF-7 and HeLa cell lines, with IC50 values of 58.11 and 66.68 v/v, respectively. LCMS analysis, shows the presence of methyl syringate, 2-hydroxycinnamic acid, and fumaric acid in SBH which were not present in IBH. Docking experiments determined that these compounds, with the exception of fumaric acid, interacted stronger with the binding sites of ER β than ER α. The comprehensive findings from antioxidant, anti-proliferative, and docking studies underscore the potential anti-cancer properties of Tetragonula travancorica honey, particularly its heightened cytotoxicity against reproductive system cancer cell lines, such as those in the breast and cervix. Keywords : Anti-proliferative, estrogen receptor, stingless bee, cytotoxicity.

Trametes villosa laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by 13C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents.