Liquid Chromatography-tandem Mass Spectrometry Analysis Research Articles

Mendelian randomization and multi-omics approach analyses reveal impaired glucose metabolism and oxidative phosphorylation in visceral adipose tissue of women with polycystic ovary syndrome.

What is the significance of visceral adipose tissue (VAT) in the pathogenesis of polycystic ovary syndrome (PCOS) and its impact on the regulation of metabolic disorders in women with PCOS? We revealed a potentially causal relationship between increased genetically predicted VAT and PCOS-related traits, and found that VAT exhibited impaired glucose metabolism and mitochondrial oxidative phosphorylation (OXPHOS) in women with PCOS. PCOS is a common reproductive endocrine disorder accompanied by many metabolic abnormalities. Adipose tissue is a metabolically active endocrine organ that regulates multiple physiological processes, and VAT has a much stronger association with metabolism than subcutaneous adipose tissue does. Mendelian randomization (MR) analysis was used to investigate the potential causal association between genetically predicted VAT and the risk of PCOS. Data for MR analysis were extracted from European population cohorts. VAT samples from sixteen PCOS patients and eight control women who underwent laparoscopic surgery were collected for proteomics and targeted metabolomics analyses. PCOS was diagnosed according to the 2003 Rotterdam Criteria. The control subjects were women who underwent laparoscopic investigation for infertility or benign indications. Proteomics was performed by TMT labeling and liquid chromatography-tandem mass spectrometry analysis, and targeted metabolomics was performed by ultra-performance liquid chromatography-tandem mass spectrometry analysis. The key differentially expressed proteins (DEPs) were validated by immunoblotting. MR analysis revealed a potentially causal relationship between increased genetically predicted VAT and PCOS, as well as related traits, such as polycystic ovaries, total testosterone, bioavailable testosterone, and anti-Müllerian hormone, while a negative relationship was found with sex hormone-binding globulin. Enrichment pathway analysis of DEPs indicated the inhibition of glycolysis and activation of mitochondrial OXPHOS in the VAT of PCOS patients. MR analysis revealed that key DEPs involved in glycolysis and OXPHOS were significantly linked to PCOS and its related traits. Dot blot assay confirmed a significant decrease in glycolysis enzymes PKM2 and HK1, and an increase in mitochondrial Complex I and III subunits, NDUFS3 and UQCR10. Moreover, metabolomics analysis confirmed down-regulated metabolites of energy metabolic pathways, in particular glycolysis. Further analysis of PCOS and control subjects of normal weight revealed that dysregulation of glucose metabolism and OXPHOS in VAT of women with PCOS was independent of obesity. The mass spectrometry proteomics data have been deposited to the iProX database (http://www.iprox.org) with the iProX accession: IPX0005774001. There may be an overlap in some exposure and outcome data, which might affect the results in the MR analysis. The changes in protein expression of key enzymes affect their activities and disrupt the energy metabolic homeostasis in VAT, providing valuable insight for identifying potential intervention targets of PCOS. This work was supported by the National Key Research and Development Project of China (2021YFC2700402), the National Natural Science Foundation of China (82071608, 82271665), the Key Clinical Projects of Peking University Third Hospital (BYSY2022043), and the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-001). All authors report no conflict of interest. N/A.

Comprehensive Coverage of Glycolysis and Pentose Phosphate Metabolic Pathways by Isomer-Selective Accurate Targeted Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Assay.

The accurate liquid chromatography-tandem mass spectrometry analysis of phosphorylated isomers from glycolysis and pentose phosphate pathways is a challenging analytical problem in metabolomics due to extraction problems from the biological matrix, adherence to stainless steel surfaces leading to tailing in LC, and incomplete separation of hexose and pentose phosphate isomers. In this study, we present a targeted HILIC-ESI-MS/MS method based on a BEH amide fully porous 1.7 μm particle column with an inert surface coating of column hardware and multiple reaction monitoring (MRM) acquisition fully covering the glycolysis and pentose phosphate pathway metabolites. To minimize contact of the phosphorylated analytes with stainless steel surfaces, a μ-ESI-MS probe with a hybrid electrode made of PEEKsil was employed. Optimized HILIC gradient elution conditions with 100 mM ammonium formate (pH 11) provided the separation of hexose monophosphate and pentose phosphate isomers. To ensure good retention time repeatability in HILIC, perfluoroalkoxy alkane bottles were used for the mobile phase (with sd over 60 runs between 0.01 and 0.02 min). For the quantitative assay, the U-13C-labeled cell extract was spiked prior to extraction by metal oxide-based affinity chromatography (MOAC) with TiO2 beads. The concentrations of the 24 targets were quantified in HeLa and human embryonic kidney (HEK293) cells. Erastin-induced ferroptosis in HEK293 cells was accompanied by enhanced levels of fructose-1,6-bis-phosphate, 2- and 3-phosphoglycerate, and 2,3-bis-phosphoglycerate.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

PFKP inhibition protects against pathological cardiac hypertrophy by regulating protein synthesis

Metabolic reprogramming precedes most alterations during pathological cardiac hypertrophy and heart failure (HF). Recent studies have revealed that Phosphofructokinase, platelet (PFKP) has a wealth of metabolic and non-metabolic functions. In this study, we explored the role of PFKP in cardiac hypertrophic growth and HF. The expression level of PFKP was elevated both in pathological cardiac remodeling mouse model challenged by transverse aortic constriction (TAC) surgery and in the neonatal rat cardiomyocytes (NRCMs) stimulated by phenylephrine (PE). In global PFKP knockout (PFKP-KO) mice, cardiac hypertrophy was ameliorated under TAC surgery, while overexpression of PFKP by intravenous injection of adeno-associated virus 9 (AAV9) under the cardiac troponin T (cTnT) promoter worsened myocardial hypertrophy and fibrosis. In NRCMs, small interfering RNA (SiRNA) knockdown or adenovirus (Adv) overexpression of PFKP was employed and the intervention of PFKP showed a similar phenotype. Mechanistically, immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP-MS/MS) analysis was used to identify the interacting proteins of PFKP. Eukaryotic translation initiation factor 2 subunit beta (EIF2S2) was identified as the downstream target of PFKP. In the PE-stimulated NRCM hypertrophy model and mouse TAC model, knocking down EIF2S2 after PFKP overexpression reduced the synthesis of new proteins and alleviated the hypertrophy phenotype. Our findings illuminate that PFKP participates in pathological cardiac hypertrophy partly by regulating protein synthesis through EIF2S2, which provides a new clue for the involvement of metabolic intermediates in signal transduction.

Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Dalbavancin in Plasma of Pediatric and Young Adult Patients.

Dalbavancin, an antimicrobial lipoglycopeptide, is authorized in Europe for treating acute bacterial infections of the skin and skin structures in adults and pediatric patients aged 3 months and older. However, off-label dosing regimens have been proposed for various indications beyond acute bacterial infections of the skin and skin structures. This study presents a novel bioanalytical method using liquid chromatography-tandem mass spectrometry to quantify dalbavancin in low-volume plasma samples (50 μL). The method underwent validation in accordance with international guidelines for bioanalytical method validation and was applied to 9 clinical samples obtained from pediatric and young adult patients undergoing dalbavancin therapy. Liquid chromatography-tandem mass spectrometry analyses were conducted at the G. Gaslini Institute in Genoa, Italy, utilizing an Ultimate 3000 ultra high performance liquid chromatography system coupled to a TSQ Quantiva Triple Quadrupole system (Thermo Fisher Scientific, Milan, Italy). The analytical procedure involved the addition of deuterated dalbavancin as internal standard and a rapid extraction from 50 µL of human plasma, followed by chromatographic separation on a Thermo Scientific Accucore Polar Premium column. Accurate quantification of the analyte was achieved through multiple reaction monitoring detection. The assay exhibited linearity within the concentration range of 0.66-400 mcg/mL in plasma, demonstrating accuracy and reproducibility in the absence of matrix effects. Stability testing was conducted on both quality controls and real samples to establish a robust protocol under real-life conditions. This fast and reliable dalbavancin quantitation method could improve current pediatric clinical practice by enabling data collection for future dose recommendations in special patient populations.

Simultaneous detection of myostatin-targeting monoclonal antibodies in dried blood spots and plasma using liquid chromatography-tandem mass spectrometry with field asymmetric ion mobility spectrometry

Transforming growth factor-β superfamily members, such as myostatin, growth/differentiation factor 11, and activin A, negatively regulate skeletal muscle mass. Inhibitors targeting these cytokines or activin receptor type IIB have the potential to treat muscular diseases and enhance physical performance. However, because of their effects on muscle mass and potential misuse, they are strictly prohibited in sports. Given the high potential for misuse as a doping agent in sports, effective analytical methods for these prohibited antibodies targeting these specific cytokines or their receptor are critically needed. In this study, we aimed to develop and validate a multitarget method to detect the prohibited transforming growth factor-β superfamily-targeting monoclonal antibodies, such as landogrozumab, domagrozumab, and the activin receptor type IIB-targeting antibody, bimagrumab, in human plasma and dried blood spot (DBS) samples using liquid chromatography-tandem mass spectrometry. Antibodies were purified from both the DBS and plasma samples using protein G magnetic beads and field-asymmetric ion mobility spectrometry (FAIMS) to minimize interference, followed by liquid chromatography-tandem mass spectrometry analysis. The validation process included tests for specificity, selectivity, linearity, limit of detection (LOD), limit of identification, precision, recovery, carryover effect, and matrix effect. The LODs for the target antibodies were identical in both DBS and plasma samples at 0.1 µg/mL for landogrozumab heavy and light chains, as well as 0.25 µg/mL for the domagrozumab light chain and 0.25 µg/mL for the bimagrumab heavy chain. However, the heavy chain of domagrozumab exhibited an LOD of 0.5 µg/mL in DBS and 1 µg/mL in plasma. The analytical method demonstrated strong linearity, with R² values greater than 0.99 for both plasma and DBS, and no carryover effect. Precision (CV%) was below 15 % at both middle (1 or 5 µg/mL; specific to the heavy chain of domagrozumab in plasma) and high (10 µg/mL) concentrations and was less than 20 % at the LOD. The selectivity and specificity indicated no interference in the analysis of target mAbs in different blood samples. Recovery was 31.6–49.8 % for DBS and 51.4–85.3 % for plasma, with no significant matrix effect. This study provides an effective method for doping analysis and novel protein detection.

Towards the non-invasive determination of estradiol levels: Development and validation of an LC-MS/MS assay for quantification of salivary estradiol at sub-pg/mL level

BackgroundEstradiol (E2) is a female sex hormone involved in several biological processes. Although E2 levels are commonly measured in blood samples, the use of non-invasive techniques (e.g. determination of salivary E2) would allow for the collection of repeated samples and the inclusion of a greater number of participants. Immunoassay-based techniques to measure salivary E2 failed to accurately mirror the variations observed in the plasmatic concentrations of E2 during the menstrual cycle probably due to the high sensitivity required (in the sub-pg/mL range). Therefore, sensitive and rugged analytical methods for the determination of salivary E2 are required. For this, we developed and validated an analytical methodology for the accurate determination of salivary E2. ResultsThe method is based on chemical derivatization with 1,2-dimethyl-1H-imidazole-5-sulphonyl chloride and liquid chromatography-tandem mass spectrometry analysis by summing highly-specific SRM transitions. This strategy allowed for increasing the sensitivity of the method. The validation of the method showed an accurate and precise quantification of E2 in 1 mL of saliva even at 250 fg/mL (97 % accuracy and 15 % RSD intra-day, and 104 % accuracy and 18 % RSD inter-day). In order to evaluate its efficacy, we analysed saliva samples from 5 healthy female volunteers collected during a whole menstrual cycle. Our analyses showed that the variations in the concentration of E2 in the measured samples mirrored those expected during a complete menstrual cycle. Additionally, we validated the suitability of our method for determining salivary E2 levels during pregnancy. SignificanceTo the best of our knowledge, this is the first method that allows to precisely and accurately measuring E2 in saliva samples along the whole menstrual cycle of healthy females. It is also suitable for the determination of estradiol during pregnancy. Its high sensitivity makes this strategy ideal for the evaluation of the role of hormone production in women's health.

Comparative metabolomic analysis of spaghetti meat and wooden breast in broiler chickens: unveiling similarities and dissimilarities.

Spaghetti meat (SM) and wooden breast (WB) are emerging myopathies in the breast meat of fast-growing broiler chickens. The purpose of the study was to investigate the metabolomic differences between normal (N), SM, and WB fillets 24h postmortem. Eight chicken breasts for each experimental group were collected from a commercial processing plant. Supernatant from tissue homogenates were subjected to ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS) analysis. A total of 3,090 metabolites were identified in the chicken breast meat. The comparison of WB and N showed 850 differential metabolites (P < 0.05), and the comparison of SM and N displayed 617 differential metabolites. The comparison of WB and SM showed 568 differential metabolites. The principal component analysis (PCA) plots showed a distinct separation between SM and N and between WB and N except for one sample, but SM and WB were not distinctly separated. Compared to N, 15-Hydroxyeicosatetraenoic acid (15-HETE) increased, and D-inositol-4-phosphate decreased in both SM and WB, indicating that cellular homeostasis and lipid metabolism can be affected in SM and WB. The abundance of nicotinamide adenine dinucleotide (NAD) + hydrogen (H) (NADH) was exclusively decreased between SM and N (P < 0.05). Purine metabolism was upregulated in SM and WB compared to N with a greater degree of upregulation in WB than SM. Folic acid levels decreased in SM and WB compared to N (P < 0.05). Steroid hormone biosynthesis was downregulated in SM compared to N (P < 0.05). Carbon metabolism was downregulated in SM and WB compared to N with greater degree of downregulation in WB than SM (P < 0.05). These data suggest both shared and unique metabolic alterations in SM and WB, indicating commonalities and differences in their underlying etiologies and meat quality traits. Dietary supplementation of deficient nutrients, such as NADH, folic acids, etc. and modulation of altered pathways in SM and WB would be strategies to reduce the incidence and severity of SM and WB.

Di(2-ethylhexyl) phthalate disrupts circadian rhythm associated with changes in metabolites and cytochrome P450 gene expression in Caenorhabditis elegans

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is a widespread environmental pollutant due to its extensive use. While circadian rhythms are inherent in most living organisms, the detrimental effects of DEHP on circadian rhythm and the underlying mechanisms remain largely unknown. This study investigated the influence of early developmental exposure to DEHP on circadian rhythm and explored the possible relationship between circadian disruption and DEHP metabolism in the model organism Caenorhabditis elegans. We observed that DEHP disrupted circadian rhythm in a dose-dependent fashion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that DEHP-induced circadian disruption accompanies with altered proportions of DEHP metabolites in C. elegans. RNA sequencing data demonstrated that DEHP-induced circadian rhythm disruption caused differential gene expression. Moreover, DEHP-induced circadian disruption coincided with attenuated inductions of DEHP-induced cytochrome P450 genes, cyp-35A2, cyp-35A3, and cyp-35A4. Notably, cyp-35A2 mRNA exhibited circadian rhythm with entrainment, but DEHP exposure disrupted this rhythm. Our findings suggest that DEHP exposure disrupts circadian rhythm, which is associated with changes in DEHP metabolites and cytochrome P450 gene expression in C. elegans. Given the ubiquitous nature of DEHP pollution and the prevalence of circadian rhythms in living organisms, this study implies a potential negative impact of DEHP on circadian rhythm and DEHP metabolism in organisms.

Synthesis of 1,2,4-triazole-based deep eutectic solvents modified nickel ferrite nanoparticles and their application in dispersive solid phase extraction of triazole pesticides prior to LC-MS/MS analysis

A dispersive solid phase extraction method using new magnetic nanoparticles based on nickel ferrite was introduced for the extraction of six triazole pesticides (penconazole, hexaconazole, tebuconazole, diniconazole, triadimefon, and difenoconazole) from water samples before liquid chromatography-tandem mass spectrometry analyses. Initially, a new deep eutectic solvent was synthesized with 1,2,4-triazole and n-octanol for surface modification of the nanoparticles easily achieved through microwave radiation. The nanoparticles morphology, magnetic properties, adsorption capacity, isotherms, and crystalline patterns of the sorbent were examined. The capability of the sorbent was evaluated by extracting the target pesticides from water samples showing significant differences in adsorption capacity and efficiency between the modified and non-modified nanoparticles. High extraction recoveries (68-86%) were achieved for the analytes using small amounts of the sorbent with low limits of detection (0.03-0.08 ng mL-1) and quantification (0.13-0.29 ng mL-1), a wide linear range (0.29-250 ng mL-1), and acceptable precision (relative standard deviations ≤6.9%).

Liquid chromatography-tandem mass spectrometry analysis of nitrogen mustard degradation products after derivatization with benzoylimidazole

Nitrogen mustards are highly toxic compounds and strictly controlled by the Chemical Weapons Convention. When those agents are used, exposure must be confirmed by analyzing biological samples from victims to allow prompt medication and reduction of secondary damage. In this study, a derivatization-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method to analyze nitrogen mustard degradation products (ethanolamines; EAs) in human urine was developed. Benzoylimidazole (BzI) was used as a derivatization reagent, and two highly hydrophilic EAs (methyldiethanolamine and ethyldiethanolamine) were analyzed by using a reversed-phase column with sufficient retention. For urine samples, after solid-phase extraction with cation exchange cartridge and derivatization, EAs were analyzed at low limits of detection (5 ng/mL). The calibration curves had a wide linear range (50–1000 ng/mL), and the quantitation results were sufficient for screening. Compared with our previous derivatization-LC-MS/MS method using pentafluorobenzoyl chloride as a derivatization reagent and hydrophilic interaction liquid chromatography (HILIC)-MS/MS method, not only the limits of detection but also the operability and throughput were improved. The performance of the newly developed derivatization reagent was also evaluated using density functional theory (DFT) calculations.

Untargeted Metabolite Profiling of Wild and In Vitro Propagated Sabah Jewel Orchid Macodes limii J.J. Wood & A.L. Lamb.

Macodes limii J.J. Wood & A.L. Lamb is a terrestrial jewel orchid native to Sabah, recognised for its sparkling golden-yellow venations, uniformly distributed on its leaves. Despite its high ornamental value, the exploration of the plant's medicinal potential remains ambiguous. The current study was conducted to gain a fundamental understanding of the metabolite composition and regulation in M. limii plants from two different growing environments: wild and in vitro cultivation, as well as to analyse their phytochemical contents and antioxidant activity. The metabolite profiling of the M . limii plant extracts through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has tentatively identified compounds from various classes including sugars, carbohydrates, sugar alcohols, amino acids, organic acids, phenolic derivatives and lipid and lipid-like compounds. Subsequently, the multivariate statistical analysis confirmed the existence of significant metabolite variations across distinct growth environments. Notably, the leaf extract derived from wild-grown plants displayed the highest levels of total phenolic and flavonoid content, contributing significantly to its higher antioxidant activity as measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The discovery has offered a fundamental understanding of the metabolites in M. limii jewel orchids, indicating that in vitro regenerated plants may represent a viable alternative for further investigating their therapeutic potential, thus helping to alleviate the impact on wild populations.

Caspase-3 interactins with calpain and cathepsin L: implications for protein stability and quality in fish fillets during postmortem storage

The degradation of structural proteins during fish postmortem storage is a critical factor affecting meat quality, leading to economic losses in the seafood industry. The complex interplay between caspase-3 and structural protein stability during fish postmortem storage remains largely unexplored. By treating grass carp fillets with a caspase-3 inhibitor, we establish a model for inhibiting apoptosis, delineating the role of caspase-3 in protein degradation. The effect of caspase-3 on grass carp proteins following postmortem storage and its cascade interaction with calpain and cathepsin L was further investigated. The result demonstrated that the destabilisation of the mitochondrial membrane during apoptosis led to modifications in the B-cell lymphoma (Bcl) family proteins, subsequently triggered the activation of caspase-9/caspase-3. Additionally, caspase-3 reduced the expression of the calpastatin and lysosomal membrane protein 1 (LAMP-1) gene, resulting in increased calpain and cathepsin L activity. SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that caspase-3 inhibitor treatment reduced actin fractures and preserved critical structural proteins. This investigation unveils the molecular mechanisms through which caspase-3 affects protein degradation in fish fillets during storage, providing valuable insights into the postmortem degradation processes.

Proteomic analysis revealing mechanisms of κ-carrageenan modulating the gelling properties of heat-induced microbial transglutaminase cross-linked myofibrillar protein gels

The effects of κ-carrageenan (KC, 0.2 %, w/w) on the gelling properties and microstructures of heat-treated myofibrillar protein (MP) gels mediated by various levels (0, 0.1, 0.3, and 0.5 %, w/w) of microbial transglutaminase (TG) were investigated. The results revealed that regardless of TG level, the existence of KC significantly promoted the forming of disulphide bonds but limited the generation of large-sized protein polymers in MP gel treated with TG alone, as well as subsequently enhanced the water retention ability and gel strength of mixed MP gel, especially in the sample adding 0.3% TG and 0.2% KC, which was clearly verified by the forming of more denser and continuous gel network. Moreover, TG-induced changes in protein structure led to the alteration in the protein profiles of different MP gels, and an obvious down-regulation trend of myosin (Q9TV63, Q9TV62, P79293, and Q9TV61) was identified in the TG-0.3 group. Furthermore, liquid chromatography–tandem mass spectrometry analysis demonstrated that TG mainly induces protein crosslinking in the light meromyosin domain of the myosin rod. Meanwhile, in the presence of KC, the crosslinking-site percentage of the TG-0.3 group decreased from 37.7% to 36.2%, indicating that KC potentially reduces TG-induced crosslinking in MP gels. Thus, our findings illustrate the critical mechanisms underlying the modulation of KC on the gelling properties of TG-mediated MP gels at the molecular level, which providing potential guideline for the combined applications of TG and KC in the meat industry.

Quantification of guanidine in environmental samples using benzoin derivatization and LC-MS analysis

The recent discovery of guanidine-dependent riboswitches in many microbes raised interest in the biological function and metabolism of this nitrogen-rich compound. However, very little is known about the concentrations of guanidine in the environment. Several methods have been published for quantifying guanidine and guanidino compounds in human urine and blood, often relying on derivatization followed by fluorescence detection. We adapted this analytical approach using benzoin as the derivatization agent to sensitively and selectively quantify guanidine in environmental samples, thereby facilitating future research on the biological and environmental roles of guanidine. This adapted method was applied to human urine, raw wastewater, and biological growth media as relevant matrices. Our liquid chromatography-tandem mass spectrometry analyses of the derivatized solutions identified a different major derivatization product than previously reported. This product was consistently observed across various substrates (guanidine, methylguanidine, and arginine) and derivatization agents (benzoin and anisoin). We observed a constant background signal, restricting our analyses to a lower limit of quantification of 50 nM. Despite this limitation, our method allowed for the quantification of guanidine concentrations significantly lower than those reported in previous derivatization-based studies.•Selective and sensitive detection of guanidine by LC-MS.•Method development and validation for robust detection of guanidine in environmental samples.•Reduction of sample preparation steps and reduced usage of toxic chemicals compared to previous methods.

Exploring the anticancer potential of Cytotoxin 10 from Naja kaouthia venom: Mechanistic insights from breast and lung cancer cell lines

Breast and lung cancers are the leading causes of cancer-related deaths in the world. Although considerable progress has been made in the field of cancer therapy, quest to discover potent, safe and cost-effective alternatives especially from natural sources is being pursued. Snake venom, which is a treasure trove of various peptides and proteins including natural toxins that specifically target tissues and receptors in the envenomated victims. Many such proteins are being explored for their therapeutic potential against various diseases including cancers. Here, we report the mechanism of cytotoxic activity of crude venom and a purified protein, Cytotoxin from the monocled cobra (Naja kaouthia), an elapid snake with neurotoxic venom prominently found in the North-East India. The crude venom showed significant cytotoxicity against breast (MCF-7and MDA-MB-231) and lung (A549, NCI–H522) cancer cell lines. Bioassay-guided fractionation using RP-HPLC showed highest cytotoxic activity in peak P9. Liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS) analysis was employed and the fraction is identified as Cytotoxin 10 which showed comparable cytotoxicity against the experimental cell lines. Cytotoxin 10 also exhibited apoptosis in MCF-7 and A549 cell lines using AO/EtBr and flow cytometry analysis. Expressions of apoptosis related proteins e.g. Bax, Bcl-2, Caspase-7 and PARP were also studied following Cytotoxin 10 treatment in both cell lines. Molecular docking experiments performed to investigate the interactions between Cytotoxin 10 and the apoptotic proteins revealed favourable binding scores compared to their corresponding inhibitors. Interestingly, Cytotoxin 10 inhibited migration and adhesion in a time and dose-dependent manner in both MCF-7 and A549 cells. This is the first report elucidating the mechanism of cytotoxic activity of Cytotoxin 10 purified from Naja kaouthia venom of North-East India origin and could pave the way for development of potential therapeutic strategies against breast and lung cancer.

Improved skeletal muscle mass and strength through Protamex-mediated hydrolysis of perilla seed cake: Elevated rosmarinic acid levels as a contributing factor

Perilla seed cake (PSC) is a byproduct of oil extraction from perilla seeds. It is rich in proteins and bioactive compounds. PSC was enzymatically hydrolyzed to form PSC hydrolysate (PSCH) to enhance the absorption of PSC, and their effects on muscle health in mice were compared. High performance liquid chromatography-tandem mass spectrometry analysis revealed that PSC contains several polyphenols, including rosmarinic acid (RA), caffeic acid, apigenin, and luteolin. The hydrolysate showed 1.44- and 7.04-fold increases in RA and caffeic acid contents, respectively, compared to those of PSC. The intake of PSC, PSCH, and RA significantly improved muscle mass and exercise performance in mice by upregulating protein synthesis, myogenic differentiation, oxidative muscle fiber formation, fatty acid oxidation, and mitochondrial biogenesis; however, PSCH had better promoting effects than PSC. In conclusion, PSCH improves muscle health through its bioactive compounds (particularly RA), indicating the potential of PSCH and RA in functional foods.

Bioaccessibility and antioxidant activity of fermented chrysanthemum rice wine on In vitro simulated digestion

The purpose of this study was to investigate the bioaccessibility of total phenolic (TPC), total flavonoid (TFC), chlorogenic acid and antioxidant activity in fermented chrysanthemum rice wine (FRW) under in vitro gastrointestinal digestion model compared with rice wine (RW). The fermented samples were digested and the bioactive components and antioxidant activity during gastric phase (GP) and intestinal phase (IP) were analyzed. The results showed that TPC, TFC, ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC) decreased during the gastrointestinal digestion stage. TFC was higher in FRW during in vitro gastrointestinal digestion stage and increased to a maximum of 0.483 mg/mL at IP stage. 2.2- azinobis-3- ethyl-benzothiazoline-6 - sulfonic acid (ABTS) reached its maximum value of 91.62% in the late stage of intestinal digestion. The hydroxyl radical scavenging activity (HRSA) increased to 77.94% in the IP-2 phase. In addition, a total of 158 (FRW) and 131 (RW) polyphenols, including flavonoids, isoflavones, phenols and coumarins, were detected and identified by Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) analysis. The biotransformation was carried out between the isomers of phenolic compounds in FRW, where 4,5-dicaffeoylquinic acid might be changed into neochlorogenic acid and chlorogenic acid. Correlation analysis showed that 46.15% of metabolites in FRW were positively correlated with the decrease of DPPH, FRAP and ORAC, while 53.85% were positively correlated with the increase of ABTS and HRSA. The results showed higher bioavailability and antioxidative activity in FRW after in vitro digestion.

SGLT2 inhibitors ameliorate NAFLD in mice via downregulating PFKFB3, suppressing glycolysis and modulating macrophage polarization.

Sodium-glucose co-transporter 2 (SGLT2) inhibitor (SGLT2i) is a novel class of anti-diabetic drug, which has displayed a promising benefit for non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effects of SGLT2i against NAFLD and the underlying mechanisms. The db/db mice and western diet-induced NAFLD mice were treated with dapagliflozin (1 mg·kg-1·d-1, i.g.) or canagliflozin (10 mg·kg-1·d-1, i.g.) for 8 weeks. We showed that the SGLT2i significantly improved NAFLD-associated metabolic indexes, and attenuated hepatic steatosis and fibrosis. Notably, SGLT2i reduced the levels of pro-inflammatory cytokines and chemokines, downregulated M1 macrophage marker expression and upregulated M2 macrophage marker expression in liver tissues. In cultured mouse bone marrow-derived macrophages and human peripheral blood mononuclear cell-derived macrophages, the SGLT2i (10, 20 and 40 μmol/L) significantly promoted macrophage polarization from M1 to M2 phenotype. RNA sequencing, Seahorse analysis and liquid chromatography-tandem mass spectrometry analysis revealed that the SGLT2i suppressed glycolysis and triggered metabolic reprogramming in macrophages. By using genetic manipulation and pharmacological inhibition, we identified that the SGLT2i targeted PFKFB3, a key enzyme of glycolysis, to modulate the macrophage polarization of M1 to M2 phenotype. Using a co-culture of macrophages with hepatocytes, we demonstrated that the SGLT2i inhibited lipogenesis in hepatocytes via crosstalk with macrophages. In conclusion, this study highlights a potential therapeutic application for repurposing SGLT2i and identifying a potential target PFKFB3 for NAFLD treatment.

Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) Analysis of Alkaloid Nitrogen Oxides at Three Different Processing Stages of Binglang

Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) Analysis of Alkaloid Nitrogen Oxides at Three Different Processing Stages of Binglang