Liquid Chromatography-tandem Mass Spectrometry Analysis Research Articles

Metabolomics-based profiling of five Salvia L. (Lamiaceae) species using untargeted data analysis workflow.

The genus Salvia L., a member of the family Lamiaceae, is a keystone genus with a wide range of medicinal properties. It possesses a rich metabolite source that has long been used to treat different disorders. Due to a deficiency of untargeted metabolomic profiling in the genus Salvia, this work attempts to investigate a comprehensive mass spectral library matching, computational data annotations, exclusive biomarkers, specific chemotypes, intraspecific metabolite profile variation, and metabolite enrichment by a case study of five medicinal species of Salvia. Aerial parts of each species were subjected to QTRAP liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis workflow based on untargeted metabolites. A comprehensive and multivariate analysis was acquired on the metabolite dataset utilizing MetaboAnalyst 6.0 and the Global Natural Products Social Molecular Networking (GNPS) Web Platform. The untargeted approach empowered the identification of 117 metabolites by library matching and 92 nodes annotated by automated matching. A machine learning algorithm as substructural topic modeling, MS2LDA, was further implemented to explore the metabolite substructures, resulting in four Mass2Motifs. The automated library newly discovered a total of 23 metabolites. In addition, 87 verified biomarkers of library matching, 58 biomarkers of GNPS annotations, and 11 specific chemotypes were screened. Integrative spectral library matching and automated annotation by the GNPS platform provide comprehensive metabolite profiling through a workflow. In addition, QTRAP LC-MS/MS with multivariate analysis unveiled reliable information about inter and intraspecific levels of differentiation. The rigorous investigation of metabolite profiling presents a large-scale overview and new insights for chemotaxonomy and pharmaceutical studies.

Characterization of atmospheric nitroaromatic compounds in Southwest China by direct injection liquid chromatography-tandem mass spectrometry analysis of aerosol extracts

Nitroaromatic compounds (NACs) in ambient particles are of great concern due to their adverse effects on human health and climate. However, investigations on the characteristics and potential sources of NACs in Southwest China are still scarce. In this study, a field sampling campaign was carried out in the winter of 2022 at a suburban site in Mianyang, Southwest China. A direct injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to rapidly determine 10 NACs in fine particulate matter (PM2.5) extracts. The method was sensitive for the quantification of the NACs, with a limit of quantification (LOQ) in the range of 0.092-0.52 ng mL-1. Then, the developed method was applied to determine the concentrations of nitrophenols (NPs), nitrocatechols (NCs), nitrosalicylic acids (NSAs), and nitronaphthol in PM2.5 in Mianyang. The average concentration of total NACs was 78.2±31.2 ng m-3, with daily concentrations ranging from 20.7 to 127.9 ng m-3. Among the measured NACs, 4-nitrocatechol was the most abundant, accounting for 57.8% of the NACs in winter. The five NPs compounds together contributed to 14% of the NACs, which was lower than in other Chinese cities due to the warm climate in winter in Southwest China. NSAs and nitronaphthol each accounted for less than 5% of the NACs. Three major sources of NACs were identified based on the principal component analysis, including vehicle emissions, biomass burning, and secondary formation. The significant correlation between individual NACs and NO2 supported their secondary formation sources. The good correlation between NPs and cloud amount further suggested that gas-phase oxidation was the possible NPs formation mechanism. Our findings revealed the important role of nitrocatechols in NACs in Southwest China, implying that more measures should be taken to control biomass burning and aromatic volatile organic compounds emissions to reduce the level of NACs.

Biocontrol of Botrytis cinerea by Streptomyces noursei C27 and preliminary identification of antimicrobial metabolites

A wide variety of soluble bioactive secondary metabolites and volatile organic compounds are produced by species of Streptomyces, making them potential candidates for controlling phytopathogenic microorganisms. In the present study, Streptomyces noursei C27, isolated from soil, was found to exhibit broad spectrum inhibitory activity against several phytopathogenic fungi. Furthermore, volatile organic compounds (VOCs) produced by S. noursei C27 exhibited total inhibition of Botrytis cinerea. An analysis of the complete genome sequence confirmed the identification of C27 as Streptomyces noursei. Liquid chromatography-tandem mass spectrometry analysis was used to characterize antimicrobial metabolites present in the fermentation broth of Streptomyces C27, that were identified as gougerotin, natamycin, nystatin, and anisomycin. Streptomyces C27 was shown to control gray mold on grape leaves and fruit in vitro, inhibiting infections by 72.5% and 71.9%, respectively. Gas chromatography-mass spectrometry analysis revealed that thirty alkenes, alcohols, esters, and alkanes were among the VOCs secreted by Streptomyces C27 and contributed to the inhibition of B. cinerea growth and development in inoculated grape leaves and fruit. The biocontrol efficacy of a wettable powder formulation of Streptomyces C27 against gray mold was tested on tomato seedlings in pot experiments and greenhouse experiment. Results indicated 61.86% and 67.25% control in the pot and field tests, respectively. In summary, Streptomyces noursei C27 has excellent potential to be a commercial biocontrol agent of plant diseases caused by pathogenic fungi, especially gray mold infections of vegetables and fruits caused by B. cinerea.

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.

Determination of nine bisphenol analogues in human urine by high-throughput solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis

Bisphenol analogues (BPs) are a class of typical environmental endocrine-disrupting chemicals (EDCs). This study aimed to establish a highly sensitive and high-throughput method utilizing 96-well solid-phase extraction (96-well SPE) in conjunction with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) employing multiple reaction monitoring (MRM), information-dependent acquisition (IDA), and enhanced product ion (EPI) scan modes for the identification and quantitative analysis of nine BPs in human urine. Urine samples were initially thawed to room temperature, followed by digestion using β-glucuronidase in an ammonium acetate buffer solution at 37 °C overnight. Subsequently, they were purified using 96-well SPE and finally analyzed by UHPLC-MS/MS. The limits of detection (LOD) for the nine BPs ranged from 0.05 μg∙kg−1 to 0.3 μg kg−1. Average recoveries fell within the range of 92.8 % to 111.7 %. Moreover, both the intra-day and inter-day precisions were satisfactory, with relative standard deviations (RSDs) ranging from 2.2 % to 6.7 % and 3.5 % to 6.3 %, respectively. The targets in the samples exhibited a perfect match, with a purity fit value exceeding 70 % from the self-built library. The analytical method developed in this study demonstrates high accuracy and sensitivity. In addition, the MRM-IDA-EPI mode can effectively identifies the target BPs and prevents false positive detection of analytes in the urine.

Plasma fatty acid esters of hydroxy fatty acids and surrogate fatty acid esters of hydroxy fatty acids hydrolysis activity in children with or without obesity and in adults with or without coronary artery disease.

Fatty acid esters of hydroxy fatty acids (FAHFA) are a class of bioactive lipids with anti-inflammatory, antidiabetic and cardioprotective properties. FAHFA hydrolysis into its fatty acid (FA) and hydroxy fatty acid (HFA) constituents can affect the bioavailability of FAHFA and its subsequent biological effects. We aimed to investigate FAHFA levels and FAHFA hydrolysis activity in children with or without obesity, and in adults with or without coronary artery disease (CAD). Our study cohort included 20 children without obesity, 40 children with obesity, 10 adults without CAD and 28 adults with CAD. We quantitated plasma levels of four families of FAHFA [palmitic acid hydroxy stearic acid (PAHSA), palmitoleic acid hydroxy stearic acid (POHSA), oleic acid hydroxy stearic acid (OAHSA), stearic acid hydroxy stearic acid] and their corresponding FA and HFA constituents using liquid chromatography-tandem mass spectrometry analysis. Surrogate FAHFA hydrolysis activity was estimated as the FA/FAHFA or HFA/FAHFA ratio. Children with obesity had lower plasma PAHSA (p = .001), OAHSA (p = .006) and total FAHFA (p = .011) levels, and higher surrogate FAHFA hydrolysis activity represented by PA/PAHSA (p = .040) and HSA/OAHSA (p = .025) compared with children without obesity. Adults with CAD and a history of myocardial infarction (MI) had lower POHSA levels (p = .026) and higher PA/PAHSA (p = .041), POA/POHSA (p = .003) and HSA/POHSA (p = .038) compared with those without MI. Altered FAHFA metabolism is associated with obesity and MI, and inhibition of FAHFA hydrolysis should be studied further as a possible therapeutic strategy in obesity and MI.

Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

Amino acid metabolomics and machine learning for assessment of post-hepatectomy liver regeneration

ObjectiveAmino acid (AA) metabolism plays a vital role in liver regeneration. However, its measuring utility for post-hepatectomy liver regeneration under different conditions remains unclear. We aimed to combine machine learning (ML) models with AA metabolomics to assess liver regeneration in health and non-alcoholic steatohepatitis (NASH).MethodsThe liver index (liver weight/body weight) was calculated following 70% hepatectomy in healthy and NASH mice. The serum levels of 39 amino acids were measured using ultra-high performance liquid chromatography–tandem mass spectrometry analysis. We used orthogonal partial least squares discriminant analysis to determine differential AAs and disturbed metabolic pathways during liver regeneration. The SHapley Additive exPlanations algorithm was performed to identify potential AA signatures, and five ML models including least absolute shrinkage and selection operator, random forest, K-nearest neighbor (KNN), support vector regression, and extreme gradient boosting were utilized to assess the liver index.ResultsEleven and twenty-two differential AAs were identified in the healthy and NASH groups, respectively. Among these metabolites, arginine and proline metabolism were commonly disturbed metabolic pathways related to liver regeneration in both groups. Five AA signatures were identified, including hydroxylysine, L-serine, 3-methylhistidine, L-tyrosine, and homocitrulline in healthy group, and L-arginine, 2-aminobutyric acid, sarcosine, beta-alanine, and L-cysteine in NASH group. The KNN model demonstrated the best evaluation performance with mean absolute error, root mean square error, and coefficient of determination values of 0.0037, 0.0047, 0.79 and 0.0028, 0.0034, 0.71 for the healthy and NASH groups, respectively.ConclusionThe KNN model based on five AA signatures performed best, which suggests that it may be a valuable tool for assessing post-hepatectomy liver regeneration in health and NASH.

Screening of substance use in pregnancy: A Danish cross-sectional study.

There is a paucity of objectively verified data on substance use among Danish pregnant women. We estimated the prevalence of substance use including alcohol and nicotine among the general population of Danish pregnant women. In this anonymous, national, cross-sectional, descriptive study, pregnant women were invited when attending an ultrasound scan between November 2019 and December 2020 at nine Danish hospitals. Women submitted a urine sample and filled out a questionnaire. Urine samples were screened on-site with a qualitative urine dipstick for 15 substances including alcohol, nicotine, opioids, amphetamines, cannabis, and benzodiazepines. All screen-positive urine samples underwent secondary quantitative analyses with gold standard, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results were compared to questionnaire information to analyze the validity of self-reporting and to examine possible cross-reactions. A total of 1903 of 2154 invited pregnant women participated (88.3%). The prevalence of dipstick-positive urine samples was 25.0%. 44.0% of these were confirmed positive, resulting in a total confirmed prevalence of 10.8%. The prevalence of nicotine use was 10.1%-and for all other substances, <0.5%. Nicotine use was more prevalent among younger pregnant women, while other substance use appeared evenly distributed over age groups. Self-reporting of use of nicotine products was high (71.1%), but low for cannabis and alcohol intake (0% and 33.3%, respectively). Prescription medication explained almost all cases of oxycodone, methylphenidate, and benzodiazepine use. Substance use among pregnant women consisted mainly of nicotine. Dipstick screening involved risks of false negatives and false positives. Except for alcohol intake and cannabis use, dipstick analyses did not seem to provide further information than self-reporting. LC-MS/MS analyses remain gold standard, and future role of dipstick screenings should be discussed.

Tanreqing injection demonstrates anti-dengue activity through the regulation of the NF-κB-ICAM-1/VCAM-1 axis

BackgroundTanreqing injection (TRQ) has been employed in clinical practice as a treatment for dengue fever (DF). Nevertheless, the precise pharmacological mechanism underlying its efficacy remains elusive. MethodNetwork pharmacology, molecular docking, transcriptome sequencing, and experimental evaluation were employed to analyze and study the inhibitory potential of TRQ against dengue virus (DENV). ResultWe found that TRQ inhibited the replication of DENV in human umbilical vein endothelial cells, Huh-7 cells, and Hep3B cells. In addition, TRQ prolonged the survival duration of AG129 mice infected with DF, decreased the viral load in serum and organs, and alleviated organ damage. Subsequently, ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of TRQ was performed to identify 314 targets associated with 36 active compounds present in TRQ. Integration of multiple databases yielded 47 DF-related genes. Then, 15 hub targets of TRQ in DF were determined by calculating the network topology parameters (Degree). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these pathways were primarily enriched in the processes of cytokine activation and leukocyte cross-endothelial migration, with significant enrichment of cell adhesion molecules. Molecular docking revealed favorable binding affinity between TRQ's key active compounds and the predicted hub targets. Transcriptome sequencing results showed TRQ's ability to restore the expression of vascular cell adhesion molecule-1 (VCAM-1) post-DENV infection. Finally, TRQ was found to modulate the immune status by regulating the nuclear factor kappa-B (NF-κB)- intercellular cell adhesion molecule-1 (ICAM-1)/VCAM-1 axis, as well as reduce immune cell alterations, inflammatory factor secretion, vascular permeability, and bleeding tendencies induced by DENV infection. ConclusionOur research suggests that TRQ exerts therapeutic effects on DF by regulating the NF-κB-ICAM-1/VCAM-1 axis.

O02 Ubiquitomics of CYLD-deficient skin tumours reveals dysregulation of hair follicle keratinocyte processes

Abstract Introduction and aims CYLD cutaneous syndrome (CCS) is an inherited hair follicle tumour syndrome where biallelic loss of function of the deubiquitinase enzyme CYLD gives rise to cylindroma skin tumours. The role of CYLD in skin biology is not well characterized but it is known to regulate several cell signalling pathways through deubiquitination of effector proteins. We aimed to determine how loss of CYLD function can lead to CCS skin tumour formation by studying the ubiquitome of CCS tumours and healthy skin. Methods Healthy skin (n = 5) and CCS tumour tissue (n = 5) were collected from flash frozen samples. Lysed homogenates were trypsin-digested and the resulting peptides underwent diglycine immunopurification for subsequent liquid chromatography–tandem mass spectrometry analysis on a Bruker Tims-TOF HT (Billerica, MA, USA) mass spectrometer. We utilized a data-independent acquisition method adapted for diglycine-containing peptides to generate mass spectrometry data. Peptide identification was performed by searching mass spectrometry data against an in silico neural network derived spectral library with diglycine modification. Results We identified 1273 ubiquitination sites across 649 proteins in CCS tumour and control samples. After performing t-tests (P &amp;lt; 0.05), we found 305 ubiquitination sites that were either unique to their respective condition or were differentially regulated. Analysis of ubiquitinated peptide sequences uncovered cell surface receptors with increased ubiquitination in CCS tumour samples including the insulin receptor, which had ubiquitination modifications at its kinase domain, suggesting increased insulin signalling in CCS tumours. Gene set enrichment analysis of CCS tumour ubiquitinated substrates revealed increased ubiquitination of secretory granules and extracellular exosomes, suggesting that loss of CYLD may disrupt regulation of protein secretion processes. Conclusions Our findings describe the ubiquitome of CCS keratinocytes, providing insights into how loss of CYLD function can affect biological processes in skin.

Efficacy and safety of sympathetic mapping and ablation of renal nerves for the treatment of hypertension (SMART): 6-month follow-up of a randomised, controlled trial

Previous trials of renal denervation (RDN) have been designed to investigate reduction of blood pressure (BP) as the primary efficacy endpoint using non-selective RDN without intraoperatively verified RDN success. It is an unmet clinical need to map renal nerves, selectively denervate renal sympathetic nerves, provide readouts for the interventionalists and avoid futile RDN. We aimed to examine the safety and efficacy of renal nerve mapping/selective renal denervation (msRDN) in patients with uncontrolled hypertension (HTN) and determine whether antihypertensive drug burden is reduced while office systolic BP (OSBP) is controlled to target level (＜140mmHg). We conducted a randomized, prospective, multicenter, single-blinded, sham-controlled trial. The study combined two efficacy endpoints at 6 months as primary outcomes: The control rate of patients with OSBP ＜140mmHg (non-inferior outcome) and change in the composite index of antihypertensive drugs (Drug Index) in the treatment versus Sham group (superior outcome). This design avoids confounding from excess drug-taking in the Sham group. Antihypertensive drug burden was assessed by a composite index constructed as: Class N (number of classes of antihypertensive drugs)×(sum of doses). 15 hospitals in China participated in the study and 220 patients were enrolled in a 1:1 ratio (msRDN vs Sham). The key inclusion criteria included: age (18-65 years old), history of essential HTN (at least 6 months), heart rate (≥70 bpm), OSBP (≥150mmHg and ≤180mmHg), ambulatory BP monitoring (ABPM, 24-h SBP ≥130mmHg or daytime SBP ≥135mmHg or nighttime SBP ≥120mmHg), renal artery stenosis (＜50%) and renal function (eGFR ＞45mL/min/1.73m2). The catheter with both stimulation and ablation functions was inserted in the distal renal main artery. The RDN site (hot spot) was selected if SBP increased (≥5mmHg) by intra-renal artery (RA) electrical stimulation; an adequate RDN was confirmed by repeated electronic stimulation if no increase in BP otherwise, a 2nd ablation was performed at the same site. At sites where there was decreased SBP (≥5mmHg, cold spot) or no BP response (neutral spot) to stimulation, no ablation was performed. The mapping, ablation and confirmation procedure was repeated until the entire renal main artery had been tested then either treated or avoided. AftermsRDN, patients had to follow a predefined, vigorous drug titration regimen in order to achieve target OSBP (＜140mmHg). Drug adherence was monitored by liquid chromatography-tandem mass spectrometry analysis using urine. This study is registered with ClinicalTrials.gov (NCT02761811) and 5-year follow-up is ongoing. Between July 8, 2016 and February 23, 2022, 611 patients were consented, 220 patients were enrolled in the study who received standardized antihypertensive drug treatments (at least two drugs) for at least 28 days, presented OSBP ≥150mmHg and ≤180mmHg and metall inclusion and exclusion criteria. In left RA and right RA, mapped sites were 8.2 (3.0) and 8.0 (2.7), hot/ablated sites were 3.7 (1.4) and 4.0 (1.6), cold spots were 2.4 (2.6) and 2.0 (2.2), neutral spots were 2.0 (2.1) and 2.0 (2.1), respectively. Hot, cold and neutral spots was 48.0%, 27.5% and 24.4% of total mapped sites, respectively. At 6M, the Control Rate of OSBP was comparable between msRDN and Sham group (95.4% vs 92.8%, p=0.429), achieved non-inferiority margin-10% (2.69%; 95% CI-4.11%, 9.83%, p ＜ 0.001 for non-inferiority); the change in Drug Index was significantly lower in msRDN group compared to Sham group (4.37 (6.65) vs 7.61 (10.31), p=0.010) and superior to Sham group (-3.25; 95% CI-5.56,-0.94, p=0.003), indicating msRDN patients need significantly fewer drugs to control OSBP ＜140mmHg. 24-hour ambulatory SBP decreased from 146.8 (13.9) mmHg by 10.8 (14.1) mmHg, and from 149.8 (12.8) mmHg by 10.0 (14.0) mmHg in msRDN and Sham groups, respectively (p<0.001 from Baseline; p>0.05 between groups). Safety profiles were comparable between msRDN and Sham groups, demonstrating the safety and efficacy of renal mapping/selective RDN to treat uncontrolled HTN. The msRDN therapy achieved the goals of reducing the drug burden of HTN patients and controlling OSBP <140mmHg, with only approximately four targeted ablations per renal main artery, much lower than in previous trials. SyMap Medical (Suzhou), LTD, Suzhou, China.

NP3 MS Workflow: An Open-Source Software System to Empower Natural Product-Based Drug Discovery Using Untargeted Metabolomics.

Natural products (or specialized metabolites) are historically the main source of new drugs. However, the current drug discovery pipelines require miniaturization and speeds that are incompatible with traditional natural product research methods, especially in the early stages of the research. This article introduces the NP3 MS Workflow, a robust open-source software system for liquid chromatography-tandem mass spectrometry (LC-MS/MS) untargeted metabolomic data processing and analysis, designed to rank bioactive natural products directly from complex mixtures of compounds, such as bioactive biota samples. NP3 MS Workflow allows minimal user intervention as well as customization of each step of LC-MS/MS data processing, with diagnostic statistics to allow interpretation and optimization of LC-MS/MS data processing by the user. NP3 MS Workflow adds improved computing of the MS2 spectra in an LC-MS/MS data set and provides tools for automatic [M + H]+ ion deconvolution using fragmentation rules; chemical structural annotation against MS2 databases; and relative quantification of the precursor ions for bioactivity correlation scoring. The software will be presented with case studies and comparisons with equivalent tools currently available. NP3 MS Workflow shows a robust and useful approach to select bioactive natural products from complex mixtures, improving the set of tools available for untargeted metabolomics. It can be easily integrated into natural product-based drug-discovery pipelines and to other fields of research at the interface of chemistry and biology.

Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy.

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T627 rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T627. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation to inhibit cell growth.

Purification, characterization, and identification of a novel bacteriocin produced by Lacticaseibacillus casei KLS1, and its antimicrobial mechanism against Staphylococcus aureus

Staphylococcus aureus (S. aureus) and its biofilm were recognized as one of the major threats to food safety and human health. Bacteriocins produced by Lacticaseibacillus species have been demonstrated to inhibit the growth of foodborne pathogenic bacteria and their biofilm formation. Herein, a novel bacteriocin KLS1 from Lacticaseibacillus casei KLS1 was extracted by ethyl acetate and purified using an AKTA Pure 25 protein chromatography purification system. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that the molecular mass of bacteriocin KLSI was 3786.91 Da and the amino acid sequence was DTSASIASNKSETNDLLKQIEAANTEVINLNKQID. Bacteriocin KLS1 was stable at pH 2.0–6.0, sensitive to proteases, and showed excellent thermal stability. Furthermore, bacteriocin KLS1 exhibited broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, even fungi. The crystal violet staining assay indicated that bacteriocin KLS1 significantly reduced the relative percentage of S. aureus biofilm (P < 0.05). Meanwhile, bacteriocin KLS1 was found to significantly decrease the metabolic activity of S. aureus by Cell Counting Kit-8 assay (P < 0.05). In addition, scanning electron microscopy and confocal laser scanning microscope showed that bacteriocin KLS1 disrupted the structure of S. aureus biofilm and cell membrane integrity, leading to cell deformation and leakage of contents. These results suggested that bacteriocin KLS1 might have the potential to serve as a biological preservative in the food industry.

4D label-free proteomics analysis of oxygen-induced retinopathy with or without anti-VEGF treatment

Oxygen-induced retinopathy (OIR) animal model is widely used for retinopathy of prematurity (ROP) researches. The purpose of this study was to identify proteins and related pathways of OIR with or without anti-vascular endothelial growth factor (VEGF) treatment, for use as biomarkers in diagnosing and treating ROP. Nine samples were subjected to proteomic analysis. Retina specimens were collected from 3 OIR mice, 3 OIR mice with anti-VEGF treatment and 3 normal mice (control group). Liquid chromatography-tandem mass spectrometry analysis was performed using the 4D label-free technique. Statistically significant differentially expressed proteins, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representations, InterPro (IPR) and protein interactions were analyzed. In total, 4585 unique proteins were identified as differentially expressed proteins (DEPs). Enrichment analysis of the GO and KEGG indicated functional clusters related to peptide biosynthetic and metabolic process, cellular macromolecule biosynthetic process and nucleic acid binding in OIR group. For anti-VEGF treatment group, DEPs were clustered in DNA replication, PI3K/Akt signaling pathway and Jak/STAT signaling pathway. Proteomic profiling is useful for the exploration of molecular mechanisms of OIR and mechanisms of anti-VEGF treatment. These findings may be useful for identification of novel biomarkers for ROP pathogenesis and treatment.

Glycosylation of recombinant adeno-associated virus serotype 6

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycan that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAVs serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity-chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn-antigen) and mono- and di-sialylated Galβ1-3GalNAc (T-antigen) at S156, T162, T194, and T201 in viral protein (VP) 2, and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%–1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Lipid matrix-specific pretreatment method for enhancing the extractability and allergenicity maintenance of bovine milk allergens in ELISA detection

The presence of various components in the food matrix makes allergen detection difficult and inaccurate, and pretreatment is an innovative breakthrough point. Food matrices were categorised based on their composition. Subsequently, a pretreatment method was established using a combination of ultrasound-assisted n-hexane degreasing and weakly alkaline extraction systems to enhance the detection accuracy of bovine milk allergens. Results showed that more allergens were obtained with less structural destruction, as demonstrated using immunological quantification and spectral analysis. Concurrently, allergenicity preservation was confirmed through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, a KU812 cell degranulation model, and western blotting. The method exhibited good accuracy (bias, 8.47%), repeatability (RSDr, 1.52%), and stability (RSDR, 5.65%). In foods with high lipid content, such as chocolate, the allergen content was 2.29-fold higher than that of commercial kits. Laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM) analyses revealed a significant decrease in fat content after post-pretreatment using our method. In addition, colloidal stability surpassed that achieved using commercial kits, as indicated through the PSA and zeta potential results. The results demonstrated the superiority of the extractability and allergenicity maintenance of lipid matrix-specific pretreatment methods for improving the accuracy of ELISA based allergen detection in real food.

Chemoselectivity Strategy Based on B-Label Integrated with Tailored COF for Targeted Metabolomic Analysis of Short-Chain Fatty Acids by UHPLC-MS/MS.

Chemoselective extraction strategy is an emerging and powerful means for targeted metabolomics analysis, which allows for the selective identification of biomarkers. Short-chain fatty acids (SCFAs) as functional metabolites for many diseases pose challenges in qualitative and quantitative analyses due to their high polarity and uneven abundance. In our study, we proposed the B-labeled method for the derivatization of SCFAs using easily available 3-aminobenzeneboronic acid as the derivatization reagent, which enables the introduction of recognition unit (boric acid groups). To analyze the B-labeled targeted metabolites accurately, cis-diol-based covalent organic framework (COF) was designed to specifically capture and release target compounds by pH-response borate affinity principle. The COF synthesized by the one-step Schiff base reaction possessed a large surface area (215.77 m2/g), excellent adsorption capacity (774.9 μmol/g), good selectivity, and strong regeneration ability (20 times). Combined with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, our results indicated that the detection sensitivities of SCFAs increased by 1.2-2500 folds compared with unlabeled method, and the retention time and isomer separation were improved. Using this strategy, we determined twenty-six SCFAs in the serum and urine of rats in four groups about osteoporosis and identified important biomarkers related to the tricarboxylic acid cycle and fatty acid metabolism pathways. In summary, UHPLC-MS/MS based on B-labeled derivatization with tailored COF strategy shows its high selectivity, excellent sensitivity, and good chromatographic behavior and has remarkable application prospect in targeted metabolomics study of biospecimens.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary Oligosaccharides and Glycoamino Acids for the Diagnosis of Mucopolysaccharidosis and Glycoproteinosis.

Mucopolysaccharidosis (MPS) and glycoproteinosis are 2 groups of heterogenous lysosomal storage disorders (LSDs) caused by defective degradation of glycosaminoglycans (GAGs) and glycoproteins, respectively. Oligosaccharides and glycoamino acids have been recognized as biomarkers for MPS and glycoproteinosis. Given that both groups of LSDs have overlapping clinical features, a multiplexed assay capable of unambiguous subtyping is desired for accurate diagnosis, and potentially for severity stratification and treatment monitoring. Urinary oligosaccharides were derivatized with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) together with the underivatized glycoamino acids. Novel biomarkers were identified with a semi-targeted approach with precursor mass scanning, the fragmentation pattern (if applicable), and the biochemical basis of the condition. A UPLC-MS/MS analysis with improved chromatographic separation was developed. Novel biomarkers for MPS-IIIA, IIIB, IIIC, and VII were identified and validated. A total of 28 oligosaccharides, 2 glycoamino acids, and 2 ratios were selected as key diagnostic biomarkers. Validation studies including linearity, lower limit of quantitation (LLOQ), and precision were carried out with the assay performance meeting the required criteria. Age-specific reference ranges were collected. In the 76 untreated patients, unambiguous diagnosis was achieved with 100% sensitivity and specificity. Additionally, the levels of disease-specific biomarkers were substantially reduced in the treated patients. A multiplexed UPLC-MS/MS assay for urinary oligosaccharides and glycoamino acids measurement was developed and validated. The assay is suitable for the accurate diagnosis and subtyping of MPS and glycoproteinosis, and potentially for severity stratification and monitoring response to treatment.