Liquid Chromatography High-resolution Tandem Mass Research Articles

UHPLC-HRMS-based Multiomics to Explore the Potential Mechanisms and Biomarkers for Colorectal Cancer

BackgroundUnderstanding the metabolic changes in colorectal cancer (CRC) and exploring potential diagnostic biomarkers is crucial for elucidating its pathogenesis and reducing mortality. Cancer cells are typically derived from cancer tissues and can be easily obtained and cultured. Systematic studies on CRC cells at different stages are still lacking. Additionally, there is a need to validate our previous findings from human serum.MethodsUltrahigh-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics were employed to comprehensively measure metabolites and lipids in CRC cells at four different stages and serum samples from normal control (NR) and CRC subjects. Univariate and multivariate statistical analyses were applied to select the differential metabolites and lipids between groups. Biomarkers with good diagnostic efficacy for CRC that existed in both cells and serum were screened by the receiver operating characteristic curve (ROC) analysis. Furthermore, potential biomarkers were validated using metabolite standards.ResultsMetabolite and lipid profiles differed significantly among CRC cells at stages A, B, C, and D. Dysregulation of glycerophospholipid (GPL), fatty acid (FA), and amino acid (AA) metabolism played a crucial role in the CRC progression, particularly GPL metabolism dominated by phosphatidylcholine (PC). A total of 46 differential metabolites and 29 differential lipids common to the four stages of CRC cells were discovered. Eight metabolites showed the same trends in CRC cells and serum from CRC patients compared to the control groups. Among them, palmitoylcarnitine and sphingosine could serve as potential biomarkers with the values of area under the curve (AUC) more than 0.80 in the serum and cells. Their panel exhibited excellent performance in discriminating CRC cells at different stages from normal cells (AUC = 1.00).ConclusionsTo our knowledge, this is the first research to attempt to validate the results of metabolism studies of serum from CRC patients using cell models. The metabolic disorders of PC, FA, and AA were closely related to the tumorigenesis of CRC, with PC being the more critical factor. The panel composed of palmitoylcarnitine and sphingosine may act as a potential biomarker for the diagnosis of CRC, aiding in its prevention.

In Vivo and In Vitro Metabolic Fate and Urinary Detectability of Five Deschloroketamine Derivatives Studied by Means of Hyphenated Mass Spectrometry.

Ketamine derivatives such as deschloroketamine and deschloro-N-ethyl-ketamine show dissociative and psychoactive properties and their abuse as new psychoactive substances (NPSs) has been reported. Though some information is available on the biotransformation of dissociative NPSs, data on deschloro-N-cyclopropyl-ketamine deschloro-N-isopropyl-ketamine and deschloro-N-propyl-ketamine concerning their biotransformation and, thus, urinary detectability are not available. The aims of the presented work were to study the in vivo phase I and II metabolism; in vitro phase I metabolism, using pooled human liver microsomes (pHLMs); and detectability, within a standard urine screening approach (SUSA), of five deschloroketamine derivatives. Metabolism studies were conducted by collecting urine samples from male Wistar rats over a period of 24 h after their administration at 2 mg/kg body weight. The samples were analyzed using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography-mass spectrometry (GC-MS). The compounds were mainly metabolized by N-dealkylation, hydroxylation, multiple oxidations, and combinations of these metabolic reactions, as well as glucuronidation and N-acetylation. In total, 29 phase I and 10 phase II metabolites were detected. For the LC-HRMS/MS SUSA, compound-specific metabolites were identified, and suitable screening targets could be recommended and confirmed in pHLMs for all derivatives except for deschloro-N-cyclopropyl-ketamine. Using the GC-MS-based SUSA approach, only non-specific acetylated N-dealkylation metabolites could be detected.

Plasma lipidome differences in patients with and without significant carotid plaque

BackgroundAtherosclerosis is a major cause of ischemic stroke, and early detection of advanced atherosclerosis in the carotid artery is important for reducing morbidity and mortality. What is even more important is not only detection of atherosclerosis but early determination whether the patients are at high risk of an event with adverse effects as the size of the plaque does not necessarily reflect its potential to trigger such events. AimWe studied whether plasma lipidomics profile can be used as a diagnostic tool for stratification of stable or unstable plaques without the need of removing the carotid plaque. MethodsThis study used liquid chromatography high-resolution tandem mass spectrometry lipidomics to characterize lipid profiles in patients' plasma and found that patients with significant and complicated (vulnerable) atherosclerotic plaque had distinct lipid profiles compared to those with insignificant plaques. ResultsThe lipid classes that were most predictive of vulnerable plaque were lysophosphoethanolamines, fatty acyl esters of hydroxy fatty acids, free fatty acids, plasmalogens, and triacylglycerols. Most of these compounds were found decreased in plasma of patients with unstable plaques which enabled sufficient performance of a statistical model used for patient stratification. ConclusionsPlasma lipidomes measured by liquid chromatography-mass spectrometry show differences in patients with stable and unstable carotid plaques, therefore these compounds could potentially be used as biomarkers for unstable plaque in future clinical diagnosis.

Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Integrative leaf anatomy structure, physiology, and metabolome analyses revealed the response to drought stress in sainfoin at the seedling stage.

Sainfoin (Onobrychis viciaefolia) is a vital legume forage, and drought is the primary element impeding sainfoin growth. The anatomical structure, physiological indexes, and metabolites of the leaves of sainfoin seedlings with a drought-resistant line of P1 (DRL) and a drought-sensitive material of 2049 (DSM) were analyzed under drought (-1.0 MPa) with polyethylene glycol-6000 (PEG-6000). The leaf anatomy was studied by the paraffin section method. The related physiological indexes were measured by the hydroxylamine oxidation method, titanium sulfate colorimetric method, thiobarbituric acid method, acidic ninhydrin colorimetric method, and Coomassie brilliant blue method. The metabolomics analysis was composed of liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS). The results revealed that the thickness of the epidermis, palisade tissue, and sponge tissue of DRL were significantly greater than those of DSM. The leaves of DRL exhibited lower levels of superoxide anion (O2 •-) production rate, hydrogen peroxide (H2O2) content, and malondialdehyde (MDA) content compared with DSM, while proline (Pro) content and soluble protein (SP) content were significantly higher than those of DSM. A total of 391 differential metabolites were identified in two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that the primary differential metabolites were concentrated into the tyrosine metabolism; isoquinoline alkaloid biosynthesis; ubiquinone and other terpenoid quinone biosynthesis; neomycin, kanamycin, and gentamicin biosynthesis; and anthocyanin biosynthesis metabolic pathways. Compared with DSM, DRL had more complete anatomical structure, lower active oxygen content, and higher antioxidant level. The results improved our insights into the drought-resistant mechanisms in sainfoin.

Abstract 1839: Proteomic and glycoproteomic differences associated with prostate cancer in urine

Abstract Background and Aims: Prostate cancer is one of the most common cancers affecting men in the United States, with ~288,300 new cases and 34,700 deaths in 2023 alone. Prostate-specific antigen (PSA), the most widely used prostate cancer biomarker, lacks specificity and sensitivity, leading to unnecessary biopsies, overdiagnosis, and overtreatment. There is an urgent unmet clinical need to find new biomarkers that can distinguish patients with benign disease from those with prostate cancer. Urine is an attractive source for these biomarkers as prostate-derived proteins are more highly concentrated in urine than in blood. To better understand both global proteomic and glycoproteomic molecular signatures that could lead to improved diagnostics for prostate cancer, we analyzed urine samples from unaffected men, men with benign prostatic hyperplasia (BPH), and men with prostate cancer. Methods: Urine samples (N = 58 total) were collected from unaffected men (N = 17), men with BPH (N = 24), and men with prostate cancer (N = 17). Samples were digested with trypsin and analyzed with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for shotgun proteomics analysis. Association with cancer was determined through supervised analysis using Pearson correlation. For glycoproteomics analysis, intact glycopeptides were enriched using strong anion exchange-electrostatic repulsion hydrophilic interaction chromatography, fractionated using high pH reversed-phase chromatography, and analyzed with LC-MS/MS. Association with the three conditions was determined through several rounds of supervised analysis using Pearson correlation. Results: In total, 3542 human proteins were identified, and 78 proteins (p < 0.01) were significantly associated with prostate cancer. The glycolysis pathway and lysosome pathway were enriched in samples from men with prostate cancer. In the glycoproteomics analysis, 36 proteins (p < 0.05) had altered glycan diversity in one of the three conditions, and the complement and coagulation cascade pathway and the lysosome pathway were highly enriched. Six proteins (p < 0.05) had significantly increased glycan diversity in the cancerous samples than in the BPH and normal samples. Conclusions: This study identifies a set of interesting proteomic and glycoproteomic differences in urine from men with prostate cancer compared to BPH and non-cancer samples, which warrant further investigation in additional sample cohorts as potential novel biomarkers. Citation Format: Nikhiya Shamsher, Fernando Garcia-Marques, Abel Bermudez, Mark R. Flory, Sharon J. Pitteri. Proteomic and glycoproteomic differences associated with prostate cancer in urine [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 1839.

Perturbations in human bile acid profiles following drug-induced liver injury investigated using semitargeted high-resolution mass spectrometry.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF) in North America. To investigate the effect of drug-induced liver injury (DILI) on circulating bile acid (BA) profiles, serum from ALF patients and healthy controls were analyzed using a semitargeted high-resolution mass spectrometry approach to measure BAs in their unconjugated and amidated forms and their glucuronide and sulfate conjugates. Human serum samples from 20 healthy volunteers and 34 ALF patients were combined with deuterated BAs and extracted, prior to liquid chromatography high-resolution tandem mass spectrometry analysis. A mix of 46 standards helped assign 26 BAs in human serum by accurate mass and retention time matching. Moreover, other isomers of unconjugated and amidated BAs, as well as glucuronide and sulfate conjugates, were assigned by accurate mass filtering. In vitro incubations of standard BAs provided increased information for certain peaks of interest. A total of 275 BA metabolites, with confirmed or putative assignments, were measured in human serum samples. APAP overdose significantly influenced the levels of most BAs, promoting glycine conjugation, and, to a lesser extent, taurine conjugation. When patient outcome was considered, 11 BAs were altered significantly, including multiple sulfated species. Although many of the BAs measured did not have exact structures assigned, several putatively identified BAs of interest were further characterized using in vitro incubations. An optimized chromatographic separation tailored to BAs of ranging polarities was combined with accurate mass measurements to investigate the effect that DILI has on their complex profiles and metabolism to a much wider extent than previously possible. The analysis of complex BA profiles enabled in-depth analysis of the BA metabolism perturbations in ALF, including certain metabolites related to patient outcomes.

LC-HRMS methods for the quantification of two artemisinin drugs and their metabolites in rat blood and plasma.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2nm) and its metabolite D-ART (18.8-751.9nm), and the linear range was 40.0-4,000.0nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

In vitro, invivo metabolism and quantification of the novel synthetic opioid N-piperidinyl etonitazene (etonitazepipne).

N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine. Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using invitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced invitro and invivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof. Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.

Predicting Retention Time in Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography High-Resolution Tandem Mass Spectrometry (Unified-HILIC/AEX/HRMS/MS) for Comprehensive Structural Annotation of Polar Metabolome.

The accuracy of the structural annotation of unidentified peaks obtained in metabolomic analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be enhanced using retention time (RT) information as well as precursor and product ions. Unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) has been recently developed as an innovative method ideal for nontargeted polar metabolomics. However, the RT prediction for unified-HILIC/AEX has not been developed because of the complex separation mechanism characterized by the continuous transition of the separation modes from HILIC to AEX. In this study, we propose an RT prediction model of unified-HILIC/AEX/HRMS/MS, which enables the comprehensive structural annotation of polar metabolites. With training data for 203 polar metabolites, we ranked the feature importance using a random forest among 12,420 molecular descriptors (MDs) and constructed an RT prediction model with 26 selected MDs. The accuracy of the RT model was evaluated using test data for 51 polar metabolites, and 86.3% of the ΔRTs (difference between measured and predicted RTs) were within ±1.50 min, with a mean absolute error of 0.80 min, indicating high RT prediction accuracy. Nontargeted metabolomic data from the NIST SRM 1950-Metabolites in frozen human plasma were analyzed using the developed RT model and in silico MS/MS prediction, resulting in a successful structural estimation of 216 polar metabolites, in addition to the 62 identified based on standards. The proposed model can help accelerate the structural annotation of unknown hydrophilic metabolites, which is a key issue in metabolomic research.

Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

Study on the inhibitory effect of fermentation extract of Microporus vernicipes on Candida albicans.

Candida albicans is one of the most common species of Candida, which cause various mucosal and systemic infectious diseases. However, the resistance rate to existing clinical antifungal drugs gradually increases in C. albicans. Therefore, new antifungal drugs must be developed to solve the current problem. This study discovered that the solid fermented ethyl acetate crude extract of Microporus vernicipes had inhibitory activity on C. albicans. This study determined that the Mv5 components had significantly inhibited the activity of C. albicansusing column chromatography separation component screening. The components included 23 compounds of fatty acids and their derivatives, alkaloids, phenols, and other classes using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HR-MS) analysis, with fatty acids constituting the primary components. The mechanism of action showed that the minimum inhibitory concentration (MIC) of Mv5 components against C. albicans was 15.63μg/mL, while minimum fungicidal concentration (MFC) was 31.25μg/mL. Mv5 components can inhibit the early biofilm formation and destroy the mature biofilm structure. It can inhibit the germ tube growth of C. albicans, thereby inhibiting the transformation of yeast morphology to hyphae. We detected 193 differentially expressed genes, including 156 upregulated and 37 downregulated genes in the Mv5 components of the MIC concentration group. We detected 391 differentially expressed genes, including 334 upregulated and 57 downregulated expression genes in the MFC concentration group. Among these differentially expressed genes, the genes related to mycelium and biofilm formation were significantly downregulated. GO enrichment analysis presented that single-organism process metabolic process, and cellular processes were the biological processes with the most gene enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG)of Mv5 components were mainly enriched in metabolic pathways, such as meiosis yeast and amino acid metabolism. Therefore, it is believed that the fermentation extract of M. vernicipes inhibits C. albicans, which can provide clues for developing effective antifungal drugs.

Untargeted metabolomics to expand the chemical space of the marine diatom Skeletonema marinoi.

Diatoms (Bacillariophyceae) are aquatic photosynthetic microalgae with an ecological role as primary producers in the aquatic food web. They account substantially for global carbon, nitrogen, and silicon cycling. Elucidating the chemical space of diatoms is crucial to understanding their physiology and ecology. To expand the known chemical space of a cosmopolitan marine diatom, Skeletonema marinoi, we performed High-Resolution Liquid Chromatography-Tandem Mass Spectrometry (LC-MS2) for untargeted metabolomics data acquisition. The spectral data from LC-MS2 was used as input for the Metabolome Annotation Workflow (MAW) to obtain putative annotations for all measured features. A suspect list of metabolites previously identified in the Skeletonema spp. was generated to verify the results. These known metabolites were then added to the putative candidate list from LC-MS2 data to represent an expanded catalog of 1970 metabolites estimated to be produced by S. marinoi. The most prevalent chemical superclasses, based on the ChemONT ontology in this expanded dataset, were organic acids and derivatives, organoheterocyclic compounds, lipids and lipid-like molecules, and organic oxygen compounds. The metabolic profile from this study can aid the bioprospecting of marine microalgae for medicine, biofuel production, agriculture, and environmental conservation. The proposed analysis can be applicable for assessing the chemical space of other microalgae, which can also provide molecular insights into the interaction between marine organisms and their role in the functioning of ecosystems.

Sample Preparation for Suspect and Non-target Screening of Xenobiotics in Human Biofluids by Liquid Chromatography – High Resolution Tandem Mass Spectrometry

The concept of exposome covers all the exposures an individual suffers from conception to death, which can be partially assessed through the monitoring of human biofluids. In there, target analytical approaches tend to focus on a limited set of xenobiotics, whereas exposomic studies need broad scopes in search of a full understanding. Given the issue, suspect and non-target screening are feasible alternatives. However, adequate sample preparation procedures should minimize interferences without significantly reducing the number of xenobiotics. Within this context, the present article aims to describe comprehensive sample preparation procedures for suspect or non-target screening of organic xenobiotics in several human biofluids, all coupled to unified separation and detection conditions based on ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). The referred biofluids consist of human urine, breast milk, saliva and ovarian follicular fluid.•Analytical methods for untargeted analysis of a wide range of xenobiotics in human biofluids are fully described in order to ensure reproducibility.•The sample preparation procedures balance selectivity and sensitivity.•Unified analysis conditions allow simultaneous analysis of diverse biofluids.

Proteomics Landscape and Machine Learning Predictor of Response to Splenectomy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an auto-immune disease characterized by antibody-mediated platelet destruction. Splenectomy remains the effective curative treatment for patients with ITP who failure to respond to TPO-RAs and/or rituximab (Mageau et al, Am J Hematol. 2022). The effectiveness rate of splenectomy for ITP is reported to be roughly 80%, with a 5-year remission rate ranging from 50% to 75% (Chaturvedi et al, Blood. 2018). For subjects presenting with severely low platelet counts or active bleeding, splenectomy represents the primary approach for rapidly increasing platelet levels and potentially saving lives. To achieve personalized and precise treatment, the prediction efficacy on splenectomy remains an urgent problem. Here, we have characterized immune features of ITP patients with splenectomy, and developed a predictive model for assessing the response to splenectomy. Methods Bone marrow plasma samples before splenectomy were collected and analyzed by high-resolution liquid chromatography-tandem mass spectrometry. Weight protein co-expression network analysis (WPCNA) was applied to generate modules from all proteins and mapped them to clinical characteristics. We used least absolute shrinkage and selection operator (Lasso) regression, recursive feature elimination (RFE) for predictors selection. Next, multivariable logistic regression (LR), support vector machine (SVM), and decision tree algorithms were used to develop a predictive model. Results 60 patients with ITP undergoing splenectomy treatment were enrolled into our study, and divided into three groups according to the response [patients with CR (n=30), NR (n=7), and Relapse (n=23)]. A pathway enrichment analysis of WPCNA modules by ClueGO revealed that megakaryocyte differentiation, humoral immune response mediated by circulating immunoglobulin, Fc receptor signaling pathway, and cGMP-PKG signaling pathway were correspondingly enriched in MEtan, MEturquoise, MEblue, and MEpink modules, which were associated with treatments with TPO-RA, IVIG, rhTPO, and post-operation platelet level, respectively. We then performed KEGG pathway enrichment analyses on the different expression proteins (DEPs) to deciphering the immune landscape among each group. For the relapse group, DEPs were primarily enriched in ribosome, spliceosome, neutrophil extracellular trap formation, and necroptosis pathways, comparing with the CR group. For the NR group, most DEPs were enriched in natural killer cell mediated cytotoxicity, NF-kappa B signaling, Fc gamma R-mediated phagocytosis pathways, comparing with the CR group. The models were based on a multi-step predictor pipeline. Inside the pipeline, features were first filtered by lasso regression and RFE algorithm. We identified five proteomic features present in the bone marrow plasm that associated with relapsed to therapy, which included CD40, COL4A3, GALNT6, GSTM3, and UBE2A (Figure). Each ensemble consisted of three algorithms acting in parallel: LR, SVM and decision tree. The AUC values of LR, SVM, and decision tree were 0.962 (95%CI: 0.915-1), 0.957 (95%CI: 0.897-1), and 0.875 (95%CI: 0.782-0.967), respectively. The three algorithms were then tested for performance. The sensitivity, specificity, positive predictive value, and negative predictive value analysis revealed that LR model reached the best performance. Targeting CD40 represents a promising therapeutic approach for enhancing clinical outcomes in patients with ITP who have shown inadequate response following splenectomy. Conclusion Our analysis revealed that ITP patients with splenectomy showed distinct immune characteristics when achieving different response. Protein features extracted from bone marrow plasma can be selected as predictors of response to splenectomy.

Large-scale phosphoproteome analysis in wheat seedling leaves provides evidence for extensive phosphorylation of regulatory proteins during CWMV infection

BackgroundChinese wheat mosaic virus (CWMV) often causes severe damage to wheat (Triticum aestivum L.) growth and yield. It is well known that a successful infection in plants depends on a complex interaction between the host plant and the pathogen. Post-translational modification (PTM) of proteins is considered to be one of the main processes that decides the outcome of the plant-pathogen arms race during this interaction. Although numerous studies have investigated PTM in various organisms, there has been no large-scale phosphoproteomic analysis of virus-infected wheat plants. We therefore aimed to investigate the CWMV infection-induced phosphoproteomics changes in wheat by high-resolution liquid chromatography-tandem mass spectroscopy (LC–MS/MS) using affinity-enriched peptides followed by comprehensive bioinformatics analysis.ResultsThrough this study, a total of 4095 phosphorylation sites have been identified in 1968 proteins, and 11.6% of the phosphorylated proteins exhibited significant changes (PSPCs) in their phosphorylation levels upon CWMV infection. The result of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that most of the PSPCs were associated with photosynthesis, plant-pathogen interactions, and MAPK signaling pathways. The protein–protein interaction (PPI) network analysis result showed that these PSPCs were mainly participated in the regulation of biosynthesis and metabolism, protein kinase activities, and transcription factors. Furthermore, the phosphorylation levels of TaChi1 and TaP5CS, two plant immunity-related enzymes, were significantly changed upon CWMV infection, resulting in a significant decrease in CWMV accumulation in the infected plants.ConclusionsOur results indicate that phosphorylation modification of protein plays a critical role in wheat resistance to CWMV infection. Upon CWMV infection, wheat plants will regulate the levels of extra- and intra-cellular signals and modifications of enzyme activities via protein phosphorylation. This novel information about the strategies used by wheat to resist CWMV infection will help researchers to breed new CWMV-resistant cultivars and to better understand the arms race between wheat and CWMV.

Evaluating membrane bioreactor treatment for the elimination of emerging contaminants using different analytical methods

Since wastewater treatment plants (WWTPs) were not originally designed to eliminate contaminants of emerging concern (CECs), alternative strategies like membrane bioreactor (MBR) technology are gaining importance in achieving effective CEC removal and minimising their environmental impact. In this study, composite wastewater samples were collected from the biggest WWTP in the Basque Country (Galindo, Biscay) and the performance of two secondary treatments (i.e. conventional activated sludge treatment, CAS, and MBR) was assessed. The combination of a suspect screening approach using liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) and multitarget analysis by gas chromatography-mass spectrometry (GC-MS) allowed the detection of approximately 200 compounds in the WWTP effluents. The estimated removal efficiencies (REs) revealed that only 16 micropollutants exhibited enhanced removal by MBR treatment (RE > 70% or 40 – 60%). The environmental risk posed by the non-eliminated compounds after both treatments remained similar, being anthracene, clarithromycin, bis(2-ethylhexyl) phthalate (DEHP) and dilantin the most concerning pollutants (RQ > 1). The Microtox® bioassay confirmed the MBR’s efficiency in removing baseline toxicity, while suggesting a similar performance of CAS treatment. These minimal differences between treatments call into question the worthiness of MBR treatment and emphasise the need to seek more efficient alternative treatment methods.