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Untargeted metabolomics reveals differential metabolic pathways and biomarkers in the acute phase of Kawasaki disease.

INTRODUCTION: Kawasaki disease (KD) is one of the most common rheumatic diseases in children and manifests with multisystem clinical features. Using untargeted metabolomics, our study investigated alterations in small-molecule metabolites in plasma of children with acute KD. Our study aimed to identify differential metabolic pathways and potential biomarkers. METHODS: Plasma samples were collected from 30 children diagnosed with KD and 30 age-matched healthy controls (HC) at Jinhua Maternal and Child Health Hospital between January 2025 and December 2025. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to analyse plasma samples. Enriched pathways were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and differential metabolic pathways were determined using MetaboAnalyst 5.0. Differential metabolites were screened using the nonparametric Mann-Whitney U-test and receiver operating characteristic curve area (AUC). The conservative average AUC from nested cross-validation was reported as the primary performance metric. Pearson correlation analysis was conducted to evaluate correlations between metabolites and clinical parameters. RESULTS: In total, 261 differential metabolites were identified between the KD and HC groups, including 87 lipids and lipid-like molecules, 69 organic heterocyclic compounds, 38 benzenoids, 34 organic acids, 14 phenylpropanoids, and 19 other compounds. Pathway analysis of these differential metabolites revealed 30 putatively enriched metabolic pathways for exploratory analysis. Of these pathways, primary bile acid biosynthesis, arginine biosynthesis, histidine metabolism, and phenylalanine-tyrosine-tryptophan biosynthesis were nominally associated with KD. Six metabolites with exploratory discriminatory performance (AUC > 0.8) were further identified: L-tyrosine, L-tryptophan, glutamine, histidine, histamine, and taurocholic acid. A combined model incorporating these metabolites achieved an apparent AUC of 0.984 in the full dataset; nested cross-validation yielded a more conservative AUC of 0.889 (95% CI 0.798-0.968), indicating promising exploratory discriminatory performance. CONCLUSION: Untargeted metabolomics enables identification of metabolically perturbed pathways during the acute phase of KD. L-tyrosine, L-tryptophan, glutamine, histidine, histamine, and taurocholic acid may serve as candidate biomarkers for acute phase of KD.

Kawasaki disease↗

Streptomyces violaceusniger WZS5-6 suppresses Fusarium oxysporum f. sp. cubense tropical race 4 via antifungal metabolites and host defense induction.

INTRODUCTION: Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), poses a serious threat to the safety and sustainable development of the banana industry. Biological control represents one of the most environmentally friendly approaches for managing this disease. METHODS: In this study, Streptomyces violaceusniger WZS5-6 antifungal activity against Foc TR4 has been investigated through an integrated approach combining antifungal assays, genome analysis, and metabolomic profiling. For the purpose, the effects of the bacterial strain and its cell-free extract on morphological and ultrastructural changes on pathogenic fungal hyphae and spores were assessed using scanning and transmission electron microscopy. LC-MS analysis was used to identify the metabolites responsible for antifungal activity. We further explored the potential of S. violaceusniger WZS5-6 against Foc TR4 through in planta validation. RESULTS: Streptomyces violaceusniger WZS5-6 exhibited a strong inhibition rate of 91.57% on Foc TR4. The cell-free extract obtained from S. violaceusniger WZS5-6 strongly inhibited Foc TR4 with an EC50 value of 91.62 µg·mL-1, indicating the presence of antifungal bioactive metabolites. The results showed that S. violaceusniger WZS5-6 significantly inhibited the mycelial growth of Foc TR4 and induced alterations in spore morphology, mycelial ultrastructure, and cell membrane leakage. Metabolomic profiling of the S. violaceusniger WZS5-6 extracts revealed numerous antifungal metabolites, among which the key metabolites, viz., citronellic acid and furanodienone, exhibited strong inhibitory effects on Foc TR4, with antifungal activity of 61.13% and 57.44%, respectively. Moreover, strain WZS5-6 not only demonstrated 61.54% control efficacy against FWB in a pot experiment but also showed promising growth-promoting effects on banana plants. DISCUSSION: This study demonstrates that S. violaceusniger WZS5-6 inhibits Foc TR4 through a multi-level mechanism involving cellular disruption, metabolic adaptation, and activation of host defense responses. These findings highlight the potential of S. violaceusniger WZS5-6 as a promising novel candidate strain to be employed as a biological control agent of FWB.

Fusarium wilt of banana↗

Isolation and Characterization of a Naturally Occurring Brevundimonas vesicularis Strain Exhibiting High Phytoene Accumulation.

Phytoene, a colorless precursor of carotenoids, has attracted increasing attention because of its favorable bioavailability, antioxidant activity, and potential applications in functional foods, nutraceuticals, and animal nutrition. However, its industrial utilization remains limited by low natural abundance and the dependence of current production strategies on genetic engineering or metabolic pathway manipulation. In this study, we identified and characterized a naturally occurring Brevundimonas vesicularis strain (Bv-xms2024) exhibiting pronounced phytoene accumulation without genetic modification. The strain was comprehensively characterized using morphological, biochemical, molecular, genomic, metabolomic, and transcriptional analyses. Quantitative LC-MS/MS analysis demonstrated that Bv-xms2024 accumulated phytoene to 420.42 ± 98.11 μg/g dry biomass after 96 h of cultivation, substantially exceeding the levels of downstream carotenoids, including β-carotene and astaxanthin. Optimization of cultivation parameters identified 25 °C, pH 7.0, and 96 h as the optimal conditions for phytoene accumulation, while serial passaging confirmed stable production over 20 generations. Genome annotation identified the carotenoid biosynthetic gene repertoire, while RT-qPCR analysis revealed a temporal shift from early upregulation of crtE and crtB to later upregulation of downstream pathway genes, consistent with the observed phytoene-dominant carotenoid profile. Short-term tolerance evaluations in mice and chickens revealed no observable adverse effects under the tested conditions. Collectively, these findings identify Bv-xms2024 as a promising natural microbial resource for phytoene production and provide a basis for further process development and strain-level safety evaluation.

Brevundimonas vesicularis↗

Integrated Optimization, Genomic Characterization, and Functional Evaluation of Biogenic Selenium Nanoparticles from Bacillus licheniformis BLN313: Antibacterial and Anticancer Potential.

Microbial synthesis of selenium nanoparticles (SeNPs) offers a sustainable alternative to chemical routes, but the genetic basis of selenium handling in Bacillus remains poorly defined, which limits rational strain selection. Here, SeNP production, physicochemical characterization, and closed-genome sequencing are combined for Bacillus licheniformis BLN313. Selenite reduction peaked at 500 µg/mL Na2SeO3 (88.8% conversion; 444 ± 27 µg/mL Se0); at higher concentrations, conversion efficiency and viability diverged, indicating that tolerance and reductive capacity are distinct traits. Purified SeNPs were spherical and partially crystalline trigonal Se0 (TEM 190 ± 52 nm; DLS 166 nm, PDI 0.03; zeta potential -20.8 mV), carrying a proteinaceous capping layer confirmed by XPS, EDS, and FTIR and shown by LC-MS to be enriched in cell wall-derived metabolites. The particles were bactericidal against Micrococcus luteus (MIC 62.5 µg/mL) and Klebsiella pneumoniae (MIC 250 µg/mL) and reduced MCF-7 viability (IC50 2.7 µg/mL) while sparing MCF-10A cells. The 4.11 Mb genome (46.3% GC; ANI 99.7%, dDDH 97.8%) encodes SulP and Pit transporters, multiple trxB copies, and sulfur-metabolism and oxidative-stress genes, defining a candidate gene set for selenium uptake, reduction and detoxification. BLN313 thus provides a genetically defined platform for SeNP production in biomedical and environmental applications.

Selenium↗

Relative Quantitative Analysis of Site-Specific N-Linked Glycosylation in Hyperglycosylated Interferon-β via Mass Spectrometry.

Glycosylation is a critical determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. R27T, an engineered variant of interferon-β1a, contains two N-glycosylation sites (Asn25 and Asn80), increasing its structural complexity and analytical requirements. In this study, we performed comprehensive total and site-specific glycan profiling of R27T using complementary analytical approaches. For total glycan analysis, the released N-glycans were fluorescently labeled with procainamide, providing enhanced sensitivity and broader glycan coverage compared with conventional 2-aminobenzamide labeling. Site-specific glycan profiling was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide mapping. Protease digestion conditions were optimized to improve recovery of site-specific glycopeptides, with chymotrypsin identified as the most effective enzyme for resolving glycopeptides from individual glycosylation sites. Total glycan distributions reconstructed from peptide-mapping data were compared with fluorescence-based glycan profiling, showing that total and site-specific glycan data can be effectively combined. Minor discrepancies were observed depending on glycan structure, mainly due to differences in ionization efficiency. Distinct glycan distributions were observed between the two N-glycosylation sites of R27T. Molecular modeling further suggested that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity. These results demonstrate an integrative strategy for accurate glycan characterization in multi-site glycoproteins relevant to biotherapeutic development.

Glycosylation↗

Multi-Omics and Integrative Analytics in Natural Products Discovery.

Natural products (NPs) have long been an essential source of new bioactive compounds for drug discovery; however, traditional methods for screening and isolating these compounds can be slow and often yield diminishing returns. Fortunately, advanced multi-omics and computational approaches present powerful solutions to these challenges. This review highlights innovative methodologies that integrate metabolomics, genomics, transcriptomics, and proteomics with bioinformatics and analytical chemistry to accelerate NP discovery. For instance, untargeted metabolomics platforms like high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Global Natural Products Social (GNPS) molecular networking allow for comprehensive profiling of new compounds, while targeted isotope-labeling strategies enhance this process. Additionally, genome and metagenome mining tools such as antibiotics and secondary metabolite analysis shell (antiSMASH), Deep Biosynthetic Gene Cluster (DeepBGC), and Pipeline for Reconstructing Integrated Syntheses of Metabolites (PRISM) quickly identify biosynthetic gene clusters (BGCs) in both cultured and uncultured organisms, often using heterologous expression to validate products. Transcriptomic analyses, including RNA sequencing (RNA-seq), co-expression networks, and fluxomics, help clarify how pathways are regulated, while quantitative proteomics techniques like tandem mass tags/isobaric tags for relative and absolute quantitation (TMT/iTRAQ) and label-free methods, along with chemoproteomics approaches such as cellular thermal shift assay and thermal proteome profiling (TPP), uncover molecular targets and their mechanisms of action. This review also places significant emphasis on the role of artificial intelligence (AI) and machine learning (ML) in integrating multi-omics data, spanning activities from constructing gene-metabolite correlation networks to leveraging knowledge graphs and graph neural networks for data fusion and functional prediction. Finally, this review concludes by discussing the synergistic benefits of multi-omics for natural-product discovery, addressing current technical challenges, and exploring future directions toward high-throughput, intelligent data integration for next-generation NP research.

Biological Products↗

Proteomic insights into azoospermia: protein differences in testicular tissue between non-obstructive and obstructive azoospermia patients.

Non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) are the main classifications of severe male infertility, but the molecular mechanism of NOA remains poorly understood. This study aimed to identify potential biomarkers and pathological mechanisms by comparing the proteomic differences in testicular tissues of NOA and OA patients. Through proteomic analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of testicular samples from 5 NOA patients and 5 OA patients, we identified 5264 proteins, among which 717 differentially expressed proteins (DEPs) were found between the two groups (242 upregulated and 475 downregulated in NOA). Bioinformatics analysis indicated that these DEPs were significantly associated with reproductive development, gametogenesis, and cell structural stability. On the basis of this, six candidate proteins, including dysferlin (DYSF), myoferlin (MYOF), mitsugumin 53 (MG53), cluster of differentiation 63 (CD63), caveolin-3 (CAV3), and calpain-3 (CAPN3), were selected from the DEPs and verified in an expanded sample set (37 NOA cases and 28 OA cases) through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, confirming their dysregulation in NOA. These findings provide new proteomic insights into NOA, highlighting the disruption of membrane repair and structural pathways, and offer potential biomarkers for understanding its pathogenesis.

Humans↗

A lipidomic study on the lens epithelial cells of patients with age related cataracts.

Age related cataracts (ARC) represent the main reason for blindness globally. The lens epithelial cells (LECs) participate not only in the metabolism of many substances in the lens but also in maintaining lens transparency. This study used lipidomics to investigate the metabolic differences in LECs of ARC patients with different severity, aiming at identifying potential metabolic biomarkers of ARC. Patients diagnosed with ARC and underwent cataract surgery at Shanghai Tongren Hospital were selected to participate in this study, which were classified as mild ARC group and severe ARC group. During their cataract surgery, anterior lens capsules(LCs) containing LECs were obtained. The lipidomics of LECs were analyzed using the liquid chromatography‑mass spectrometry (LC-MS). Potential pathways of lipids were searched for using databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst platform. In LEC lipids, 26 lipids have been identified as potential biomarkers between mild ARC and severe ARC, with AUC values of 0.67-0.94. The pathway analysis results revealed that the Glycerophospholipid (GPL) metabolism was significantly influenced, indicating that these metabolic markers contribute significantly to regulating this pathway. The LEC metabolic spectrum demonstrates a proficient ability to differentiate between patients with varying levels of cataracts. Herein, we have successfully identified potential metabolic biomarkers and pathways that have proven to be valuable in enhancing our understanding of ARC pathogenesis. The finding has translational value for developing new cataract treatment methods in the future.

Humans↗

Urinary metabolic profiles in human and rat of 1,2-dimethyl- and 1,2-diethyl-substituted 3-hydroxypyridin-4-ones.

The urinary metabolic profiles of two novel orally active iron chelators, 1,2-dimethyl-3-hydroxypyridin-4-one (CP20 or L1) and 1,2-diethyl-3-hydroxypyridin-4-one (CP94), have been studied in rats. The metabolism of CP20 was also studied in humans. Four novel metabolites of CP20, and a further three metabolites of CP94 were characterized. CP20 was found to undergo extensive phase II metabolism at the 3-hydroxy position, forming predominantly the O-glucuronide, which accounted for 44% of the dose administered in rat and greater than 85% of the dose administered in man. The 3-O-methylated CP20 metabolite (metabolite I) accounted for 1% of the administered dose in both species, whereas the unmetabolized CP20 amounted to 10.5% and 4% of the dose administered in the rats and man, respectively. In contrast, CP94 was extensively hydroxylated at the 2-ethyl position to give its 2-(1-hydroxyethyl) metabolite in the rat, which accounted for 40% of the administered dose. The O-glucuronide metabolite of CP94 accounted for 13.8% of the administered dose, whereas the unmetabolized CP94 amounted to 6.9% of the administered dose. At 72 hr, urinary levels of CP20 and CP94 and their metabolites in the rat accounted for about 55-60% of the administered dose. A large portion of the dose is therefore probably eliminated via the bile. The identity of the above metabolites was established using a combination of two or more of the following techniques: fast atom bombardment-mass spectroscopy, LC-MS, UV-VIS spectroscopy, NMR spectroscopy, specific enzyme hydrolysis assays, and chemical synthesis of compounds.

Animals↗

Pharmacokinetics and metabolism of oral diosmin in healthy volunteers.

Pharmacokinetic studies of diosmin were performed after an oral administration to healthy volunteers. Diosmin and its aglycone, diosmetin, were determined by HPLC and LC-MS techniques. At least, at the level of sensitivity of our method, no parent compound was present in the plasma but only its aglycone, diosmetin. Analysis of the pharmacokinetic parameters showed that the drug was rapidly absorbed. Diosmetin presents a long plasma elimination half-life ranging from 26 to 43 hours. Our data show the total absence of urinary elimination for both diosmin and its aglycone diosmetin, while its minor metabolites are eliminated in the urine, mainly as glucuronic acid conjugates. The presence of degradation products such as alkyl-phenolic acids confirms a metabolic pattern similar to other flavonoids.

Absorption↗

Isolation and identification of 6-desmethylnaproxen sulfate as a new metabolite of naproxen in human plasma.

A new metabolite of naproxen, 6-desmethylnaproxen sulfate (6-DMNS), has been identified in plasma from normal and uremic subjects after oral administration of a single 500 mg dose of naproxen. Tentative identification was achieved by the finding of an increase in the concentration of 6-DMN upon incubation of the plasma with arylsulfatase from Helix pomatia or from Aerobacter aerogenes. More definitive identification was established through demonstration that the HPLC retention time of the conjugate is identical to that of an authentic reference sample of 6-DMNS. Unequivocal identification was accomplished by means of LC-MS after the metabolite was isolated from the plasma by protein precipitation with acetonitrile and further purified by anion-exchange and reversed phase HPLC. Plasma profiles of 6-DMNS for normal and uremic subjects, obtained by a procedure involving differential enzymatic hydrolysis using arylsulfatase from H. pomatia, revealed that 6-DMNS was present in plasma from all subjects but in relatively high concentrations only in subjects with impaired renal function and that the extent of the conjugation is related directly to the severity of the dysfunction. No evidence was found for the presence of glucuronide or sulfate conjugates of naproxen in plasma.

Glucuronates↗

Metabolism of bambuterol in rat liver microsomes: identification of hydroxylated and demethylated products by liquid chromatography mass spectrometry.

The oxidative metabolism of (R,S)-bambuterol in rat liver microsomes was studied. Metabolite fractions were analyzed by thermospray LC-MS. The use of an equimolar mixture of deuterium-labeled and unlabeled bambuterol facilitated the mass spectrometric identification of the metabolites. Six metabolites, formed via hydroxylation, demethylation, and hydrolytic reactions, were identified. The demethylated metabolites were found to be chemically unstable under physiological conditions. It is likely that the complex biotransformation of bambuterol into terbutaline is one factor contributing to the long duration of action of bambuterol.

Animals↗

Unveiling crosstalk regulations within the polyamine pathway and between polyamine and purine pathways in Aedes aegypti females.

We previously demonstrated that ornithine decarboxylase (ODC) deficiency critically impairs nitrogen metabolism and survival in Aedes aegypti. To further examine the role of the polyamine pathway in Ae. aegypti nitrogen metabolism, we evaluated the expression of three additional genes encoding proteins involved in the biosynthetic pathway: S-adenosylmethionine decarboxylase, spermidine synthase (SdS), spermine synthase (SmS), and seven genes encoding proteins involved in the catabolic pathway in fat body, midgut and Malpighian tubules by qPCR. Distinct transcriptional profiles were observed in mosquito tissues during the first gonotrophic cycle. SdS and SmS showed a differential protein expression pattern in fat body of sugar- and blood-fed mosquitoes. Genetic silencing of SdS, SmS or SdS and SmS by RNA interference (RNAi) decreased female survival. Mosquitoes with SdS or SmS deficiency exhibited a reduction of 5G1 trypsin level in the midgut at 24 h post-blood meal (PBM) , a delay in blood digestion, and a decrease in uric acid concentration in the excreta at 48 h PBM. RNAi-mediated SdS knockdown also caused a decrease in SmS protein level and vice-versa, RNAi-driven SmS deficiency resulted in a decrease in SdS protein abundance. Notably, ODC knockdown reduced SdS, SmS, xanthine dehydrogenase-1 protein levels, and decreased specific metabolite concentrations in fat body at 24 h PBM. In addition, RNAi-mediated ODC, SdS and SmS knockdown impacted transcript levels of genes involved in polyamine and purine pathways in fat body at 24 h PBM. Our findings uncover unique crosstalk regulations within the polyamine pathway and between polyamine and purine pathways.

Ammonia metabolism↗

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba↗

Improved comprehensive profiling of fecal bile acids through chemical derivatization combined with HPLC-MS/MS analysis.

Bile acids (BAs) facilitate the digestion and absorption of fats and influence lipid and glucose homeostasis, making them potential therapeutic targets for obesity and related metabolic disorders. The liver and intestinal microbiota modify BAs structurally, generating diverse chemical forms and isomers. Comprehensive profiling of the BA pool is critical for understanding their key biological functions and as a therapeutic approach for related diseases. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is usually chosen as the preferred method for BA detection due to the complex chemical structures, the wide range of actual concentrations and the complexity of fecal sample matrices. However, free BAs are difficult to ionize, resulting in low detection signals and a lack of characteristic structural fragments to assist in structural identification. In this method, the labeling reagent (2-aminoethyl) trimethylammonium (AETMA) is employed to label the carboxyl group of BAs. Compared with underivatized BAs, the detection sensitivity of unconjugated BAs was enhanced by 25-180 fold, while that of conjugated BAs increased by 6-160 fold. It also generates unique fragment ions and enhances MS response, facilitating the discovery of potential BAs. Methodological parameters were validated using 38 BAs as representatives. Through methodological validation, it was verified that the precision, recovery, matrix effect and stability parameters of the method met acceptable criteria. We also identified 61 confirmed BAs and 55 additional candidate BAs in human pooled fecal samples. It has been successfully applied to fecal BA analysis in obese populations, providing valuable insights into potential therapeutic strategies for obesity.

Tandem Mass Spectrometry↗

On-filter fractionation by empFASP improves identification of membrane peptides in proteomic experiments.

Membrane proteins remain among the most analytically challenging targets in bottom-up proteomics due to their limited solubility and low abundance of protease-accessible sites within transmembrane domains. In addition, hydrophobic peptides are frequently lost during detergent removal and the on-filter processing steps. Here, we present empFASP, a straightforward on-filter-fractionation-based modification of the enhanced filter-aided sample preparation (eFASP) workflow that enhances recovery of membrane-embedded peptides otherwise lost during digestion and cleanup. The method combines controlled on-filter inversion with sequential ethyl acetate extraction at defined pH values, enabling recovery of peptide material retained on the filter and redistributed into detergent micelles. Compared with SP3 and SP4 in HEK293T lysates, empFASP increased unique hydrophobic peptide identifications by up to 48% and increased the proportion of detected transmembrane peptides. Application to mouse mitochondrial membranes and phosphatidylethanolamine-deficient and PE-containing Escherichia coli membranes showed that the additional fractions of empFASP contribute complementary recovery of hydrophobic and membrane-associated peptides, with the strongest gains observed at the peptide level. Because empFASP requires no specialized reagents or instrumentation, it can be readily implemented in standard proteomics workflows to improve coverage of membrane-embedded regions. SIGNIFICANCE: The empFASP (enhanced membrane peptide) workflow offers a practical solution to one of the persistent limitations in membrane proteomics-the underrepresentation of hydrophobic and transmembrane peptides in standard digests. By integrating simple pH-controlled extractions into an on-filter format, empFASP recovers peptides otherwise lost through adsorption or detergent micelle retention, substantially improving coverage of the membrane proteome. This method expands the analytical reach of bottom-up proteomics without requiring specialized instrumentation, making it immediately applicable for studies of membrane topology, protein-lipid interactions, and the structural consequences of altered membrane composition.

Proteomics↗

Quantitative N-glycoproteomic analysis reveals glycosylation signatures of plasma immunoglobulin G in sepsis.

INTRODUCTION: Sepsis is a life-threatening condition resulting from organ dysfunction due to a dysregulated immune response to infection. Immunoglobulin G (IgG) plays a role in modulating immune responses. However, the precise IgG subclass-specific N-glycosylation profiles in patients with sepsis remain poorly characterized. METHODS: This study aimed to define the site-specific N-glycosylation signatures of plasma IgG subclasses in sepsis patients with different prognoses using quantitative glycoproteomics. By employing our established GlycoQuant strategy, we quantified the intact N-glycopeptides (IGPs) of IgG subclasses in 40 healthy controls and 40 sepsis patients with a clear prognosis. RESULTS: We identified 12 IGPs with altered abundances between patients with sepsis and healthy controls. After Benjamini-Hochberg (BH) correction of the 31 outcome-stratified IGP comparisons, IGP24 and IGP25 remained significant and met the prespecified fold-change criterion. Global BH correction across 124 IGP-clinical parameter correlations retained positive associations of IGP19, IGP22, and IGP23 with procalcitonin (PCT). In exploratory outcome-stratified ROC analyses, candidates were selected using the original unadjusted P-value and fold-change screen; five IGPs were evaluated, with IGP25 and IGP24 yielding the highest individual AUCs. Collectively, our findings underscore the potential of IgG subclass-specific glycosylation profiling as a novel translational approach for clinical applications in sepsis management. SIGNIFICANCE: Sepsis remains a leading cause of global mortality, with patient outcomes heavily dependent on timely diagnosis and accurate prognosis. The dysregulated host immune response, particularly involving immunoglobulins, is central to its pathophysiology. This study provides a significant advance in the field of clinical glycoproteomics by applying a quantitative, site-specific strategy to delineate the plasma IgG subclass N-glycosylation landscape in sepsis. We report, for the first time, a panel of subclass-specific intact IgG N-glycopeptides (IGPs) that are significantly altered in sepsis patients compared to healthy controls. The identified IGPs not only demonstrate diagnostic and prognostic potential but also show a significant correlation with procalcitonin, a key clinical severity index. These findings bridge a critical knowledge gap by moving beyond bulk IgG glycosylation analysis to subclass-resolved profiling, offering novel molecular insights into sepsis immunopathology. The identified glycosylation signatures hold substantial translational promise as a foundation for developing innovative, glycan-based biomarker panels to improve the precision management of this heterogeneous and life-threatening syndrome.

Humans↗

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression.

Cirrhosis, advanced liver disease, affects 2 to 5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery top-down proteomics to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring.

Humans↗