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PubMed2024

Proteomics analysis of deep fascia in acute compartment syndrome.

Acute compartment syndrome (ACS) is a syndrome in which local circulation is affected due to increased pressure within the compartment. We previously found in patients with calf fractures, the pressure of fascial compartment could be sharply reduced upon the appearance of tension blisters. Deep fascia, as the important structure for compartment, might play key role in this process. Therefore, the aim of the present study was to examine the differences in gene profile in deep fascia tissue in fracture patients of the calf with or without tension blisters, and to explore the role of fascia in pressure improvement in ACS. Patients with lower leg fracture were enrolled and divided into control group (CON group, n = 10) without tension blister, and tension blister group (TB group, n = 10). Deep fascia tissues were collected and LC-MS/MS label-free quantitative proteomics were performed. Genes involved in fascia structure and fibroblast function were further validated by Western blot. The differentially expressed proteins were found to be mainly enriched in pathways related to protein synthesis and processing, stress fiber assembly, cell-substrate adhesion, leukocyte mediated cytotoxicity, and cellular response to stress. Compared with the CON group, the expression of Peroxidasin homolog (PXDN), which promotes the function of fibroblasts, and Leukocyte differentiation antigen 74 (CD74), which enhances the proliferation of fibroblasts, were significantly upregulated (p all <0.05), while the expression of Matrix metalloproteinase-9 (MMP9), which is involved in collagen hydrolysis, and Neutrophil elastase (ELANE), which is involved in elastin hydrolysis, were significantly reduced in the TB group (p all <0.05), indicating fascia tissue underwent microenvironment reconstruction during ACS. In summary, the ACS accompanied by blisters is associated with the enhanced function and proliferation of fibroblasts and reduced hydrolysis of collagen and elastin. The adaptive alterations in the stiffness and elasticity of the deep fascia might be crucial for pressure release of ACS.

Humans↗

PubMed2025

Mutations in the transcriptional regulator MAB_2885 confer tedizolid and linezolid resistance through the MmpS-MmpL efflux pump MAB_2302-MAB_2303 in Mycobacterium abscessus.

Mycobacterium abscessus (MAB) is a clinically significant multidrug-resistant (MDR) pathogen, particularly implicated in pulmonary infections among cystic fibrosis (CF) patients. Tedizolid (TZD), an oxazolidinone-class antibacterial drug, has been recommended as an alternative treatment for MAB-infected patients who are intolerant to or whose isolate is resistant to first-line drugs including linezolid (LZD). To investigate the TZD resistance mechanisms in MAB, we isolated 23 TZD-resistant MAB mutants and performed whole-genome sequencing (WGS) to identify resistance-associated genes. Frequent mutations were identified in MAB_2885, encoding a putative TetR transcriptional regulator, and MAB_2303, encoding a putative mycobacterial membrane protein large (MmpL). Drug susceptibility testing confirmed that MAB_2885 mutations contribute to both TZD and LZD resistance in MAB. RNA-seq analysis revealed that restoring wild-type MAB_2885 in mutants downregulated the MAB_2302-MAB_2303. Electrophoretic mobility shift assay (EMSA) showed the MAB_2885 protein binds to its target sequence upstream of MAB_2302-MAB_2303, further confirming their regulatory relationship. The W91R mutation in the MAB_2885 protein was found to impair its DNA-binding activity compared to the wild-type. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed that MAB_2302-MAB_2303 functions as a TZD efflux pump. Additionally, overexpression of MAB_2885 in M. abscessus subsp. bolletii and M. abscessus subsp. massiliense also increased their TZD susceptibility and downregulated their respective MmpS-MmpL orthologs. Overall, our study demonstrates that mutations in MAB_ 2885 contribute to TZD and LZD resistance by disrupting the negative regulation of the downstream MAB_2302-MAB_2303, which functions as a direct efflux pump for TZD. These findings provide new insights into oxazolidinone resistance mechanisms in MAB and identify potential biomarkers for detecting drug resistance.

Mycobacterium abscessus↗

PubMed2026

hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA.

Translation of key viral replicative proteins in coronaviruses requires a programmed -1 ribosomal frameshifting (-1 PRF) event controlled by the viral frameshift-stimulatory element (FSE). Although previous studies have analyzed host factor dependencies of coronaviruses, how host cellular factors alter -1 PRF efficiency and affect viral replication remains poorly understood. Here, using RNA pull-down combined with LC-MS/MS analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a major interacting protein of FSE RNA. Coronavirus infection triggers hnRNPC mRNA decay, alters hnRNPC protein levels, and induces its cytoplasmic relocalization, where it appears to bind directly to FSE RNA through residues Asn7 and Asn83. This binding is associated with increased -1 PRF efficiency and may facilitate coronavirus replication. Deletion mapping analysis shows that hnRNPC preferentially binds U-rich regions of the FSE RNA. Finally, we demonstrated that the small molecule Elbasvir directly binds hnRNPC, disrupting the interaction between hnRNPC and FSE RNA and inhibiting coronavirus replication by decreasing -1 PRF efficiency. Collectively, our study identifies hnRNPC as a key host cofactor for coronaviruses and provides a novel target for broad-spectrum antiviral drug development.

RNA, Viral↗

PubMed2026

A novel 2D and 3D model for primary adrenocortical carcinoma of advanced and metastasized stage co-secreting cortisol, aldosterone, testosterone, 18-oxocortisol and 18-hydroxycortisol.

Adrenocortical carcinoma (ACC) is a highly aggressive malignancy with poor survival rates and few treatment options. Preclinical models are indispensable to further strengthen our understanding of disease progression and development of novel therapeutic treatments. Here, we report the establishment of a new cell line named ZUC-1 originating from the resection of an advanced primary ACC and its characterization at the genomic, cellular and molecular level. ZUC-1 cells were successfully propagated as monolayer cultures and three-dimensional spheroids. LC-MS/MS analysis revealed for ZUC-1 cells co-secretion of cortisol, aldosterone and testosterone, and the model represented in direct comparison with other current ACC pre-clinical models furthermore significantly elevated expression of SF-1, CYP11B1 and CYP11B2 genes. Whole genome sequencing identified various mutations in genes linked to DNA repair/stress response, stemness, and also steroidogenesis. Interestingly, ZUC-1 represents genotypic and phenotypic variations that might be of interest beyond ACC, including congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS). Moreover, 18-oxocortisol and 18-hydroxycortisol release was detected in ZUC-1, conditions which are often linked to hyperaldosteronism, but forskolin, potassium and, at higher concentration, angiotensin II modulability of CYP11B2 for this model is retained. ZUC-1 spheroids exhibited furthermore an intra-spheroidal heterogeneous mix of canonical and non-canonical Wnt pathway activation. We conclude that due to its origin and unique geno- and phenotypes, ZUC-1 represents an intriguing model to further gain a basic understanding of adrenal function, the pathogenesis of ACC, but it might be also of interest in the context of CAH and PCOS.

Humans↗

PubMed2026

Non-invasive embryo assessment: Cell-free DNA-based genetic testing and amino acid metabolomics in relation to morphology: A case-control study.

BACKGROUND: Cell-free DNA (cfDNA) in spent culture medium (SCM) offers a non-invasive option for preimplantation genetic testing, but its low concentration and fragmentation reduce clinical reliability. Combining genetic assessment with metabolomic profiling may provide complementary information about embryo competence. OBJECTIVE: This study assessed pre-analytical cfDNA processing workflows and examined whether SCM amino acid metabolic patterns could act as practical markers of embryo quality. MATERIALS AND METHODS: In this case-control study (2021-2023), 90 embryos were evaluated using fluorescence in situ hybridization or array comparative genomic hybridization. SCM samples underwent rapid boiling, silica-based purification, or whole-genome amplification (WGA). Sex determination was performed using quantitative polymerase chain reaction (qPCR). For cfDNA quality control and aneuploidy screening, the multiplex IRFiling kit and quantitative fluorescent polymerase chain reaction (QF-PCR) were used. Amino acid profiles across embryonic developmental stages and quality grades were quantified via liquid chromatography-tandem mass spectrometry. RESULTS: Rapid boiling resulted in complete failure of DNA amplification. Conversely, silica-based purification yielded 70.0% concordance for qPCR-based sexing and 56.7% for QF-PCR. WGA achieved the highest efficacy (73.3% qPCR and 56.7% QF-PCR concordance), although quality control checks flagged occasional misclassifications. LC-MS/MS profiling revealed significantly elevated alanine and arginine levels in tripronuclear embryos. Furthermore, high-quality blastocysts exhibited elevated glutamic acid levels alongside a pronounced overall depletion of extracellular amino acids compared to low-quality counterparts and controls. CONCLUSION: WGA improves cfDNA detectability and qPCR accuracy compared with boiling or purification, but remains inadequate as a standalone screening approach. SCM amino acid profiling provides informative, complementary metabolic signatures of developmental competence, supporting a multimodal strategy for non-invasive embryo assessment.

Amino acid metabolism↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2025

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2026

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↗

PubMed2024

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↗

PubMed2026

Subcellular Proteomic Analyses Reveal REEP5 Knockdown in the Mouse Heart Disrupts Mitochondrial Networks.

Receptor Expression-Enhancing Protein 5 (REEP5) is a cardiac-enriched, membrane-shaping protein localized to the sarco(endo)plasmic reticulum (SR/ER), where it supports membrane network architecture and cardiomyocyte function. While REEP5 has been implicated in calcium handling and contractility, its role in regulating inter-organelle communication and mitochondrial homeostasis remains less well-understood. In this study, we used recombinant adeno-associated virus serotype 9-mediated shRNA knockdown of Reep5 in mouse hearts, combined with subcellular fractionation and data-independent acquisition mass spectrometry, to define proteomic remodeling across microsomal (SR/ER), mitochondrial, and cytosolic compartments. Loss of REEP5 altered the composition of SR/ER membrane-shaping proteins, including upregulation of RTN4, ATL3, and CKAP4, suggesting a partial compensatory response. Microsomal, mitochondrial and cytosolic proteomes exhibited broad reorganization, with enrichment of proteins involved in redox adaptation and proteostasis, alongside depletion of mitochondrial import machinery and antioxidant enzymes. Imaging of isolated cardiomyocytes confirmed fragmented mitochondrial networks and increased reactive oxygen species, consistent with proteomic signatures of disrupted mitochondrial dynamics and oxidative stress. Gene ontology enrichment across all fractions highlighted widespread dysregulation in organelle-specific processes, including translation, protein localization, and metabolic remodeling. Notably, several altered pathways converged on mitochondria-associated membranes, suggesting that REEP5 may support SR/ER-mitochondria tethering and functional crosstalk. These findings position REEP5 as a key regulator of organelle homeostasis in the heart and underscore how its loss disrupts mitochondrial integrity and inter-organelle communication across cellular compartments.

Animals↗

PubMed2025

Mass spectrometry-based ligand binding assays in biomedical research.

INTRODUCTION: Ligand binding assays combining immunoaffinity enrichment steps with mass spectrometry (MS) readout have gained attention as a highly specific and sensitive tool for protein quantification. These techniques typically combine enzymatic fragmentation of the sample or enriched protein with capture on the protein or peptide-level for quantification. Antibodies ensure specific target recognition, while MS offers quantitative accuracy with isotopically labeled internal standards. This dual approach supports a broad dynamic range, enabling protein measurements from picomolar to nanomolar levels. These methods have diverse applications, from quantifying signaling proteins in basic research to biomarker monitoring in clinical trials and analyzing the pharmacokinetics of therapeutic proteins. AREAS COVERED: This review delves into the diverse workflows of immunoaffinity-MS, shedding light on the innovative strategies employed, their practical applications, efficacy, and inherent limitations in the realm of protein quantification. EXPERT OPINION: Immunoaffinity-MS has transformed protein analysis, but widespread adoption is hindered by complex workflows, high instrument costs, and limited capture molecule availability. Efforts to enhance automation, standardize workflows, and advance technological innovation aim to overcome these barriers. Improvements in mass spectrometer sensitivity, advances in recombinant capture technologies, and support from public initiatives are poised to further improve the reliability and accessibility of this method.

Mass Spectrometry↗