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PubMed2026

Relaxin-2: Shaping the Proteomic Landscape of Skeletal Muscle Physiology, Glucose Trafficking, and Mitochondrial Function in Rat.

Relaxin-2 is a hormone with robust beneficial effects on the heart and blood vessels and potential as a therapy for cardiovascular (CV) disease. Considering the interorgan communication between skeletal muscle and heart, and the relation between muscle quality/composition and CV events, we hypothesize that relaxin-2 may regulate skeletal muscle physiology and metabolism. We aim to evaluate the impact of relaxin-2 on the proteome of skeletal muscle from healthy Sprague-Dawley rats. Animals were treated with 0.4 mg/kg/day of serelaxin (recombinant form of human relaxin-2) or vehicle (PBS) for 2 weeks employing subcutaneous osmotic minipumps. Skeletal muscle protein identification and quantification were performed by LC-MS/MS using a Data-Independent Acquisition (DIA)-Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH) method. SWATH/MS quantitative analysis identified that relaxin-2 significantly decreased 95 proteins and significantly increased 32 proteins in rat skeletal muscle when compared to control rats. From these, 34 proteins were associated with muscle function, myogenesis, muscle differentiation and/or regeneration, 20 are mitochondrial proteins (six from the complexes of the electron transport chain), and 10 proteins participate in glucose metabolism. Qualitative data-dependent workflow analysis identified 35 proteins exclusive to the skeletal muscle of the relaxin-2-treated group: eight proteins related to processes of skeletal muscle function (size, ion homeostasis or organization of caveolae structures and cytoskeleton) and myogenesis, and two proteins involved in muscle differentiation. Our work highlighted for the first time the role of relaxin-2 in crucial processes of muscle physiology and energetic metabolism, which could influence several processes involved in myopathy and CV.

Animals↗

PubMed2026

Transcriptomic and proteomic signatures underlying nymphal adaptation and foam production in the forage pest Mahanarva spectabilis.

The spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) is an important pest of forage grasses in South America, where its nymphs cause pasture damage by feeding on xylem sap and producing a characteristic foam that protects them against environmental stressors. To investigate the molecular basis of this adaptation, we integrated RNA-seq analysis of nymphs with LC-MS/MS proteomics of the Batelli gland, the primary source of foam secretion. De novo assembly of 100,666 unigenes revealed broad functional diversity, with strong representation of detoxification enzymes (CYP450s, GSTs, UGTs, carboxylesterases), transporters and ion pumps, cuticle proteins, and stress- and immunity-related genes. Nearly 16% of loci exhibited alternative splicing, particularly within detoxification, chemosensory and osmoregulatory gene families, highlighting evidence of transcriptomic variability. Signal peptide and secreted protein predictions identified 168 high-confidence candidate secreted proteins, including detoxification enzymes, proteases, structural proteins and immune-related factors, several of which are consistent with antimicrobial and surfactant-related functions. Proteomic profiling of the Batelli gland confirmed 500 proteins, enriched in chaperones, metabolic enzymes, detoxification pathways and osmoregulatory components, with the most abundant proteins corresponding to Hsp70 chaperones, ATP synthases, cuticle proteins and carbonic anhydrases. Together, these results provide an integrative transcriptomic and proteomic overview for M. spectabilis nymphs, highlighting genes and proteins associated with xylem feeding, foam production and responses potentially related to environmental stress tolerance. This comprehensive dataset not only advances the understanding of spittlebug biology but also identifies candidate molecular targets that may inform innovative strategies for controlling nymphal stages and mitigating spittlebug damage in forage systems.

Animals↗

PubMed2026

Comparison of Proteomic Analysis of Cerebrospinal Fluid From Neurological Patients With and Without Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by progressive muscle weakness in both bulbar and extremity muscles, leading to a diverse clinical phenotype with motor and non-motor symptoms. Approximately 85% of ALS cases are sporadic (sALS), while the remaining 10%-15% are familial (fALS). Biological biomarkers of sporadic ALS remain poorly understood, hindering precise patient screening, delaying diagnosis and negatively affecting prognosis. This study aims to identify potential proteomic biomarkers by comparing the cerebrospinal fluid (CSF) of sALS patients with that of patients suffering from other neurological diseases. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for proteomic profiling of CSF samples from 24 sALS patients and 26 patients with other neurological diseases. The complete protein expression profiles were compared using a two-tailed Student's t-test, with a p < 0.05 considered statistically significant with additional FDR correction at the 0.1 level. Proteomic analysis of CSF samples identified significant quantitative changes in 96 proteins with threshold p < 0.05 and 74 proteins with FDR < 0.1 between sALS and non-ALS patients, including alterations in proteins associated with neurodegenerative processes, such as amyloid precursor proteins and inflammatory markers. CSF proteomic analysis reveals altered inflammatory and neurodegenerative metabolic pathways, providing valuable insights into the proteomic landscape of sALS. Several dysregulated proteins were consistent with the disease mechanisms highlighted in previous studies. These findings represent a step forward in developing personalised approaches for diagnosing and managing the disease.

Humans↗

PubMed2026

Comparative Genome-Wide Association Studies of Metabolites and Grain-Related Traits in Common Wheat.

The metabolome is highly diverse and the closest layer to phenotype; therefore, it is commonly regarded as a bridge between the genome and phenome in plants. Here, we performed large-scale metabolome analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 33 grain-related traits in a diverse panel of natural accessions and a recombinant inbred line (RIL) population. We identified a new network of 2286 associations between 947 metabolites and 33 grain-related traits. Systematic integration of metabolic genome-wide association study (mGWAS) and metabolic quantitative trait locus (mQTL) analyses identified 33 566 significant single-nucleotide polymorphisms (SNPs) and 3128 mQTL. Thirteen annotated metabolites co-localized within a physical interval on 7A. Integration of metabolite-based and phenotype-based GWAS and QTL revealed an overlapped region for gibberellin A4 (GA4) content and grain roundness on 4A. Phenotyping of an ethyl methanesulfonate (EMS)-induced mutant confirmed the role of TaSDR in regulating GA4 content and grain morphology. These findings provide novel insights into the metabolic pathways influencing key grain-related traits and advance our understanding of the complex molecular mechanisms regulating grain metabolites and phenotypes in wheat. The identified metabolic markers and candidate genes provide valuable targets for molecular breeding programs aimed at improving wheat yield and quality.

QTL↗

PubMed2026

Time-resolved proteomic adaptation of multidrug-resistant Acinetobacter baumannii to antimicrobial stress induced by partially purified fraction from Caesalpinia pulcherrima flower using DEqMS.

UNLABELLED: The global prevalence of multidrug-resistant (MDR) bacteria represents an urgent public health challenge, emphasizing the critical need for novel antimicrobial agents. MDR Acinetobacter baumannii, a nosocomial pathogen of critical global concern owing to its capacity to acquire and disseminate antimicrobial resistance, was employed as a bacterial model to investigate the antimicrobial potential of natural products derived from Caesalpinia pulcherrima (L.) Sw. (Fabaceae). This medicinal plant represents a promising reservoir of novel bioactive compounds; however, its molecular effects on the A. baumannii proteome had not previously been characterized. The partially purified ethyl acetate fraction of C. pulcherrima flowers (CPF4) exhibited potent bactericidal activity against susceptible A. baumannii (minimum inhibitory concentration and minimum bactericidal concentration = 31.25 µg/mL), and time-resolved label-free quantitative LC-MS/MS proteomics was subsequently performed on MDR A. baumannii cultures treated with CPF4 at 24 h, 48 h, and 72 h post-treatment alongside untreated controls in biological triplicate, with differential protein expression assessed using differential expression of quantified mass spectrometry data. No significantly differentially expressed proteins were detected at 24 h or 48 h relative to the control, indicating that the proteomic effects of CPF4 manifest predominantly at the late treatment stage. In contrast, a robust late-phase response was identified at 72 h, comprising the coordinated induction of proteins associated with DNA damage repair, transcriptional regulation, and cell surface glycosylation remodeling. The sensor histidine kinase PmrB was significantly upregulated at 72 h vs 48 h (adjusted P = 0.029), implicating the PmrA/PmrB two-component system in late-phase colistin tolerance acquisition under sustained CPF4 exposure. IMPORTANCE: These findings provide mechanistic insight into the adaptive survival strategies employed by multidrug-resistant Acinetobacter baumannii in response to plant-derived antimicrobial challenge and support the further development of Caesalpinia pulcherrima-derived natural products as candidate antimicrobial agents.

Acinetobacter baumannii↗

PubMed2026

Machine Learning and Metabolomics to Characterize Warburg-Like Metabolic Subtypes in Human Retinal Endothelial Cells Exposed to Risk Factors Associated With Proliferative Diabetic Retinopathy.

PURPOSE: High glucose (HG), hypoxia (Hyp), and their combination are major risk factors for proliferative diabetic retinopathy (PDR). Although these conditions induce features of the Warburg-like metabolic reprogramming in human retinal endothelial cells (HRECs), it remains unclear whether they produce distinct metabolic and angiogenic subtypes. This study aimed to characterize the Warburg-like-associated metabolic heterogeneity induced by these PDR-related risk factors and evaluate the ability of supervised machine-learning models to distinguish these subtypes. METHODS: HRECs were cultured under normoglycemic, HG, Hyp (2% O2), and combined HG-Hyp conditions. Untargeted LC-MS/MS metabolomics quantified metabolites spanning carbohydrates, amino acids, nucleotides, and lipids. Principal component analysis (PCA) assessed overall metabolic variation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified metabolic pathways associated with angiogenesis. In vitro angiogenesis assays measured endothelial tube formation and branching. Nine supervised classifiers (decision tree, logistic regression, naïve Bayes, random forest, K-Nearest Neighbors, neural network, gradient boosting, AdaBoost, and Support Vector Machine) were trained on the highest-ranked metabolites selected by the Information Gain Ratio feature-ranking approach. Model performance was evaluated using 10-fold cross-validation, leave-one-out cross-validation (LOOCV), permutation testing, and a classifier stability analysis under biologically meaningful distributional shift using an independent chemically induced hypoxia model (CoCl2). RESULTS: PCA revealed partial separation of metabolic profiles across conditions, indicating different Warburg-like metabolic subtypes. The combined HG-Hyp condition exhibited enhanced angiogenic potential relative to either HG or Hyp alone. KEGG pathway enrichment analysis identified fatty acid biosynthesis and elongation among the most significantly enriched pathways in HRECs under combined HG-Hyp conditions, alongside amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, the pentose phosphate pathway, and glycolysis/gluconeogenesis. Supervised machine-learning classifiers distinguished these metabolic subtypes, with AdaBoost and gradient Boosting showing the most balanced, reproducible performance across 10-fold cross-validation, LOOCV, and permutation testing, and remaining the most reliable classifiers under domain-shift testing (area under the curve = 0.88, P = 0.0061). CONCLUSIONS: In this exploratory analysis, HG, Hyp, and their combination drive metabolically and functionally distinct subtypes of Warburg-like metabolic reprogramming in HRECs, with HG-Hyp in combination producing a highly angiogenic phenotype. Boosting-based ensemble classifiers provide a promising framework for detecting these subtypes even under domain-shift conditions, warranting validation in larger independent datasets. TRANSLATIONAL RELEVANCE: Integrating metabolomics with machine-learning classification offers a strategy to identify Warburg-like metabolic subtypes in retinal endothelial cells, providing insights into angiogenic mechanisms and guiding the development of targeted diagnostics or therapeutics for PDR.

Humans↗

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

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

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

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↗