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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

Adaptive proteomic remodeling and eNOS upregulation in luminal endothelium and perivascular adipose tissue of patent saphenous vein grafts after CABG.

OBJECTIVE: Long-term patency of saphenous vein grafts (SVGs) remains a significant challenge in coronary artery bypass grafting (CABG). The biological factors underlying successful human grafts are poorly understood. We aimed to characterize the structural and molecular features associated with successful graft function. METHODS: Patent and occluded SVG and internal thoracic artery (ITA) grafts were obtained from explanted hearts of CABG patients undergoing heart transplantation for end-stage heart failure not attributable to graft failure, along with freshly harvested ITA and SVG controls. Samples underwent histomorphological analysis, immunohistochemistry (IHC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics. RESULTS: Patent ITA (ITA-P) showed minimal intimal hyperplasia with medial reinforcement, whereas patent SVGs (SVG-P) had organized, α-smooth muscle actin (αSMA)-positive myofibroblast-rich neointima. Endothelial nitric oxide synthase (eNOS) was markedly upregulated in patent grafts at two sites-the luminal endothelium and adventitial microvessels within perivascular adipose tissue (PVAT)-and lost at both sites in occluded SVG (SVG-O). Adventitial CD31-positive microvessels were significantly increased in patent grafts. Proteomically, ITA-P and SVG-P shared a largely common adaptive proteome enriched in translation, RNA processing, and extracellular matrix (ECM) organization, with shared upstream activation of NR4A3, EGFR, and STAT1, and conduit-specific signatures (IGF-1/RUNX2 in ITA-P; RETN/SRC/PTGES in SVG-P). PTGES was strongly expressed in the adventitia of SVG-P. CONCLUSIONS: Patent arterial and venous bypass grafts exhibited a shared adaptive phenotype characterized by dual-site upregulation of eNOS in both the luminal endothelium and the perivascular microvessels/PVAT. In SVG-P, PTGES was co-upregulated alongside eNOS, indicating a mechanistic link between the proteomic and IHC findings. These findings highlight the perivascular compartment as a site of adaptive, eNOS-associated changes in patent vein grafts.

Humans↗

PubMed2026

Acidic Stress Induces Proteomic Reprogramming and Virulence-Associated Adaptation in Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis whose etiologic agents must adapt to acidic host niches such as phagolysosomes. Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to define the response of Paracoccidioides brasiliensis to acidic stress (pH 4.5) versus control pH (6.5) after 5 and 24 h. We identified and quantified 4374 proteins, including 327 and 722 differentially abundant proteins at 5 and 24 h, respectively, revealing time-dependent proteomic reprogramming. Enrichment analyses highlighted proteolysis, protein metabolism, organonitrogen metabolism, MAPK- and SNF1-like signaling, central carbon metabolism, tyrosine metabolism, and ergosterol biosynthesis as major acid-responsive processes. Complementary assays showed pH-dependent extracellular proteolytic activity, increased adhesion to A549 pulmonary epithelial cells, and dynamic ergosterol remodeling. The proteomic data further indicated increased abundance of moonlighting proteins linked to adhesion and metabolic enzymes associated with ATP generation and melanin precursor production. Together, these findings indicate that P. brasiliensis adapts to acidic environments through coordinated regulation of proteostasis, metabolism, signaling, host-cell interaction, and membrane homeostasis, supporting survival and virulence potential in acidic host microenvironments.

Paracoccidioides↗

PubMed2026

Targeted Modulation of Abundant Proteins Enhances Proteomic Profiling of Ovarian Cancer Ascites: A Pilot Technical Workflow Comparison.

Ascites from ovarian cancer patients are increasingly recognized as a valuable biofluid for cancer research, as its protein composition reflects the disease state and may reveal biomarkers of treatment sensitivity and response. However, the detection of low-abundance proteins is hindered by the presence of highly abundant proteins such as albumin. In this study, we evaluated five protein preparation methods for their effectiveness in depleting high-abundance or enriching low-abundance proteins in ovarian cancer ascites. The Norgen (Nor), Minutes (Min), and Perchloric acid (PerCA) methods were based on abundant protein depletion, while the Urine (Uri) and Nanomics (Nano) kits focused on low-abundance protein enrichment. Processed samples were analyzed using label-free quantitative bottom-up proteomics by LC-MS/MS, followed by a bioinformatics assessment. Compared with undepleted ascites (UnD), Min, Nor, Nano, and PerCA increased protein identifications, whereas Uri produced profiles similar to those of UnD. Notably, PerCA and Nano enabled the identification of distinct protein subsets associated with cancer-related pathways, including immune responses and autophagy. PerCA enriched transmembrane and secreted immunomodulatory glycoproteins, whereas Nano enrichment primarily captured secreted, nuclear, and cytoplasmic soluble proteins. Overall, our results show that both high-abundance protein depletion and low-abundance enrichment improve ascites proteome coverage, each offering distinct advantages in identifying biologically relevant low-abundance proteins.

Female↗

PubMed2026

Benchmarking the OptiSpray-μPAC Workflow against a Traditional Nanospray Capillary Interface for Multiplexed Quantitative Proteomics.

Nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) underpins modern quantitative proteomics, yet the column-to-mass spectrometer interface remains an important yet often underappreciated determinant of analytical depth, sensitivity, and reproducibility. Here, we benchmark an integrated workflow comprising the newly developed OptiSpray ion source and a micropillar array column (μPAC) cartridge against a conventional Nanospray Flex Source with an Accucore resin-packed capillary column. We performed a TMTpro 18-plex experiment across nine human cell lines on a FAIMS Pro-equipped Orbitrap Exploris 480. Following basic-pH reversed-phase fractionation, 12 fractions were analyzed on both workflow configurations under matched chromatographic gradient and acquisition conditions. Across both configurations, we quantified >9000 protein groups with highly comparable quantitative reproducibility and principal component clustering. Direct comparison of protein abundance ratios across cell lines showed agreement (Pearson R2 ≈ 0.7-0.8) without systematic bias. These results were achieved without workflow-specific optimization of the OptiSpray-μPAC platform, enabling direct transfer of established acquisition methods. Despite differences in column architecture, both configurations delivered comparable proteome coverage and quantitative fidelity. These findings establish the OptiSpray-μPAC workflow as a standardized alternative to conventional capillary-based interfaces, offering simplified operation while preserving quantitative performance.

Humans↗

PubMed2026

Proteomic insights into hepatic responses to high soybean meal inclusion with citric acid supplementation in hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) juveniles.

This study evaluated the growth performance and hepatic proteomic responses of juvenile hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) juveniles fed diets containing 30% or 40% soybean meal (SBM), with or without 3% citric acid supplementation, and compared with a fishmeal (FM) control diet over a 10-week feeding trial. Fish fed 30% SBM (LSBM) showed a final body weight comparable to the FM group, whereas fish fed 40% SBM (HSBM) recorded significantly reduced growth and net protein utilization (NPU) (p < 0.05). Supplementation of citric acid in the 40% SBM diet (HSBM + CA) significantly improved NPU and partially restored growth relative to HSBM. LC-MS/MS based quantitative proteomics identified 1544 hepatic proteins and showed clear dietary segregation. Differentially expressed proteins were predominantly associated with reduced abundance of proteins involved in lipid metabolism, amino acid metabolism, pyruvate metabolism, and the tricarboxylic cycle across all SBM diets, suggesting a shift in oxidative metabolic capacity. In contrast, LSBM induced upregulation of spliceosome-associated proteins and RNA processing components, suggesting adaptive post-transcriptional regulation under moderate SBM inclusion. In contrast, HSBM exhibited greater reduction in metabolic protein abundance without comparable upregulation of adaptive regulatory components, coinciding with reduced nutrient retention. Protein-protein interaction analysis further confirms the correlation between the regulation of spliceosomal clusters and mitochondrial lipid-catabolic modules. Citric acid supplementation did not markedly reverse hepatic metabolic protein changes but improved protein retention. Data are available via ProteomeXchange with identifier PXD081994. These findings suggest a tolerance limit between 30% and 40% SBM inclusion, with 30% inducing adaptive hepatic proteomic responses compatible with maintained growth performance.

Animals↗

PubMed2026

An Integrated Proteomics and Genomics Approach to Identify Essential Protein Kinases During Human Trophoblast Development.

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrete human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile the proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1, and TNIK, that are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKCζ are essential for STB development, whereas PTK2B, SRC, TRIO, and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

Humans↗

PubMed2026

Evaluation of pilocarpine effects on sweat proteome.

BACKGROUND: Sweat is increasingly recognized as a valuable, non-invasive biofluid for biomarker discovery, yet its composition depends on the stimulation method. This study aimed to determine how pharmacological induction with pilocarpine compares to physiologically induced sweat through exercise in shaping the sweat proteome. RESULTS: We analyzed thermoregulatory sweat from exercise, pilocarpine-induced sweat, and combined pilocarpine plus exercise sweat. Total protein concentrations were similar across conditions, but pilocarpine markedly increased proteomic diversity, with combined pilocarpine plus exercise sweat showing the highest number of identifications. The core sweat proteome remained stable, while pilocarpine selectively enriched low-abundance proteins involved in vesicular trafficking, cytoskeletal remodelling, and metabolism. Proteins linked to the canonical M3-Gq-PLC-Ca2+ pathway, including AQP5, CALML5, and CLIC1, were consistently enriched, confirming cholinergic activation. Pilocarpine-induced sweat also contained plasma-derived and immune-related proteins, reflecting enhanced secretion and reduced ductal reabsorption. CONCLUSIONS: Exercise yields a physiologically relevant but less complex proteome, pilocarpine-induced sweat produces a pharmacologically enriched yet biased profile, and combined pilocarpine plus exercise sweat maximizes protein detection at the expense of interpretability. These findings highlight the critical impact of stimulation paradigm on sweat proteomics and provide a reference framework for biomarker research. SIGNIFICANCE: This study employed LC-MS/MS to systematically characterize eccrine sweat and delineate how stimulation paradigms-exercise, pilocarpine, and their combination-shape its proteomic landscape. By demonstrating that pharmacological induction profoundly alters protein diversity and composition compared to physiologically induced sweat, these findings establish a critical benchmark for sweat-based biomarker research and highlight the need for paradigm-aware sampling strategies in clinical and translational contexts. Nonetheless, several methodological constraints warrant consideration: the limited sample size (five individuals per group), the exclusive inclusion of women under combined oral contraceptive treatment (21 active pills followed by 7 pill-free days), which restricts extrapolation to naturally cycling women, and the focus on healthy young adults (18-25 years), limiting generalizability to older or clinically heterogeneous populations. Despite these limitations, this work provides a foundational framework for optimizing sweat collection protocols and advancing precision approaches in non-invasive diagnostics.

Pilocarpine↗

PubMed2026

Mass Spectrometry-Based Profiling of Personalized Immunopeptidomes in Thai Renal Cell Carcinoma.

This study profiles the personalized immunopeptidomes of 13 Thai patients with renal cell carcinoma (RCC), addressing a critical knowledge gap in Southeast Asian populations characterized by distinct HLA allele distributions. We combined whole-exome sequencing (WES)-based personalized proteome construction with liquid chromatography-tandem mass spectrometry (LC-MS/MS), using both database-driven searches and de novo peptide sequencing. HLA typing identified several class I allotypes that are underrepresented in publicly available immunopeptidome resources, including seven alleles not previously represented in the databases examined; HLA-A*11:01 was the most frequent allele in this cohort. Database-based analysis identified a single tumor-specific neoantigen derived from a mutant JADE2 peptide in the patient with the highest tumor mutational burden, which was validated by a mutant-specific ELISPOT response. In contrast, de novo sequencing revealed numerous noncanonical peptides, a subset of which were supported by proteogenomic validation using PepQuery and detected exclusively in cancer proteomes but not in normal tissue data sets, indicating their potential as tumor-associated antigen candidates. Together, these results establish an integrated and scalable framework for identifying HLA-presented tumor-derived peptides and provide a foundational immunopeptidome resource to support personalized cancer immunotherapy development in Southeast Asia.

Humans↗

PubMed2026

Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Blood Platelets↗

PubMed2026

A streamlined workflow for high throughput metaproteomic analysis of the rumen microbiome.

Metaproteomics can provide direct functional insights into complex microbial communities, yet its application in rumen research remains limited due to labor-intensive and low-throughput sample preparation workflows before the MS analysis. This work aimed to develop and characterize a streamlined, high throughput metaproteomic workflow optimized for rumen samples. Key steps, including microbial cell extraction, cell lysis, protein digestion, and LC-MS/MS acquisition, were systematically assessed and optimized to reduce hands-on time while maintaining deep proteome coverage. The optimized workflow integrates a minimized cell extraction protocol using 0.5 g starting material and in-solution tryptic digestion. Application of the final workflow to 72 samples from in vitro fermentation revealed that biological variability between inocula dominated technical variability, which remained moderate (median CV of 21-24% across batches). Overall, the optimized workflow supports robust taxonomic and functional characterization of the rumen microbiome with improved scalability. These advances provide a foundation for applying metaproteomics to larger experimental designs, including nutritional trials and cohort studies, thereby enabling broader functional interrogation of rumen microbial ecosystems. SIGNIFICANCE: This study addresses current limitations in the application of metaproteomics to rumen microbiome research by developing a streamlined and scalable sample preparation workflow. By optimizing key steps and reducing sample input while maintaining reproducibility and proteome coverage, this work enables more efficient processing of larger sample sets. These advances support the broader use of metaproteomics in rumen studies and facilitate functional investigations relevant to animal nutrition and sustainable livestock production.

Animals↗

PubMed2026

Bioepidemiology of cardiac amyloidosis.

BACKGROUND: Cardiac amyloidosis, primarily due to immunoglobulin light chain (AL) or transthyretin (ATTR) amyloid, is an increasingly recognized cause of heart failure. Modern diagnostic advances suggest that ATTR, particularly in older adults, may be more prevalent than historically reported. METHODS: All Olmsted County decedents aged ≥40 years from 1970 to 1976 were identified. Available ventricular myocardium from retained paraffin blocks was screened histologically for amyloid using sulfated Alcian blue staining; positive cases underwent grading and proteomic typing by laser microdissection coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Beyond prevalence estimation, this analysis characterizes amyloid type, deposition grade and distribution, associated comorbidities, and cause-of-death patterns, comparing amyloid-positive decedents with age- and sex-matched controls. RESULTS: Of 2,566 eligible deaths, 1,028 autopsy cases with evaluable myocardium formed the study cohort (mean age 70.5 years; 61.1% male; 97% White). Cardiac amyloid was present in 52 cases giving an overall prevalence of 5.1% (95% CI: 3.8-6.6, which rose from 0% under age 60 to 37.5% (95% CI: 21.1-56.3) in those ≥90 years (p < 0.001). While prevalence estimates were higher in men above age 80 compared to women, there was no evidence of an interaction of age and sex (p = 0.90). The quantity of amyloid was sufficient for typing in 38 cases: ATTR (84.2%), AL (7.9%), serum amyloid A (5.3%), and apolipoprotein A-IV (2.6%). Adjusted estimates assuming untyped mild cases were ATTR-type increased ATTR prevalence to 4.5% (95% CI: 3.3-5.9) overall. Comorbidity profiles were similar between amyloid-positive and negative groups, though syncope and leg weakness were more common in amyloid-positive decedents. CONCLUSIONS: In an unselected autopsy cohort, cardiac amyloid was common, particularly ATTR in older adults. Prevalence increased steeply after age 80. These findings suggest that ATTR amyloidosis is not rare and is likely underdiagnosed and has similar prevalence in women and men, despite the male predominance reported in the literature.

Humans↗

PubMed2026

Antivenomic and Proteomic Assessment of Inter- and Intrapopulation Venom Variations in Nikolsky's Adder Vipera nikolskii: Comparison to Common Adder Vipera berus.

Snake venom variation has important clinical implications, yet individual-level venomics remains limited. We investigated inter- and intrapopulation variability in forest-steppe adder Vipera nikolskii and its recognition by commercial V. berus antivenom using proteomic and immunological approaches. Venoms from 12 individual V. nikolskii specimens representing two geographically distinct populations (BG and KM), together with three pooled V. nikolskii and one pooled V. berus samples were analyzed by LC-MS/MS, ELISA, Western blot, and pull-down assays. Multivariate analysis revealed relative homogeneity in BG and pronounced heterogeneity in KM venoms. Area-based proteomics revealed V. berus venom enrichment in PLA2 (34.6%), SVMP (14.6%), and CRiSP (15.6%), whereas V. nikolskii venoms were more variable. Pooled V. nikolskii venoms showed SVMP abundance (35.2-41.1%), contrasting with lower levels in individual samples. Antivenom binding was stronger for V. berus but weaker and more variable across individual and pooled V. nikolskii samples. Antivenom targeted PLA2/VEGF, CRiSP (only in V. berus), and Kunitz-type proteins. In vivo neutralization assay demonstrated strong protection against V. berus but not V. nikolskii venom. These findings reveal substantial compositional and antigenic variability in V. nikolskii venoms, highlight discrepancies between pooled and individual ones, and underscore the need for region-specific and functionally validated antivenom evaluation.

Animals↗

PubMed2026

The composition of the periostracum in the razor clam Sinonovacula constricta and the mantle's response to sulfide.

The razor clam Sinonovacula constricta inhabits sulfide-rich intertidal sediments and exhibits remarkable tolerance to this toxicant, yet the role of its periostracum in sulfide adaptation remains poorly understood. In this study, we investigated the composition and structure of the periostracum proteins, and the response of the mantle to sulfide stress. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed that the periostracum is approximately 10 μm thick and contains 1.43 wt% sulfur, and proteomic analysis further confirmed the presence of organic sulfur (Cys/Met-rich proteins), suggesting its involvement in sulfur deposition. Using LC-MS/MS, we identified 77 high-confidence proteins from the periostracum, which were classified into six functional categories: enzymes, framework proteins, immune-related proteins, calcium ion-related proteins, other proteins, and proteins with unknown functions. Phylogenetic analyses of representative proteins revealed bivalve-specific evolutionary patterns, with several proteins exclusively present in Bivalvia, such as Unknown protein 2 and 7, which possess signal peptides and low-complexity domains. For the sulfide exposure experiment, razor clams were subjected to three Na2S concentrations (0, 10, and 100 μM). qPCR analysis showed that, compared with the control group, Chitin-binding protein 3 and Tyrosinase were significantly upregulated in the mantle, peaking in the 100 μM group at 48 h (5677.84-fold and 157.20-fold, respectively), whereas Collagen and Cadherin 3 were generally suppressed. This study represents one of the most comprehensive proteomic profiles of the razor clam periostracum and highlights the mantle's potential role in sulfide tolerance, offering insights for sulfur-tolerant aquaculture breeding and bioremediation applications.

Animals↗

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↗

PubMed2026

Development of a High-Sensitivity Glycoproteomics Approach for Fc-Specific Quantification of IgG Core Fucosylation in Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers complex neuroinflammatory cascades that involve sustained immune activation and dysregulated antibody effector functions. Immunoglobulin G (IgG) Fc N-glycosylation, particularly core fucosylation, critically modulates immune signaling through altered Fcγ receptor (FcγR) interactions; however, its role in TBI remains unexplored. Here, we developed a high-sensitivity, mass spectrometry-based glycoproteomics method for the systematic analysis of IgG Fc core fucosylation dynamics following TBI. The approach integrates Fc-specific enzymatic truncation with GlycINATOR (EndoS2) and tryptic digestion, followed by high-resolution LC-MS/MS profiling, enabling confident identification of truncated Fc glycopeptides. Furthermore, a targeted parallel reaction monitoring (PRM) strategy allowed direct quantification of core fucosylated and afucosylated glycopeptides from 10 μg of crude serum protein, eliminating the need for IgG purification. Our results reveal time-dependent and subclass-specific remodeling of IgG Fc fucosylation postinjury, characterized by an overall reduction in fucosylated species and a relative increase in afucosylation. Collectively, this study establishes a scalable analytical platform for Fc-specific glycosylation profiling and identifies IgG core fucosylation as a candidate molecular indicator of immune dysregulation in TBI, providing new insights into post-traumatic immune regulation.

Brain Injuries, Traumatic↗

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

Schisantherin B mitigates cisplatin-induced ototoxicity by modulating the CNPY2-PERK/CHOP signaling axis.

Irreversible cisplatin-induced hearing loss (CIHL) is a refractory chemotherapy-related adverse effect with limited clinical treatments. Schisantherin B (STB), a lignan isolated from Schisandra chinensis, is widely recognized for its neuroprotective properties, while its role in auditory injury remains unclear. Herein, we found that STB alleviated cisplatin-induced ototoxicity in House Ear Institute Organ of Corti 1 (HEI-OC1) cells and guinea pig models, protecting cochlear hair cells, synaptic ribbons and spiral ganglion neurons, and partially restoring auditory brainstem response (ABR) thresholds. Furthermore, combined drug affinity responsive target stability (DARTS) assay, the cellular thermal shift assay (CETSA), and the surface plasmon resonance (SPR) assay, we confirmed STB directly binds to the canopy FGF signaling regulator 2 (CNPY2), a key initiator of endoplasmic reticulum (ER) stress. Notably, consistent dual in vitro and in vivo validation confirmed that STB exerts no regulatory effect on CNPY2 protein abundance, yet suppressed the downstream Protein kinase R-like endoplasmic reticulum kinase / C/EBP homologous protein (PERK/CHOP) signaling cascade and ER stress-mediated apoptosis. Moreover, molecular docking and co-immunoprecipitation (co-IP) validated the physical binding of STB to CNPY2 and the endogenous interaction between CNPY2 and PERK. Additionally, CNPY2 overexpression and shRNA knockdown further verified this functional relationship. Integrated proteomic and transcriptomic analyses showed STB partially reversed cisplatin-triggered inflammation and excessive ER stress. Collectively, our results suggest STB may serve as a potential otoprotective agent. The CNPY2-PERK/CHOP axis is closely linked to cisplatin-induced cochlear damage and offers a feasible target for intervention against CIHL. Abbreviations: CIHL, cisplatin-induced hearing loss; STB, Schisantherin B; HEI-OC1, house ear institute organ of corti 1; ABR, auditory brainstem response; DARTS, drug affinity responsive target stability; CETSA, cellular thermal shift assay; SPR, surface plasmon resonance; CNPY2, canopy FGF signaling regulator 2; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; co-IP, co-immunoprecipitation; STA, Schisantherin A; STC, Schisantherin C; dB SPL, decibels sound pressure level; EDTA, ethylenediaminetetraacetic acid; dB SPL, decibels sound pressure level; SGN, spiral ganglion neuron; IHCs, inner hair cells; OHCs, outer hair cells; CCK-8, Cell Counting Kit-8; OD, optical density; ODb, blank sample, ODc, control sample; NC, negative control; PVDF, polyvinylidene difluoride; RT, room temperature; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; DMSO, dimethyl sulfoxide; KDs, equilibrium dissociation constants; SP, standard precision; SEM, standard error of the mean; HSD, honestly significant difference; Ctrl, control group; CV, cell viability; Kd, dissociation rate constant; Ka, association rate constant; STS, sodium thiosulfate; UPR, unfolded protein response; BLB, blood-labyrinth barrier.

Apoptosis↗