Search Results (209)

We conducted systematic glycomic and glycoproteomic profiling to characterize the dynamic N-glycosylation landscape of rat serum, with particular focus on sex- and time-dependent variations. MALDI-TOF-MS analysis revealed that rat serum N-glycans are predominantly biantennary, disialylated complex-type structures with extensive O-acetylation of Neu5Ac residues, especially in females. LC-MS/MS-based glycoproteomic analysis of albumin/IgG-depleted serum identified 87 glycoproteins enriched in protease inhibitors (e.g., serine protease inhibitor A3K) and immune-related proteins such as complement C3. Temporal analyses revealed stable sialylation in males but pronounced daily fluctuations in females, suggesting hormonal influence. Neu5Gc-containing glycans were rare and mainly derived from residual IgG, as confirmed by glycomic analysis. In contrast to liver-derived glycoproteins, purified IgG exhibited Neu5Gc-only sialylation without O-acetylation, underscoring distinct sialylation profiles characteristic of B cell-derived glycoproteins. Region-specific glycosylation patterns were observed in IgG, with the Fab region carrying more disialylated structures than Fc. These findings highlight cell-type and sex-specific differences in sialylation patterns between hepatic and immune tissues, with implications for hormonal regulation and biomarker research. This study provides a valuable dataset on rat serum glycoproteins and underscores the distinctive glycosylation features of rats, reinforcing their utility as model organisms in glycobiology and disease research. Full article

Compositional Data Analysis (CoDA) is a powerful statistical approach for analyzing 24 h time-use data, effectively addressing the interdependence of sleep, sedentary behavior, and physical activity. Unlike traditional methods that struggle with perfect multicollinearity, CoDA handles time use as proportions of a whole, providing biologically meaningful insights into how daily activity patterns relate to health. Applications in epidemiology have linked variations in time allocation between behaviors to key health outcomes, including adiposity, cardiometabolic health, cognitive function, fitness, quality of life, glycomics, clinical psychometrics, and mental well-being. Research consistently shows that reallocating time from sedentary behavior to sleep or moderate-to-vigorous physical activity (MVPA) improves health outcomes. Importantly, CoDA reveals that optimal activity patterns vary across populations, supporting the need for personalized, context-specific recommendations rather than one-size-fits-all guidelines. By overcoming challenges in implementation and interpretation, CoDA has the potential to transform healthcare analytics and deepen our understanding of lifestyle behaviors’ impact on health. Full article

Saccharides critically influence tobacco quality. To elucidate the effects of postharvest shading (PS) pre-curing on saccharide metabolic flux, a targeted glycomics analysis was conducted. Compared to light exposure (CK), PS delayed chlorophyll degradation during pre-curing but accelerated yellowing, ultimately resulting in similar pigment levels. Additionally, PS inhibited photosynthesis, leading to reduced starch content and increased soluble sugar content before curing. Furthermore, PS altered the starch-to-sugar conversion, ultimately resulting in significantly higher soluble sugar content and lower starch content. Targeted glycomics analysis identified 21 saccharides, with glucose, D-fructose, and sucrose being dominant. Notably, PS ultimately increased glucose, D-fructose, and sucrose levels by 74.09%, 66.49%, and 17.36%, respectively. Pairwise comparisons revealed 6, 12, 5, 13, 10, and 11 differentially expressed metabolites before curing and at 38, 40, 42, 54, and 68 °C during curing, respectively, between PS and CK. Conjoint analysis identified methylgalactoside and three oligosaccharides (sucrose, raffinose, and maltose) as the central metabolites of saccharide metabolism during curing. D-mannose, D-sorbitol, and D-glucuronic acid were identified as biomarkers for assessing storage-induced metabolic perturbations using random forest algorithms. Collectively, these findings suggest that PS might enhance tobacco quality via carbohydrate metabolism modulation, providing a scientific basis for pre-curing protocol optimization and industrial application. Full article

Breast cancer (BC) is a major global health concern, and early detection is key to improving patient outcomes. Aberrant glycosylation, particularly the sulfation of glycans, is implicated in cancer progression; however, analyzing these low-abundance glycans is challenging. This study aimed to profile serum sulfated N-glycans in Ethiopian patients with BC to identify novel biomarkers for the early detection of BC. Using a glycoblotting-based sulphoglycomics workflow, including high-throughput glycoblotting enrichment, weak anion exchange (WAX) separation, and MALDI-TOF MS, serum samples from 76 BC patients and 20 healthy controls were analyzed. Statistical evaluation revealed significant differences in the sulfated N-glycan profiles. Seven mono-sulfated N-glycans were markedly elevated in patients with BC, demonstrating high diagnostic accuracy (AUC ≥ 0.8) in this internal cohort. Terminal Lewis-type glycan epitopes were prominent in sulfated glycans but were absent in their non-sulfated counterparts. The increased fucosylation and sialylation of sulfated glycans are statistically significant markers of early-stage BC. The preservation of sialic acid groups during the analysis ensured detailed structural insight. This pioneering study quantitatively examined sulfated N-glycans in BC and identified potential glyco-biomarkers for early detection. Validation in larger, diverse cohorts is needed to establish their broader diagnostic relevance and improve our understanding of cancer-associated glycomic alterations. Full article

Aberrant glycosylation is a hallmark of cancer, offering opportunities to enhance clinical decision-making and enable precise targeting of cancer cells. Nevertheless, alterations in the bladder urothelial carcinoma (BLCA) N-glycome remain poorly characterized. Here, we used in situ N-deglycosylation and mass spectrometry, revealing a marked enrichment of oligomannose-type N-glycans in non-invasive Ta tumors, which diminished with disease progression. A complementary analysis of The Cancer Genome Atlas (TCGA) transcriptomic data revealed downregulation of the key mannosidases in BLCA, suggesting a mechanistic basis for oligomannose accumulation, though this requires further validation. Then, targeted glycoproteomic profiling identified potential stage-specific carriers of oligomannoses. Exploratory functional annotation suggests stage-dependent differences among detected glycoproteins, ranging from metabolic regulation in Ta tumors to oxidative stress adaptation in muscle-invasive disease, highlighting glycosylation’s contribution to tumor progression. Furthermore, myeloperoxidase (MPO) was enriched in more aggressive stages. Spatial validation confirmed MPO overexpression in tumor-infiltrating immune cells and its correlation with oligomannose content. Importantly, high MPO expression combined with low mannosidase levels was linked to poor survival, suggesting biological relevance. This study suggests a dynamic, stage-specific N-glycome in BLCA and identifies oligomannose-bearing glycoproteins as exploratory leads for biomarker and therapeutic target discovery, providing a N-glycomic resource for further investigation towards glycan-based precision oncology. Full article

Metastatic melanoma is an aggressive skin cancer with a five-year survival rate of only 35%. Despite recent advances in immunotherapy, there is still an urgent need for the development of innovative therapeutic approaches to improve clinical outcomes of patients with metastatic melanoma. Prior research from our laboratory revealed that loss of the I-branching enzyme β1,6 N-acetylglucosaminyltransferase 2 (GCNT2), with consequent substitution of melanoma surface I-branched poly-N-acetyllactosamines (poly-LacNAcs) with i-linear poly-LacNAcs, is implicated in driving melanoma metastasis. In the current study, we explored the role of galectin-3 (Gal-3), a lectin that avidly binds surface poly-LacNAcs, in dictating melanoma aggressive behavior. Our results show that Gal-3 favors binding to i-linear poly-LacNAcs, while enforced GCNT2/I-branching disrupts this interaction, thereby suppressing Gal-3-dependent malignant characteristics, including extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway activation, BCL2 expression, cell proliferation, and migration. This report establishes the crucial role of extracellular Gal-3 interactions with i-linear glycans in promoting melanoma cell aggressiveness, placing GCNT2 as a tumor suppressor protein and suggesting both extracellular Gal-3 and i-linear glycans as potential therapeutic targets for metastatic melanoma. Full article

Gynecologic tumors are a leading cause of cancer-related mortality in women worldwide, with endometrial, ovarian, and cervical types being the most prevalent. Aberrant glycosylation, a key post-translational modification, plays a crucial role in tumor development, metastasis, and immune evasion. Specific glycosylation changes, such as altered sialylation and fucosylation, have been identified in gynecologic cancers and are associated with disease progression and prognosis. Understanding glycosylation alterations in gynecologic cancers holds promise for novel diagnostic and therapeutic approaches, ultimately enhancing patient outcomes. In this study, the serum N-glycome was analyzed in patients with myoma uteri, endometriosis, and cervical carcinoma by hydrophilic-interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection in order to identify their biomarker potential. Individual serum samples were deglycosylated by PNGase F digestion followed by procainamide labeling and solid-phase-extraction-based purification. All disease groups exhibited consistently higher levels of specific bi-antennary glycans (A2G2 and A2G2S1) compared to control patients. Additionally, significantly higher levels of agalactosylated and mono-sialylated glycans were found in cervical cancer, while a notable decrease in bisected N-glycans, alongside an increase in highly branched tetra-sialylated glycans, was found in endometriosis. Our study serves as proof-of-concept, demonstrating that discovering biomarkers within the serum N-glycome is a promising approach for identifying non-invasive indicators of gynecologic conditions. Full article

Proteins represent a significant portion of the global therapeutics market, surpassing hundreds of billions of dollars annually. Among the various post-translational modifications, glycosylation plays a crucial role in influencing protein structure, stability, and function. This modification is especially important in biotherapeutics, where the precise characterization of glycans is vital for ensuring product efficacy and safety. Although mass spectrometry-based techniques have become essential tools for glycomic analysis due to their high sensitivity and resolution, their complexity and lengthy processing times limit their practical application. In contrast, electrochemical methods provide a rapid, cost-effective, and sensitive alternative for glycosylation assessment, enabling the real-time analysis of glycan structures on biotherapeutic proteins. These electrochemical techniques, often used in conjunction with complementary methods, offer valuable insights into the glycosylation profiles of both isolated glycoproteins and intact cells. This review examines the latest advancements in electrochemical biosensors for glycosylation analysis, highlighting their potential in enhancing the characterization of biotherapeutics and advancing the field of precision medicine. Full article

Mucomics is the study of mucus and its biochemical properties. This discipline has gained increasing attention due to the critical roles mucus plays in protection, adhesion, and communication across species. Ethical restrictions on vertebrate research have driven the interest in invertebrate models such as mollusks. Mollusks produce large amounts of mucus which has several functions, including immune defense, digestion, and environmental adaptation. Mollusks—terrestrial, freshwater, or marine—are valuable models for investigating mucus composition and its responses to environmental stressors, including heavy metal contamination. Histochemical and glycomic techniques have revealed variations in mucin glycosylation patterns that influence mucus functionality, such as its viscoelastic and adhesive properties. Bivalves and gastropods, widely used as bioindicators and generally not subject to regulatory constraints in experimental use, accumulate pollutants in their mucus, reflecting environmental health. Investigative techniques such as lectin histochemistry, proteomic, and glycomic analyses provide insights into the impact of contaminants on mucus composition. Further research on molluscan mucins can enhance understanding of their physiological roles, environmental interactions, and potential biomedical applications. By integrating molecular and histochemical approaches, mucomic studies offer a comprehensive perspective on mucus function, advancing both ecological monitoring and biotechnological applications. Full article

Glycosaminoglycans (GAGs) are a diverse family of ancient biomolecules that evolved over millennia as key components in the glycocalyx that surrounds all cells. GAGs have molecular recognition and cell instructive properties when attached to cell surface and extracellular matrix (ECM) proteoglycans (PGs), which act as effector molecules that regulate cellular behavior. The perception of mechanical cues which arise from perturbations in the ECM microenvironment allow the cell to undertake appropriate biosynthetic responses to maintain ECM composition and tissue function. ECM PGs substituted with GAGs provide structural support to weight-bearing tissues and an ability to withstand shear forces in some tissue contexts. This review outlines the structural complexity of GAGs and the diverse functional properties they convey to cellular and ECM PGs. PGs have important roles in cartilaginous weight-bearing tissues and fibrocartilages subject to tension and high shear forces and also have important roles in vascular and neural tissues. Specific PGs have roles in synaptic stabilization and convey specificity and plasticity in the regulation of neurophysiological responses in the CNS/PNS that control tissue function. A better understanding of GAG instructional roles over cellular behavior may be insightful for the development of GAG-based biotherapeutics designed to treat tissue dysfunction in disease processes and in novel tissue repair strategies following trauma. GAGs have a significant level of sophistication over the control of cellular behavior in many tissue contexts, which needs to be fully deciphered in order to achieve a useful therapeutic product. GAG biotherapeutics offers exciting opportunities in the modern glycomics arena. Full article

Introduction: Obesity is a rapidly growing common health problem worldwide that can lead to the development of gestational diabetes mellitus (GDM). However, GDM not only affects women with obesity but can also develop at any time, even after the OGTT test; therefore, an increasing number of complications related to GDM can be seen in both mothers and their children. It is necessary to discover biomarkers capable of indicating the development of GDM or complications during/after pregnancy. Since the N-glycosylation motif of human IgG has been described to change under many physiological and pathological conditions, it is a promising target for biomarker research. In our study, the effects of obesity and GDM were investigated on human serum IgG N-linked glycosylation patterns during human pregnancy. Materials and Methods: The study participants were categorized into four groups according to their body mass index (BMI) and GDM status: normal weight as control, obese (BMI > 30 kg/m²), normal weight with GDM, and obese with GDM. The released N-glycan components of IgG were separated with capillary electrophoresis and detected using a laser-induced fluorescence detector. Results: The result revealed several differences between the N-glycosylation patterns of the four study groups. Of this, 17 of the 20 identified structures differed significantly between the groups. The ratios of sialylated to non-sialylated structures were not changed significantly, but the core fucosylation level showed a significant decrease in the GDM and obese GDM groups compared to the control subjects. The lowest degree of core fucosylation was observed in the GDM group. Conclusions: The findings indicate that obesity in isolation does not have a significant impact on the IgG N-glycosylation pattern in pregnancy. Conversely, alterations in the N-glycan profile of antibodies may serve as biomarkers for the diagnosis of GDM in mothers with a normal BMI, although more evidence is needed. By incorporating glycan-based biomarkers into clinical practice, healthcare providers can improve early detection, personalize management strategies, and potentially mitigate adverse pregnancy outcomes associated with obesity and GDM. Full article

The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected. Full article

Background: Subarachnoid hemorrhage is a serious condition caused by ruptured intracranial aneurysms, resulting in severe disability mainly in young adults. Cerebral vasospasm is one of the most common complication of subarachnoid hemorrhage; thus, active prevention is key to improve the prognosis. The glycosylation of proteins is a critical quality attribute which is reportedly altered in patients diagnosed with acute ischemic stroke. In this study, we examined the N-glycosylation profile of serum glycoproteins in patients with subarachnoid hemorrhage without vasospasm compared to patients with vasospasm. Methods: The serum N-glycans were released by PNGase F (Peptide: N-glycosidase F) digestion and subsequently labeled by procainamide via reductive amination. The samples were analyzed by hydrophilic-interaction liquid chromatography after solid-phase extraction-based sample purification. Results: Besides the glycosylation pattern, we also investigated the biomarkers following subarachnoid hemorrhage. Multiple statistical analyses were performed in order to find significant differences and identify potential prediction factors of cerebral vasospasm. Significant differences were identified such as higher sialylation on bi-, tri-, and tetra-antennary structures in patients with subarachnoid hemorrhage and cerebral vasospasm. Conclusions: Our results suggest that glycosylation analysis can improve the identification of patients with cerebral vasospasm in combination with laboratory parameters. Full article

N-glycome analysis of individual proteins and tissues is crucial for fundamental and applied biomedical research and medical diagnosis and plays an important role in the evaluation of the quality of biopharmaceutical and biotechnological products. The interest in this research area continues to grow annually, thereby increasing the demand for the high-throughput profiling of human blood plasma N-glycome. In response to this need, we have developed an optimized, simple, and rapid protocol for the N-glycome profiling of human plasma proteins. This protocol encompasses the entire analysis cycle, from plasma isolation to N-glycan spectrum quantification. While the proposed method may have lower efficiency compared to already published high-throughput methods, its adaptability makes it suitable for implementation in virtually any molecular biological laboratory. Full article

Aberrant protein glycosylation is a hallmark alteration of cancer and is highly associated with cancer progression. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, but the N-glycosylation of its glycoproteins has not been well characterized. In this work, we analyzed multiple freshly prepared PTC specimens along with paired normal tissue obtained from thyroidectomies. Glycomic analyses focused on Asn-linked (N)-glycans and employed mass spectrometry (MS), along with Western blot approaches of total solubilized materials that were examined for binding by specific lectins and a monoclonal antibody (mAb) O6, specific for 3-O-sulfated galactose residues. We observed major differences in PTC versus paired normal specimens, as PTC specimens exhibited higher levels of N-glycan branching and bisection with N-acetylglucosamine residues, consistent with RNAseq data. We also found that 3-O-sulfated galactose was present in N-glycans of multiple glycoproteins from both PTC and control specimens, as recognized by the O6 mAb and as confirmed by MS analyses. These results provide new insights into the N-glycans present in glycoproteins of thyroid cancer and context for further studies of these altered glycans as biomarkers and targets for therapeutics. Full article