Search Results (104)

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

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

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

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

The hypoxic tumor microenvironment significantly impacts cellular behavior and intercellular communication, with extracellular vesicles (EVs) playing a crucial role in promoting angiogenesis, metastasis, and host immunosuppression, and presumed cancer progression and metastasis are closely associated with the aberrant surface N-glycan expression in EVs. We hypothesize that hypoxic tumors synthesize specific hypoxia-induced N-glycans in response to or as a consequence of hypoxia. This study utilized nano-LC–MS/MS to integrate quantitative proteomic and N-glycomic analyses of both cells and EVs derived from the MDA-MB-231 breast cancer cell line cultured under normoxic and hypoxic conditions. Whole N-glycome and proteome profiling revealed that hypoxia has an impact on the asparagine N-linked glycosylation patterns and on the glycolysis/gluconeogenesis proteins in cells in terms of altered N-glycosylation for their adaptation to low-oxygen conditions. Distinct N-glycan types, high-mannose glycans like Man3 and Man9, were highly abundant in the hypoxic cells. On the other hand, alterations in the sialylation and fucosylation patterns were observed in the hypoxic cells. Furthermore, hypoxia-induced EVs exhibit a signature consisting of mono-antennary structures and specific N-glycans (H4N3F1S2, H3N3F1S0, and H7N4F3S2; H8N4F1S0 and H8N6F1S2), which are significantly associated with poor prognoses for breast tumors, presumably altering the interactions within the tumor microenvironment to promote tumorigenesis and metastasis. Our findings provide an overview of the N-glycan profiles, particularly under hypoxic conditions, and offer insights into the potential biomarkers for tracking tumor microenvironment dynamics and for developing precision medicine approaches in oncology. Full article

In immunoglobulin G (IgG), N-glycosylation plays a pivotal role in structure and function. It is often altered in different diseases, suggesting that it could be a promising health biomarker. Studies indicate that IgG glycosylation not only associates with various diseases but also has predictive capabilities. Additionally, changes in IgG glycosylation correlate with physiological and biochemical traits known to reflect overall health state. This study aimed to investigate the power of IgG glycans to predict physiological and biochemical parameters. We developed two models using IgG N-glycan data as an input: a regression model using elastic net and a machine learning model using deep learning. Data were obtained from the Korčula and Vis cohorts. The Korčula cohort data were used to train both models, while the Vis cohort was used exclusively for validation. Our results demonstrated that IgG glycome composition effectively predicts several biochemical and physiological parameters, especially those related to lipid and glucose metabolism and cardiovascular events. Both models performed similarly on the Korčula cohort; however, the deep learning model showed a higher potential for generalization when validated on the Vis cohort. This study reinforces the idea that IgG glycosylation reflects individuals’ health state and brings us one step closer to implementing glycan-based diagnostics in personalized medicine. Additionally, it shows that the predictive power of IgG glycans can be used for imputing missing covariate data in deep learning frameworks. Full article

Glyphosate, the active ingredient in many herbicides, has been widely used in agriculture since the 1970s. Despite initial beliefs in its safety for humans and animals due to the absence of the shikimate pathway, recent studies have raised concerns about its potential health effects. This study aimed to identify glycomic changes indicative of glyphosate-induced toxicity. Specifically, the study focused on profiling N-glycosylation, a protein post-translational modification increasingly recognized for its involvement in various disorders, including neurological conditions. A comprehensive analysis of rat serum N-glycomics following chronic exposure to glyphosate-based herbicides (GBH) was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results revealed significant changes in the N-glycan profile, particularly in sialylated and sialofucosylated N-glycans. The analysis of N-glycans across gender subgroups provided insights into gender-specific responses to GBH exposure, with the male rats exhibiting a higher susceptibility to these N-glycan changes compared to females. The validation of significantly altered N-glycans using parallel reaction monitoring (PRM) confirmed their expression patterns. This study provides novel insights into the impact of chronic GBH exposure on serum N-glycan composition, with implications for assessing glyphosate toxicity and its potential neurological implications. Full article

The attachment of sugar to proteins and lipids is a basic modification needed for organismal survival, and perturbations in glycosylation cause severe developmental and neurological difficulties. Here, we investigated the neurological consequences of N-glycan populations in the spinal cord of Wt AB and mgat1b mutant zebrafish. Mutant fish have reduced N-acetylglucosaminyltransferase-I (GnT-I) activity as mgat1a remains intact. GnT-I converts oligomannose N-glycans to hybrid N-glycans, which is needed for complex N-glycan production. MALDI-TOF MS profiles identified N-glycans in the spinal cord for the first time and revealed reduced amounts of complex N-glycans in mutant fish, supporting a lesion in mgat1b. Further lectin blotting showed that oligomannose N-glycans were more prevalent in the spinal cord, skeletal muscle, heart, swim bladder, skin, and testis in mutant fish relative to WT AB, supporting lowered GnT- I activity in a global manner. Developmental delays were noted in hatching and in the swim bladder. Microscopic images of caudal primary (CaP) motor neurons of the spinal cord transiently expressing EGFP in mutant fish were abnormal with significant reductions in collateral branches. Further motor coordination skills were impaired in mutant fish. We conclude that identifying the neurological consequences of aberrant N-glycan processing will enhance our understanding of the role of complex N-glycans in development and nervous system health. Full article

The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as abdominal pain, ultrasonography and altered laboratory parameters (C reactive protein, absolute neutrophil cell number and white blood cell number). The glycosylation pattern of serum N-glycome was analyzed in this study of 38 controls and 40 patients with pediatric appendicitis. The glycans were released by enzymatic deglycosylation followed by fluorescent labeling and solid-phase extraction. The prepared samples were analyzed by hydrophilic interaction liquid chromatography with fluorescence and mass-spectrometric detection. The generated data were analyzed by multiple statistical tests involving the most important laboratory parameters as well. Significant differences associated with the examined patient groups were revealed suggesting the potential use of glycosylation analysis supporting the detection of pediatric appendicitis. Full article

Glycans of MVs are proposed to be candidates for mediating targeting specificity or at least promoting it. In contrast to exosomes, glycomic studies of MVs are largely absent. We studied the glycoprofile of endothelial cell-derived MVs using 21 plant lectins, and the results show the dominance of oligolactosamines and their α2-6-sialylated forms as N-glycans and low levels of α2-3-sialylated glycans. The low levels of α2-3-sialosides could not be explained by the action of extracellular glycosidases. Additionally, the level of some Man-containing glycans was also decreased in MVs. Spatial masking as the causative relationship between these low level glycans (as glycosphingolipids) by integral proteins or proteoglycans (thus, their lack of interaction with lectins) seems unlikely. The results suggest that integral proteins do not pass randomly into MVs, but instead only some types, differing in terms of their specific glycosylation, are integrated into MVs. Full article