Deep analysis of total serum N-glycome suggests glyco-signatures for phospholipase A2 receptor 1-related idiopathic membranous nephropathy diagnosis.

Idiopathic membranous nephropathy (IMN) is an antibody-mediated and kidney-specific autoimmune disease, with the antigen phospholipase A2 receptor 1 (PLA2R1) accounting for approximately 70% of IMN cases. Although a variety of new podocyte target antigens and their autoantibodies have been identified, they are still of limited diagnostic and therapeutic value due to lack of high specificity and sensitivity. N-glycans play vital roles in renal system and their pathobiological relevance has become increasingly recognized in many kidney diseases, but not fully explored in IMN. To find possible glyco-signatures for PLA2R1-related IMN diagnosis, we herein established a comprehensive workflow for total serum N-glycome analysis based on our recently developed mass spectrometry (MS)-based N-glycan purification method, named Ultrafast Glycoprotein Immobilization for Glycan extraction (UltraGIG). A total of 191 N-glycans were identified from IMN patients, representing the largest N-glycome dataset in IMN. Compared to healthy controls, up-regulation of sialylation and core-fucosylation as well as down-regulation of galactosylation were observed in PLA2R1-positive IMN patients, and up-regulation of hyper-galactosylation was specific for PLA2R1-negative IMN patients. A six-glycan marker panel consisting of H4N3S1, H4N3F1, H6N4S2, H6H5F1S2, H6N5 and H6N6F1S1, was proposed to aid in the accurate diagnosis of PLA2R1-related IMN, which provided new insights into IMN biomarker study. SIGNIFICANCE: PLA2R1-related IMN is a kidney-specific autoimmune disease with a high risk of developing end-stage renal disease (ESRD) and even kidney failure. Current biomarkers are still of limited diagnostic and therapeutic value due to lack of high specificity and sensitivity. An in-depth MS analysis of total serum N-glycome of PLA2R1-related IMN patients was conducted for the first time. We generated the largest dataset of serum N-glycome for IMN to date, and proposed a novel six-glycan marker panel that may help the accurate diagnosis of PLA2R1-related IMN.

Influence of plasma collection tubes on N-glycome in human blood samples.

Background and aims: Quantitative analysis of plasma N-glycome is a promising method for identifying disease biomarkers. This study aimed to investigate the impact of using blood collection tubes with different anticoagulants on plasma N-glycome. Materials and methods: We used a robust mass spectrometry method to profile plasma N-glycomes in two cohorts of healthy volunteers (cohort 1, n = 16; cohort 2, n = 53). The influence of three commonly used blood collection tubes on fully characterized N-glycomic profiles were explored. Results: Principal component analysis revealed distinct clustering of blood samples based on the collection tubes. Pairwise comparisons demonstrated significant differences between EDTA and heparin plasma in 55 out of 82 quantified N-glycan traits, and between EDTA and citrate plasma in 62 out of 82 traits. These differences encompassed various N-glycan features, including glycan type, sialylation, galactosylation, fucosylation, and bisection. Trends in N-glycan variations in citrate and heparin plasma were largely consistent compared to EDTA plasma. In correlation analysis (EDTA vs. heparin; EDTA vs. citrate), Pearson's correlation coefficients were consistently higher than 0.7 for the majority of N-glycan traits. Conclusion: Sample matrix variations impact plasma N-glycome measurements. Caution is crucial when comparing samples from different plasma collection tubes in glycomics projects.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

Noninvasive serum N-glycans associated with ovarian cancer diagnosis and precancerous lesion prediction.

BACKGROUND: Ovarian cancer (OC) is one of the most common gynecological tumors with high morbidity and mortality. Altered serum N-glycome has been observed in many diseases, while the association between serum protein N-glycosylation and OC progression remains unclear, particularly for the onset of carcinogenesis from benign neoplasms to cancer. METHODS: Herein, a mass spectrometry based high-throughput technique was applied to characterize serum N-glycome profile in individuals with healthy controls, benign neoplasms and different stages of OC. To elucidate the alterations of glycan features in OC progression, an orthogonal strategy with lectin-based ELISA was performed. RESULTS: It was observed that the initiation and development of OC was associated with increased high-mannosylationand agalactosylation, concurrently with decreased total sialylation of serum, each of which gained at least moderately accurate merits. The most important individual N-glycans in each glycan group was H7N2, H3N5 and H5N4S2F1, respectively. Notably, serum N-glycome could be used to accurately discriminate OC patients from benign cohorts, with a comparable or even higher diagnostic score compared to CA125 and HE4. Furthermore, bioinformatics analysis based discriminative model verified the diagnostic performance of serum N-glycome for OC in two independent sets. CONCLUSIONS: These findings demonstrated the great potential of serum N-glycome for OC diagnosis and precancerous lesion prediction, paving a new way for OC screening and monitoring.

Plasma protein N-glycome composition associates with postprandial lipaemic response.

BACKGROUND: A dysregulated postprandial metabolic response is a risk factor for chronic diseases, including type 2 diabetes mellitus (T2DM). The plasma protein N-glycome is implicated in both lipid metabolism and T2DM risk. Hence, we first investigate the relationship between the N-glycome and postprandial metabolism and then explore the mediatory role of the plasma N-glycome in the relationship between postprandial lipaemia and T2DM. METHODS: We included 995 individuals from the ZOE-PREDICT 1 study with plasma N-glycans measured by ultra-performance liquid chromatography at fasting and triglyceride, insulin, and glucose levels measured at fasting and following a mixed-meal challenge. Linear mixed models were used to investigate the associations between plasma protein N-glycosylation and metabolic response (fasting, postprandial (Cmax), or change from fasting). A mediation analysis was used to further explore the relationship of the N-glycome in the prediabetes (HbA1c = 39-47 mmol/mol (5.7-6.5%))-postprandial lipaemia association. RESULTS: We identified 36 out of 55 glycans significantly associated with postprandial triglycerides (Cmax ß ranging from -0.28 for low-branched glycans to 0.30 for GP26) after adjusting for covariates and multiple testing (padjusted < 0.05). N-glycome composition explained 12.6% of the variance in postprandial triglycerides not already explained by traditional risk factors. Twenty-seven glycans were also associated with postprandial glucose and 12 with postprandial insulin. Additionally, 3 of the postprandial triglyceride-associated glycans (GP9, GP11, and GP32) also correlate with prediabetes and partially mediate the relationship between prediabetes and postprandial triglycerides. CONCLUSIONS: This study provides a comprehensive overview of the interconnections between plasma protein N-glycosylation and postprandial responses, demonstrating the incremental predictive benefit of N-glycans. We also suggest a considerable proportion of the effect of prediabetes on postprandial triglycerides is mediated by some plasma N-glycans.

Comprehensive review of MS-based studies on N-glycoproteome and N-glycome of extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles that can be released by all type of cells. Whereas, as one of the most common post-translational modifications, glycosylation plays a vital role in various biological functions of EVs, such as EV biogenesis, sorting, and cellular recognition. Nevertheless, compared with studies on RNAs or proteins, those investigating the glycoconjugates of EVs are limited. An in-depth investigation of N-glycosylation of EVs can improve the understanding of the biological functions of EVs and help to exploit EVs from different perspectives. The general focus of studies on glycosylation of EVs primarily includes isolation and characterization of EVs, preparation of glycoproteome/glycome samples and MS analysis. However, the low content of EVs and non-standard separation methods for downstream analysis are the main limitations of these studies. In this review, we highlight the importance of glycopeptide/glycan enrichment and derivatization owing to the low abundance of glycoproteins and the low ionization efficiency of glycans. Diverse fragmentation patterns and professional analytical software are indispensable for analysing glycosylation via MS. Altogether, this review summarises recent studies on glycosylation of EVs, revealing the role of EVs in disease progression and their remarkable potential as biomarkers.

Serum protein N-glycome patterns reveal alterations associated with endometrial cancer and its phenotypes of differentiation.

Background: Aberrant N-glycosylation and its involvement in pathogenesis have been reported in endometrial cancer (EC). Nevertheless, the serum N-glycomic signature of EC remains unknown. Here, we investigated serum N-glycome patterns of EC to identify candidate biomarkers. Methods: This study enrolled 34 untreated EC patients and 34 matched healthy controls (HC) from Peking Union Medical College Hospital. State-of-the-art MS-based methods were employed for N-glycans profiling. Multivariate and univariate statistical analyses were used to identify discriminative N-glycans driving classification. Receiver operating characteristic analyses were performed to evaluate classification accuracy. Results: EC patients displayed distinct differences in serum N-glycome and had abnormal high-mannose and hybrid-type N-glycans, fucosylation, galactosylation, and linkage-specific sialylation compared with HC. The glycan panel built with the four most discriminative and biologically important derived N-glycan traits could accurately identify EC (random forest model, the area under the curve [AUC]=0.993 [95%CI 0.955-1]). The performance was validated by two other models. Total hybrid-type N-glycans significantly associated with the differentiation types of EC could effectively stratify EC into well- or poorly-differentiated subgroups (AUC>0.8). Conclusion: This study provides the initial evidence supporting the utility of serum N-glycomic signature as potential markers for the diagnosis and phenotyping of EC.

Not-So-Sweet Dreams: Plasma and IgG N-Glycome in the Severe Form of the Obstructive Sleep Apnea.

Obstructive sleep apnea (OSA) is a prevalent disease associated with increased risk for cardiovascular and metabolic diseases and shortened lifespan. The aim of this study was to explore the possibility of using N-glycome as a biomarker for the severe form of OSA. Seventy subjects who underwent a whole-night polysomnography/polygraphy and had apnea-hypopnea index (AHI) over 30 were compared to 23 controls (AHI under 5). Plasma samples were used to extract 39 glycan peaks using ultra-high-performance liquid chromatography (UPLC) and 27 IgG peaks using capillary gel electrophoresis (CGE). We also measured glycan age, a molecular proxy for biological aging. Three plasma and one IgG peaks were significant in a multivariate model controlling for the effects of age, sex, and body mass index. These included decreased GP24 (disialylated triantennary glycans as major structure) and GP28 (trigalactosylated, triantennary, disialylated, and trisialylated glycans), and increased GP32 (trisialylated triantennary glycan). Only one IgG glycan peak was significantly increased (P26), which contains biantennary digalactosylated glycans with core fucose. Patients with severe OSA exhibited accelerated biological aging, with a median of 6.9 years more than their chronological age (p < 0.001). Plasma N-glycome can be used as a biomarker for severe OSA.

IgG glycans in health and disease: Prediction, intervention, prognosis, and therapy.

Immunoglobulin (IgG) glycosylation is a complex enzymatically controlled process, essential for the structure and function of IgG. IgG glycome is relatively stable in the state of homeostasis, yet its alterations have been associated with aging, pollution and toxic exposure, as well as various diseases, including autoimmune and inflammatory diseases, cardiometabolic diseases, infectious diseases and cancer. IgG is also an effector molecule directly involved in the inflammation processes included in the pathogenesis of many diseases. Numerous recently published studies support the idea that IgG N-glycosylation fine-tunes the immune response and plays a significant role in chronic inflammation. This makes it a promising novel biomarker of biological age, and a prognostic, diagnostic and treatment evaluation tool. Here we provide an overview of the current state of knowledge regarding the IgG glycosylation in health and disease, and its potential applications in pro-active prevention and monitoring of various health interventions.

Age-Related Changes in Serum N-Glycome in Men and Women-Clusters Associated with Comorbidity.

(1) Aim: To describe, in a general adult population, the serum N-glycome in relation to age in men and women, and investigate the association of N-glycome patterns with age-related comorbidity; (2) Methods: The serum N-glycome was studied by hydrophilic interaction chromatography with ultra-performance liquid chromatography in 1516 randomly selected adults (55.3% women; age range 18-91 years). Covariates included lifestyle factors, metabolic disorders, inflammatory markers, and an index of comorbidity. Principal component analysis was used to define clusters of individuals based on the 46 glycan peaks obtained in chromatograms; (3) Results: The serum N-glycome changed with ageing, with significant differences between men and women, both in individual N-glycan peaks and in groups defined by common features (branching, galactosylation, sialylation, fucosylation, and oligomannose). Through K-means clustering algorithm, the individuals were grouped into a cluster characterized by abundance of simpler N-glycans and a cluster characterized by abundance of higher-order N-glycans. The individuals of the first cluster were older, showed higher concentrations of glucose and glycation markers, higher levels of some inflammatory markers, lower glomerular filtration rate, and greater comorbidity index; (4) Conclusions: The serum N-glycome changes with ageing with sex dimorphism. The N-glycome could be, in line with the inflammaging hypothesis, a marker of unhealthy aging.

The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis.

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

In-depth profiling of urinary N-glycome in diabetic kidney disease by ultrafast glycoprotein immobilization for glycan extraction (UltraGIG).

Diabetic kidney disease (DKD) is a common and serious kidney-related complication of diabetic mellitus. Although albuminuria and estimated glomerular filtration rate (eGFR) are commonly used diagnostic biomarkers, they have limitations in the diagnosis of DKD due to the lack of specificity and sensitivity. Urinary N-glycans are emerging as novel biomarkers for predicting renal prognosis in DKD. However, most of the current N-glycan profiling methods for DKD were based on lectin affinity enrichment or hydrophilic interaction chromatography (HILIC) with optical detection, which provided limited N-glycome coverage in low throughput. Herein, we developed a novel N-glycan purification method, termed Ultrafast Glycoprotein Immobilization for Glycan extraction (UltraGIG), for in-depth profiling of urinary N-glycome in DKD using mass spectrometry (MS). In UltraGIG, proteins were rapidly (within 40 min) immobilized to resin with high efficiency (over 98%) through NHS-reactive chemistry, and then N-glycans were enzymatically released from the resin by PNGase F. Owing to good efficiency of immobilization of proteins to resin, the subsequent washing steps to remove proteins and other impurities could be easily performed. The UltraGIG showed good selectivity towards glycans (extracting N-glycans from the mixture of IgG and BSA at a 1:100 M ratio) and samples loss was minimized (detection sensitivity at fmol level). A total of 237 N-glycan compositions were identified from urine samples with DKD and healthy controls, which representing the largest data set of N-glycome from DKD. Compared to healthy controls, 3 N-glycans were up-regulated and 14 N-glycans were down-regulated in DKD patients. Collectively, UltraGIG offers a competitive sample purification method for in-depth analysis of urinary N-glycome by MS and provide new insights into biomarker study for the diagnosis of DKD.

IgG N-Glycosylation from Patients with Pemphigus Treated with Rituximab.

Pemphigus is a life-threatening auto-immune blistering disease of the skin and mucous membrane that is caused by the production of auto-antibodies (auto-Abs) directed against adhesion proteins: desmoglein 1 and 3. We demonstrated in the "Ritux3" trial, the high efficacy of rituximab, an anti-CD20 recombinant monoclonal antibody, as the first-line treatment for pemphigus. However, 25% of patients relapsed during the six-month period after rituximab treatment. These early relapses were associated with a lower decrease in anti-desmoglein auto-Abs after the initial cycle of rituximab. The N-glycosylation of immunoglobulin-G (IgG) can affect their affinity for Fc receptors and their serum half-life. We hypothesized that the extended half-life of Abs could be related to modifications of IgG N-glycans. The IgG N-glycome from pemphigus patients and its evolution under rituximab treatment were analyzed. Pemphigus patients presented a different IgG N-glycome than healthy donors, with less galactosylated, sialylated N-glycans, as well as a lower level of N-glycans bearing an additional N-acetylglucosamine. IgG N-glycome from patients who achieved clinical remission was not different to the one observed at baseline. Moreover, our study did not identify the N-glycans profile as discriminating between relapsing and non-relapsing patients. We report that pemphigus patients present a specific IgG N-glycome. The changes observed in these patients could be a biomarker of autoimmunity susceptibility rather than a sign of inflammation.

Association Between Human Gut Microbiome and N-Glycan Composition of Total Plasma Proteome.

Being one of the most dynamic entities in the human body, glycosylation of proteins fine-tunes the activity of the organismal machinery, including the immune system, and mediates the interaction with the human microbial consortium, typically represented by the gut microbiome. Using data from 194 healthy individuals, we conducted an associational study to uncover potential relations between the gut microbiome and the blood plasma N-glycome, including N-glycome of immunoglobulin G. While lacking strong linkages on the multivariate level, we were able to identify associations between alpha and beta microbiome diversity and the blood plasma N-glycome profile. Moreover, for two bacterial genera, namely, Bilophila and Clostridium innocuum, significant associations with specific glycans were also shown. The study's results suggest a non-trivial, possibly weak link between the total plasma N-glycome and the gut microbiome, predominantly involving glycans related to the immune system proteins, including immunoglobulin G. Further studies of glycans linked to microbiome-related proteins in well-selected patient groups are required to conclusively establish specific associations.

The dynamic brain N-glycome.

The attachment of carbohydrates to other macromolecules, such as proteins or lipids, is an important regulatory mechanism termed glycosylation. One subtype of protein glycosylation is asparagine-linked glycosylation (N-glycosylation) which plays a key role in the development and normal functioning of the vertebrate brain. To better understand the role of N-glycans in neurobiology, it's imperative we analyse not only the functional roles of individual structures, but also the collective impact of large-scale changes in the brain N-glycome. The systematic study of the brain N-glycome is still in its infancy and data are relatively scarce. Nevertheless, the prevailing view has been that the neuroglycome is inherently restricted with limited capacity for variation. The development of improved methods for N-glycomics analysis of brain tissue has facilitated comprehensive characterisation of the complete brain N-glycome under various experimental conditions on a larger scale. Consequently, accumulating data suggest that it's more dynamic than previously recognised and that, within a general framework, it has a given capacity to change in response to both intrinsic and extrinsic stimuli. Here, we provide an overview of the many factors that can alter the brain N-glycome, including neurodevelopment, ageing, diet, stress, neuroinflammation, injury, and disease. Given this emerging evidence, we propose that the neuroglycome has a hitherto underappreciated plasticity and we discuss the therapeutic implications of this regarding the possible reversal of pathological changes via interventions. We also briefly review the merits and limitations of N-glycomics as an analytical method before reflecting on some of the outstanding questions in the field.

Changes in Serum N-Glycome for Risk Drinkers: A Comparison with Standard Markers for Alcohol Abuse in Men and Women.

Background and aim: Glycomic alterations serve as biomarker tools for different diseases. The present study aims to evaluate the diagnostic capability of serum N-glycosylation to identify alcohol risk drinking in comparison with standard markers. Methods: We included 1516 adult individuals (age range 18-91 years; 55.3% women), randomly selected from a general population. A total of 143 (21.0%) men and 50 (5.9%) women were classified as risk drinkers after quantification of daily alcohol consumption and the Alcohol Use Disorders Identification Test (AUDIT). Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) was used for the quantification of 46 serum N-glycan peaks. Serum gamma-glutamyltransferase (GGT), carbohydrate-deficient transferrin (CDT), and red blood cell mean corpuscular volume (MCV) were measured by standard clinical laboratory methods. Results: Variations in serum N-glycome associated risk drinking were more prominent in men compared to women. A unique combination of N-glycan peaks selected by the selbal algorithm shows good discrimination between risk-drinkers and non-risk drinkers for men and women. Receiver operating characteristics (ROC) curves show accuracy for the diagnosis of risk drinking, which is comparable to that of the golden standards, GGT, MCV and CDT markers for men and women. Additionally, the inclusion of N-glycan peaks improves the diagnostic accuracy of the standard markers, although it remains relatively low, due to low sensitivity. For men, the area under the ROC curve using N-glycome data is 0.75, 0.76, and 0.77 when combined with GGT, MCV, and CDT, respectively. In women, the areas were 0.76, 0.73, and 0.73, respectively. Conclusion: Risk drinking is associated with significant variations in the serum N-glycome, which highlights its potential diagnostic utility.

Detailed Characterization of Antipathogenic Properties of Human Milk N-Glycome, against Staphylococcus aureus, Indicating Its Targeting on Cell Surface Proteins.

Human milk N-glycome was previously identified to have strong antipathogenic activities. This study is aimed to characterize the detailed antibacterial properties and the potential function mechanism of human milk N-glycome against Staphylococcus aureus. A serials of traditional antibacterial assays showed that human milk N-glycome possessed both bacteriostatic and bactericidal activities, which was further confirmed by the cell structure disruption including the change of transmembrane potential and leakage of intracellular contents. The results of the bacterial surface zeta potential and hydrophobicity, bacterial binding assay, gel shift assay, and fluorescence spectra and the different synergistic effects of human milk N-glycome combined with different antibiotics indicated that the bacterial surface proteins could be the targets of human milk N-glycome. Moreover, human milk N-glycome also showed antibiofilm activity. In conclusion, human milk N-glycome exhibited good potential for acting as an antibacterial substance against S. aureus and the antibacterial mechanism was a cell surface targeting action.

The mechanism of probiotic action of human milk N-glycome towards B. infantis ATCC 15697 and identification of the principal functional components.

Human milk N-glycome promotes the growth of Bifidobacterium longum subsp. infantis ATCC 15697. However, the action mode of, and the major functional components for, the bifidogenic function of human milk N-glycome remain unclear. In this study, we demonstrated that milk N-glycome was transferred in an intact form from culture into the bacterial cell and then decomposed intracellularly, evidenced by the following facts: (1) No UHPLC peak shift of N-glycome recovered from culture was observed. (2) No milk N-glycan specific monosugar was detected in culture supernatant. (3) High intracellular exoglycosidase activities were detected. (4) Fluorescently labeled N-glycans were found to be located intracellularly using Laser Scanning Confocal Microscopy (LSCM). Regarding the principal components identification, a novel sequential deglycosylation-based strategy was established. Degalactosylation, defucosylation-desialylation, and defucosylation-desialylation-degalactosylation treatments of human milk N-glycome showed that galactose-containing glycans were the principal components for the probiotic function of human milk N-glycome towards B. infantis ATCC 15697.

Validation of diagnostic and predictive biomarkers for hereditary angioedema via plasma N-glycomics.

BACKGROUND: Hereditary angioedema (HAE) is a rare disease with heterogeneous clinical symptoms. It is vitally important to predict whether an HAE patient will develop severe symptoms in clinical practice, but there are currently no predictive biomarkers for HAE stratification. Plasma N-glycomes are disease-specific and have great potential for the discovery of non-invasive biomarkers. In this study, we profiled the plasma N-glycome of HAE patients from two independent cohorts to identify candidate biomarkers. METHODS: Linkage-specific sialylation derivatization combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry detection and automated data processing was employed to analyze the plasma N-glycome of two independent type-1 HAE cohorts. RESULTS: HAE patients had abnormal glycan complexity, galactosylation, and α2,3- and α2,6-linked sialylation compared to healthy controls (HC). The classification models based on dysregulated glycan traits could successfully discriminate between HAE and HC with area under the curves (AUCs) being greater than 0.9. Some of the aberrant glycans showed response to therapy. Moreover, we identified a series of glycan traits with strong associations with the occurrence of laryngeal or gastrointestinal angioedema or disease severity score. Predictive models based on these traits could be used to predict disease severity (AUC > 0.9). These results were replicated in an independent cohort. CONCLUSIONS: We reported the full plasma N-glycomic signature of HAE for the first time, and identified potential biomarkers. These findings may play a critical role in predicting disease severity and guide the treatment of HAE in clinical practice. Further protein-specific and prospective studies are needed to validate our findings.

The Rapidly Expanding Nexus of Immunoglobulin G N-Glycomics, Suboptimal Health Status, and Precision Medicine.

Immunoglobulin G is a prevalent glycoprotein, whose downstream immune responses are partially mediated by the N-glycans within the fragment crystallisable domain. Collectively termed the N-glycome, it is considered a complex intermediate phenotype: an amalgamation of genetic predisposition, environmental exposure, and health behaviours over the life-course. Thus, the immunoglobulin G N-glycome may provide an indication of health status on the spectrum from health to disease and infirmary. Although variability exists within and between populations, composition of the immunoglobulin G N-glycome remains stable over short periods of time. This underscores the potential of harnessing the immunoglobulin G N-glycome as an ideal tool for preclinical disease risk prediction, stratification, and prognosis through the development of precise dynamic biomarkers.