N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N-glycopeptides in seminal plasma, we identified 92 intact N-glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility.

Gestational diabetes mellitus (GDM) has negative effects on mothers and offspring, which may be related to the glycosylation level of milk proteins. Here, the human milk N/O-glycome of healthy and GDM individuals was analyzed by HILIC-MS/MS. A total of 56 putative N-glycans were detected, among which 12 N-glycans were significantly different between GDM and healthy milk. A total of 25 putative O-glycans were detected, and 11 of them varied greatly between GDM and healthy milk, especially H1N1S1 and H2N2S1. Overall, the relative content of N/O-glycans in GDM milk was significantly lower than that of healthy milk. In GDM milk, fucosylated N-glycans present higher proportion, whereas the proportion of sialylated O-glycans were lower. These findings would provide a foundation for in-depth study on the structure-activity relationship of milk N/O-glycans and are expected to drive the design of infant formula for newborns.

Food allergy is a serious health problem affecting more than 10% of the human population worldwide. Medical treatments for food allergy remain limited because immune therapy is risky and costly, and anti-allergic drugs have many harmful side effects and can cause drug dependence. In this paper, we review natural bioactive substances capable of alleviating food allergy. The sources of the anti-allergic substances reviewed include plants, animals, and microbes, and the types of substances include polysaccharides, oligosaccharides, polyphenols, phycocyanin, polyunsaturated fatty acids, flavonoids, terpenoids, quinones, alkaloids, phenylpropanoids, and probiotics. We describe five mechanisms involved in anti-allergic activities, including binding with epitopes located in allergens, affecting the gut microbiota, influencing intestinal epithelial cells, altering antigen presentation and T cell differentiation, and inhibiting the degranulation of effector cells. In the discussion, we present the limitations of existing researches as well as promising advances in the development of anti-allergic foods and/or immunomodulating food ingredients that can effectively prevent or alleviate food allergy. This review provides a reference for further research on anti-allergic materials and their hyposensitizing mechanisms.

β-Conglycinin is one of the major allergens existed in soybean. N-Glycans attached to the β-conglycinin influenced the immunoreactivity and antigen presenting efficiency of β-conglycinin. In this study, we described a new method used to release and collect the N-glycans from β-conglycinin, and the N-glycans existed in linear epitopes of β-conglycinin were identified. Glycopeptides hydrolyzed from β-conglycinin were purified by cotton hydrophilic chromatography. Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides, and new glycopeptides containing one single N-acetyl-D-glucosamine (GlcNAc) moiety were then utilized for mass spectrometry. Five glycosylation sites (Asn-199, Asn-455, Asn-215, Asn-489 and Asn-326) and 22 kinds of glycopeptides were identified. It is noteworthy that the peptide VVN#ATSNL (where # represents for the glycosylation site) was analyzed to be both glycopeptide and linear epitope. Our results provided a new method for the N-glycoform analysis of food allergens, and laid a foundation for understanding the relationship between glycosylation and food allergy.

Normal liquefaction of semen is one of the key steps to ensure the smooth progress of fertilization, and glycosylation has been reported to be involved in the whole process of fertilization. Till now, it is still unclear whether and how glycosylation changes during the liquefaction process of semen. In this study, by performing a glycoproteomic analysis of human semen with the liquefaction process (liquefaction time of semen: 0 min vs 30 min) using our recently developed StrucGP software combined with the Tandem Mass Tags (TMT) based quantification, we identified 25 intact glycopeptides (IGPs) from 10 glycoproteins in semen that were significantly changed during liquefaction, including 23 up-regulated and two down-regulated. Among the 23 up-regulated glycopeptides, half were modified with sialylated glycans, suggesting that sialylated glycans may play a key role in the semen liquefaction process. The data provide an invaluable resource for further studies on the role of glycosylation during semen liquefaction.

Previous research studies have shown that sulfated polysaccharides can inhibit food allergy, but the detailed mechanism remains largely unknown. In this study, RBL-2H3 cells were used to compare the anti-allergic activities of four sulfated polysaccharides, and an ovalbumin (OVA)-sensitized allergic mouse experiment was used to explore their desensitization effect, with regard to the alteration in gut microbiota and immune cell differentiation. Compared with the shark, bovine and porcine chondroitin sulfate, sea cucumber chondroitin sulfate (SCCS) significantly inhibited the degranulation of RBL-2H3 cells. SCCS reduced allergic symptoms and protected the jejunum from injury in mice. Furthermore, SCCS increased the relative abundance of Lachnospiraceae NK4A136, decreased the relative proportion of Prevotellaceae NK3B31, and up-regulated the secretion of short chain fatty acids such as butyric acid in the feces, resulting in an increase in the mucin 2 (MUC2) secretion by goblet cells HT-29. Meanwhile, SCCS induced the differentiation of regulatory T cells in the mesenteric lymph nodes of mice. This study provides a deeper understanding of the functioning mechanism of SCCS in alleviating food allergy and may guide the development and production of anti-allergy active ingredients.

Extensive cross-allergic reactivity exists among food-borne plants. As most allergens are glycoproteins, protein sensitization has been extensively studied. However, little attention has been given to the glycan component of glycoproteins. Here, we qualitatively and quantitatively compared the N-glycans of eight cross-reactive plant glycoproteins derived from peach pollen, soy, pine nut, cashew nut, pistachio, walnut, almond, and hazelnut. Cross-reactive carbohydrate determinants (CCDs) were widely present in the allergic glycoproteins according to the following order: pollen > soybean > pistachios > pine nuts > walnuts > cashews > hazelnuts > almonds. The N-glycan structure presented clear differences between the germplasm and vegetative tissue. Importantly, fucosylation and xylosylation levels correlated positively with extensive cross-reactivity between plants, which suggests that CCDs may be one of the causes of cross-allergy. This result provides the foundation for mechanistic studies on plant-derived CCDs as a source of food cross-allergies.

N-Linked glycoproteins are rich in seminal plasma, playing various essential roles in supporting sperm function and the fertilization process. However, the detailed information on these glycoproteins, particularly site-specific glycan structures, is still limited. In this study, a precision site-specific N-glycoproteome map of human seminal plasma was established by employing the site-specific glycoproteomic approach and a recently developed glycan structure interpretation software, StrucGP. A total of 9567 unique glycopeptides identified in human seminal plasma were composed of 773 N-linked glycan structures and 1019 N-glycosites from 620 glycoproteins. These glycans were comprised of four types of core structures and 13 branch structures. The majority of identified glycoproteins functioned in response to stimulus and immunity. As we reported in human spermatozoa, heavy fucosylation (fucose residues ≥6 per glycan) was also detected on seminal plasma glycoproteins such as clusterin and galectin-3-binding protein, which were involved in the immune response of biological processes and reactome pathways. Comparison of site-specific glycans between seminal plasma and spermatozoa revealed more complicated glycan structures in seminal plasma than in spermatozoa, even on their shared glycoproteins. These present data will be greatly beneficial for the in-depth structural and functional study of glycosylation in the male reproduction system.

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.

Human milk oligosaccharides (HMOs) are the second most abundant carbohydrates in colostrum. In this study, we performed a quantitative analysis of 13 oligosaccharides in 99 colostrum samples obtained from mothers living in Northwest China. The analysis combined liquid chromatography-mass spectrometry (LC-MS) with 2-amino-N-(2-aminoethyl)benzamide (AEAB) labeling and nonsecretors accounted for 17%. Compared with healthy secretor mothers, those with gestational diabetes mellitus presented lower levels of sialylated oligosaccharides, especially 3'-sialyllactose. Colostrum from mothers with pregnancy-induced hypertension had higher levels of fucosylated oligosaccharides, but the difference was not significant, and hypothyroidism appeared to have no effect on HMOs. Most HMOs (especially 6'-sialyllactose) were more abundant in colostrum from mothers who underwent vaginal delivery than a C-section. These findings show that the concentration of total or individual HMOs is affected by multiple factors. These findings provide a reference for evaluating variations in HMO expression among different populations and potential guidance for providing personalized clinical nutrition.

Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80

Intrahepatic cholangiocarcinoma (ICC) is the second major subtype of primary liver cancer and has caused more and more attention with increasing incidence and mortality worldwide. Our previous study found that bisecting N-glycans are commonly increased in ICC, while the effects and potential functions of bisecting GlcNAc in ICC are still largely unclear. In this study, we further confirmed that the structures of bisecting GlcNAc were significantly up-regulated in ICC compared with paracancer tissues by glycoproteomic data and lectin histochemistry. The expression of its glycosyltransferase MGAT3 was also up-regulated in ICC tissues at both mRNA and protein levels, and expression of MGAT3 is negatively correlated with overall survival explored by bioinformatic analyses and published datasets from 255 patients. Next, the silencing of MGAT3 could inhibit the growth and invasion of ICC cells, and overexpressing of MGAT3 only promoted ICC cell invasion. Further glycoproteomic analysis showed that the commonly glycoproteins modified by bisecting GlcNAc after MGAT3-overexpression in two ICC cell lines were mainly involved in cell movement-related biological processes, such as cell adhesion, integrin-related and ECM-receptor interaction. This study sheds light on the potential effects of bisecting GlcNAc in ICC cells and suggests that MGAT3 might be used as a potential target in the therapy of ICC.

The study of carbohydrates requires large amounts of glycans. N-Glycans can be synthesized but generating large quantities of N-glycans with diverse structures remains difficult. In this study, we aimed to obtain large amounts of glycans using an optimized procedure. Two types of reductive N-glycans were released from chicken egg albumin (ovalbumin) and soy protein using an ammonia catalysis method and labeled with benzenesulfonyl hydrazide (BSH). After preliminary separation by preparative HPLC, N-glycan-BSH components were de-labeled separately and reducing N-glycans were recovered. The de-labeled reducing N-glycans were derived with different labeling reagents and further separated and purified with two/multi-dimensional HPLC for various studies. We selected the bifunctional reagent 2-amino-N-(2-aminoethyl)-benzamide (AEAB) as a labeling reagent combined with C18 column for two-dimensional HPLC separation. A total of 21 and 8 N-glycan-AEAB conjugates were obtained from ovalbumin and soy protein, respectively. A reactive primary alkylamine of N-glycan-AEAB conjugates can be effectively immobilized on microarray surfaces, allowing for subsequent functional studies of glycans.

•Glycoqueuing strategy was used to quantify sialylated whey N-glycans in human milk.•23 α2,6-linked sialylated N-glycan isomers were detected; 72% were fucosylated.•3 mono- and 4 bi-sialylated glycan isomers were first identified in human milk.•Sialylated whey N-glycan content was 86.43% lower in mature than in colostrum milk.

The peptide portion of the sialylglycopeptide (SGP) in egg yolk is composed of six amino acids (KVANKT), among which asparagine (N) is modified by a complex sialylated N-glycan. Sialylglycopeptides are mainly obtained from egg yolk through a cumbersome, expensive, and poorly scalable process. In this study, a simple and low-cost method for the separation and purification of sialylglycopeptide from egg yolk was developed based on hydrophilic interaction chromatography with medical absorbent cotton as stationary phase. Fifty egg yolks were treated with phenol to obtain the crude sialylglycopeptide. The latter was then formulated into a 150 mg/mL solution and loaded on a cotton hydrophilic chromatographic column, followed by treatment with 100%, 95%, 85% and 75% acetonitrile (ACN) aqueous solution to remove impurities. Finally, deionized water was applied for the final elution step. By this way, 300 mg of sialylglycopeptide was yielded and its purity was determined by high-performance liquid chromatography to be 95%. The glycan composition and glycosylation site of the sialylglycopeptide were validated by electrospray ionization mass spectrometry and tandem mass spectrometry.

The glycoproteins in Ginkgo seeds were separated and isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The N-glycans released from glycoproteins were qualitatively and quantitatively analyzed by electrospray ionization mass spectrometry (ESI-MS) and on-line liquid chromatography-mass spectrometry(LC-UV-MS/MS2). As a result, eleven glycoproteins containing oligomannose type glycans(4. 88%) and complex type glycans (95. 12%) were obtained. And most of the N-glycans were modified by the core alpha-1, 3- and beta-1, 2-xylose in the 21000, 36000 and 50000 glycoproteins, which accounted for 68. 23% , 64. 37% and 75. 09% , respectively. This study can highlight the understanding of protein glycosylation in Ginkgo and provide foundation in the research of Ginkgo biloba seed allergens.

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.