Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.

Influence of centroid acceleration acquisition and filtering class on head injury criterion evaluation.

BACKGROUND: Although the Head Injury Criteria (HIC) has been widely applied to assess head impact injuries, it faces two outstanding problems: 1) HIC is affected strongly by the cut-off frequency when processing acceleration signals. And these cut-off frequencies are experiential and lack unified guidelines; 2) If the head was impacted on a different part, should the corresponding HIC threshold be the same? If these problems are not resolved, it could potentially lead to a critical misinterpretation of the safety assessment. METHODS: Finite element method was used to reconstruct head impacts. The head model includes tissues like skull, brainstem, cerebrospinal fluid, etc. The head model was impacted in the frontal, occipital, parietal or lateral direction with different impact velocities. Acceleration signals of the head model were extracted directly from the skull and the head centroid node. To obtain a robust HIC, the filtering class of acceleration signals were analyzed carefully. Then, the relation between rigid body HIC and the centroid node HIC were studied systematically. RESULTS: When the filtering class of rigid body acceleration and centroid node acceleration reached the cut-off frequency, the corresponding derivative of HIC tended to change smoothly. Using these cut-off frequencies, robust HICs were obtained. The rigid body HIC far exceeded that of centroid node HIC, such as 8, 9, 14 and 31 times exceeded in the frontal, occipital, parietal and lateral impact conditions, respectively. Moreover, approximate linear relations were found between the rigid body HIC and the centroid node HIC in different impact directions, respectively. From these relations, the injury thresholds of rigid body HIC of various directions were given quantitatively. CONCLUSIONS: The rational filtering class like CFC 800 and CFC 700 were given for rigid body HIC and centroid node HIC, respectively. The rigid body HIC had a significant discrepancy from the centroid node HIC. Linear relations between the rigid body HIC and centroid node HIC were found, and their slopes changed with impact directions. From these relations, we can adjust the injury thresholds reasonably if the head receives different impacts. These findings can effectively enhance the applicability of HIC.

Site-specific N-glycan changes during semen liquefaction.

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.

Maximal performance of intact N-glycopeptide enrichment using sequential HILIC and MAX columns.

Low abundance and heterogeneity of N-glycosylation at the peptide level poses a great challenge to the structural and functional analysis of glycosylation in the field of glycobiology. Solving this conundrum requires a sufficient and specific method for intact N-glycopeptide enrichment. Using the C18 or HLB desalting column followed by the mixed-mode strong anion exchange (MAX) or hydrophilic interaction chromatography (HILIC) glycopeptide enrichment column are commonly applied approaches for sample preparation of intact N-glycopeptides from complex samples. Herein, we compared the effects of different combinations of two desalting columns and two enrichment columns using equal amounts of mouse brain tissues from the same source. The results revealed the C18 column was a bit superior to the HLB column, and the MAX and HILIC columns were complementary on intact N-glycopeptides enrichment. Additionally, the results also demonstrated that enriching glycopeptides using a HILIC column followed by a MAX column from the flow-through solution got a better enrichment performance than the reversed order. Based on these results, the sequential enrichment of glycopeptides using HILIC and then MAX columns could maximize the enrichment performance of intact N-glycopeptides, and therefore is an option for in-depth analysis of site-specific glycoproteome.

Precision Characterization of Site-Specific O-Acetylated Sialic Acids on N-Glycoproteins.

O-Acetylation is a common modification of sialic acid, playing a significant role in glycoprotein stability, immune response, and cell development. Due to the lack of efficient methods for direct analysis of O-acetylated sialoglycopeptides (O-AcSGPs), the majority of identified O-acetylated sialic acids (O-AcSia) until now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of O-AcSGPs with probability estimation by recognizing the characteristic B and Y ions of O-AcSias. With further optimization of mass spectrometry parameters, the method allowed us to identify a total of 171 unique O-AcSGPs in mouse serum. Although the majority of these O-AcSGPs were at a relatively low abundance compared with their non-O-acetylated states, they were mainly involved in peptidase/endopeptidase inhibitor activities. The method paves the way for large-scale structural and functional analyses of site-specific O-AcSias in various complex samples as well as further identification of many other similar chemical modifications on glycoproteins.

Application of StrucGP in medical immunology: site-specific N-glycoproteomic analysis of macrophages.

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc4Hex5Fuc1Neu5Ac1, N4H5F1S1) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.

Risk factors, prediction model, and prognosis analysis of myocardial injury after acute upper gastrointestinal bleeding.

Background: Cardiovascular complications in patients with acute upper gastrointestinal bleeding (AUGIB) have been associated with a high-risk of subsequent adverse consequences. This study aimed to analyze the risk factors for myocardial injury in AUGIB patients, predict the risk of myocardial injury, and explore the clinical prognosis and influencing factors in AUGIB patients with myocardial injury. Materials and methods: A retrospective case-control study based on AUGIB patients in the First Affiliated Hospital of Xi'an Jiaotong University from 2016 to 2020 was performed. We divided the enrolled patients into a myocardial injury group and a control group according to whether they developed myocardial injury. The variables significant in the univariate analysis were subjected to binary logistic regression for risk factor analysis and were used to establish a nomogram for predicting myocardial injury. In addition, logistic regression analysis was performed to better understand the risk factors for in-hospital mortality after myocardial injury. Result: Of the 989 AUGIB patients enrolled, 10.2% (101/989) developed myocardial injury. Logistic regression analysis showed that the strong predictors of myocardial injury were a history of hypertension (OR: 4.252, 95% CI: 1.149-15.730, P = 0.030), blood urea nitrogen (BUN) (OR: 1.159, 95% CI: 1.026-1.309, P = 0.018) and left ventricular ejection fraction (LVEF) <68% (OR: 3.667, 95% CI: 1.085-12.398, P = 0.037). The patients with a tumor history (digestive system tumors and non-digestive system tumors) had no significant difference between the myocardial injury group and the control group (P = 0.246). A prognostic nomogram model was established based on these factors with an area under the receiver operator characteristic curve of 0.823 (95% CI: 0.730-0.916). The patients with myocardial injury had a much higher in-hospital mortality rate (10.9% vs. 2.0%, P < 0.001), and an elevated D-dimer level was related to in-hospital mortality among the AUGIB patients with myocardial injury (OR: 1.273, 95% CI: 1.085-1.494, P = 0.003). Conclusion: A history of hypertension, renal dysfunction, and cardiac function with LVEF <68% were strong predictors of myocardial injury. Coagulopathy was found to be associated with poor prognosis in AUGIB patients with myocardial injury.

Recognition of Core-Fucosylated Glycopeptides Based on the Y1+Fuc/Y1 Ratio in Low-Energy HCD Spectra.

Accurate identification of core fucosylation on N-glycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8-/- mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8+/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.

Glycoproteomic analysis reveals the effects of bisecting GlcNAc in intrahepatic cholangiocarcinoma.

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.

Evaluation of absorbent cotton for glycopeptide enrichment.

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% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200µL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.

Structural- and Site-Specific N-Glycosylation Characterization of COVID-19 Virus Spike with StrucGP.

The spike (S) protein plays a key role in COVID-19 (SARS-CoV-2) infection and host-cell entry. Previous studies have systematically analyzed site-specific glycan compositions as well as many important structural motifs of the S protein. Here, we further provide structural-clear N-glycosylation of the S protein at a site-specific level by using our recently developed structural- and site-specific N-glycoproteomics sequencing algorithm, StrucGP. In addition to the common N-glycans as detected in previous studies, many uncommon glycosylation structures such as LacdiNAc structures, Lewis structures, Mannose 6-phosphate (M6P) residues, and bisected core structures were unambiguously mapped at a total of 20 glycosites in the S protein trimer and protomer. These data further support the glycosylation structural-functional investigations of the COVID-19 virus spike.

De-sialylation of glycopeptides by acid treatment: enhancing sialic acid removal without reducing the identification.

Sialic acid, a common terminal monosaccharide on many glycoconjugates, plays essential roles in many biological processes such as immune responses, pathogen recognition, and cancer development. For various purposes, sialic acids may need to be removed from glycopeptides or glycans, mainly using enzymatical or chemical approaches. In this study, we found that most commonly used chemical methods couldn't completely remove sialic acids from glycopeptides. Although the de-sialylation efficiency could be further enhanced by increasing the treatment time or acid concentration, the undesirable side reactions on the peptide portion would decrease glycopeptide identification. By adding the deamidation on carbamidomethyl-cysteine (C), asparagine (N), and glutamine (Q) residues as a variable modification during database search, most of the unidentified spectra could be recovered. This optional acid-treatment and database search method for the complete removal of sialic acids without losing much spectral identification should be quite useful for many glycomic and glycoproteomic studies.

Precision Structural Interpretation of Site-Specific N-Glycans in Seminal Plasma.

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.

Formylation: an undesirable modification on glycopeptides and glycans during storage in formic acid solution.

In glycomic and glycoproteomic studies, solutions containing diluted organic acids such as formic acid (FA) have been widely used for dissolving intact glycopeptide and glycan samples prior to mass spectrometry analysis. Here, we show that an undesirable + 28 Da modification occurred in a time-dependent manner when the glycan and glycopeptide samples were stored in FA solution at - 20 °C. We confirmed that this unexpected modification was caused by formylation between the hydroxyl groups of glycans and FA with a relatively low reaction rate. As this incomplete modification affected the glycan and glycopeptide identification and quantification in glycomic and glycoproteomic studies, the storage at - 20 °C should be avoided once the glycan and glycopeptide samples have been dissolved in FA solution.

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa.

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.

An Empirical Study on the Promotion of Students' Physiological and Psychological Recovery in Green Space on Campuses in the Post-Epidemic Era.

Human health is closely related to the environment; a relaxing and pleasant landscape environment can make people feel less stressed and more energetic. To investigate the restorative potential of landscape types and landscape elements in the post-epidemic era from the perspective of visual perception, this study selected Sichuan Agricultural University's Laoban hill, Jiuqu bridge, and the ginkgo garden to carry out physiological and psychological measurement experiments with college students. Research results on the psycho-biological and perceptual recovery vary with the types of landscape spaces. The results of the physiological data showed that all three space types had no significant effect on the recovery of blood pressure and heart rate; Laoban hill and Jiuqu bridge had some impact on concentration, while the ginkgo garden had no significant effect; and all three space types had some effect on the relaxation of the subjects' mental state. The results of the psychological data showed that the subjects' emotions were significantly improved in the three different landscape space types. The space with the strongest restorative effect on negative emotions was the ginkgo garden, followed by Jiuqu bridge and Laoban hill. The spaces with the strongest restorative potential for positive emotions were Jiuqu bridge and the ginkgo garden, followed by Laoban hill. The results of the perceptual restoration data showed that the Laoban hill space had the most effective restorative potential on the human body, followed by Jiuqu bridge, with the ginkgo garden having the least effective restorative potential. The results of the study on the difference between the aesthetic preference of different landscape elements and the perception restorative effect of a space showed that "the harmony between artificial structures such as garden pieces and the environment", "plant species", "waterscape state", and "boundary clarity" were identified as significant landscape elements with perception-restorative effects. These findings summarize campus landscape types and elements with optimal restorative potential. In the future, in campus landscape design-an active approach with a scientific combination and configuration of campus landscape types and elements-can provide a feasible solution to enhance the potential of campus landscape restorative effects.

Identifying intact N-glycopeptides from tandem mass spectrometry data using StrucGP.

Protein glycosylation is of great importance in many biological processes. Glycosylation has been increasingly analyzed at the intact glycopeptide level using mass spectrometry to study site-specific glycosylation changes under different physiological and pathological conditions. StrucGP is a glycan database-independent search engine for the structural interpretation of N-glycoproteins at the site-specific level. To ensure the accuracy of results, two collision energies are implemented in instrument settings for each precursor to separate fragments of peptides and glycans. In addition, the false discovery rates (FDR) of peptides and glycans as well as probabilities of detailed structures are estimated. In this protocol, the use of StrucGP is demonstrated, including environment configuration, data preprocessing as well as result inspection and visualization using our in-house software "GlycoVisualTool". The described workflow should be able to be performed by anyone with basic proteomic knowledge.

StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy.

Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes. Owing to the database-independent glycan mapping strategy, StrucGP also facilitates the identification of rare/new glycan structures. The approach will be greatly beneficial for in-depth structural and functional study of glycoproteins in the biomedical research.

Screening of folate-producing lactic acid bacteria and modulatory effects of folate-biofortified yogurt on gut dysbacteriosis of folate-deficient rats.

Folate deficiency is accompanied by gut dysbacteriosis. To understand dietary intervention in folate deficiency, a folate-deficient rat model was used to evaluate the modulatory effects of folate-producing lactic acid bacteria (LAB) and biofortified yogurt on gut dysbacteriosis. The high folate-producing strain was screened from 12 LABs, and its variant, namely Lactobacillus plantarum GSLP-7 V, with folate productivity in yogurt at 3.72 µg mL-1, was obtained by stressing with 5.0 mg L-1 methotrexate and 100.00 mg L-1 Ca2+. To our knowledge, this is the highest folate productivity in yogurt by LAB strains ever reported. To further examine the folate supplement effect in vivo, a folate-deficient rat model was established and fed a folate-free diet for 8 weeks. Also, the effects of L. plantrum GSLP-7 V, yogurt fermented with L. plantrum GSLP-7 V, plain yogurt, and chemical folic acid on folate deficiency and gut dysbacteriosis were examined. Analysis of the change in gut microbiota showed that the gut dysbacteriosis was significantly correlated with folate deficiency. Administration of L. plantrum GSLP-7 V and its fermented yogurt for 10 days restored the disrupted gut microbiota and recovered the serum folate and homocysteine to normal levels, while chemical folic acid worsened the gut dysbacteriosis. Chemical folic acid only enriched Akkermansia, while L. plantrum GSLP-7 V and its fermented yogurt modulated the gut microbiota comprehensively through 7 and 10 key genera, respectively. This study confirmed the effectiveness of dietary intervention with folate-biofortified yogurt through modulating gut microbiota, suggesting the potential of the folate-producing LAB as an agent for the treatment of folate-deficiency related diseases.