ABSTRACT
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.
Subject(s)
Asthenozoospermia , Fucose , Semen , Humans , Male , Asthenozoospermia/metabolism , Semen/metabolism , Semen/chemistry , Fucose/metabolism , Glycoproteins/metabolism , Proteomics , Adult , Up-Regulation , Polysaccharides/metabolism , Polysaccharides/chemistry , Glycosylation , Glycopeptides/metabolism , Glycopeptides/analysisABSTRACT
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.
Subject(s)
Glycoproteins , Liver , Humans , Glycoproteins/chemistry , Glycosylation , Liver/metabolism , Polysaccharides/chemistry , Fucose/chemistryABSTRACT
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.
Subject(s)
Algorithms , Glycopeptides/analysis , Glycoproteins/analysis , Polysaccharides/analysis , Animals , Glycopeptides/chemistry , Glycoproteins/chemistry , Glycosylation , Male , Mice , Mice, Inbred C57BL , Polysaccharides/chemistryABSTRACT
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.
Subject(s)
Acrosome Reaction , Spermatozoa , Acrosome/metabolism , Glycoproteins/metabolism , Glycosylation , Humans , Male , Proteomics , Sperm Capacitation , Spermatozoa/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Glycopeptides , Proteome , Animals , Mice , Chromatography, Liquid/methods , Glycopeptides/chemistry , Glycosylation , Hydrophobic and Hydrophilic InteractionsABSTRACT
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.
Subject(s)
Polysaccharides , Semen , Glycopeptides/chemistry , Glycoproteins/metabolism , Glycosylation , Humans , Male , Polysaccharides/chemistry , Semen/metabolismABSTRACT
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.
Subject(s)
COVID-19 , SARS-CoV-2 , Glycosylation , Humans , Polysaccharides/chemistryABSTRACT
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.
Subject(s)
Glycopeptides , Tandem Mass Spectrometry , Animals , Mice , Glycopeptides/analysis , Tandem Mass Spectrometry/methods , Fucose/chemistry , Glycosylation , Glycoproteins/chemistryABSTRACT
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.
Subject(s)
Acetylglucosamine , N-Acetylglucosaminyltransferases , Humans , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Acetylglucosamine/metabolism , Polysaccharides/chemistry , Glycoproteins/genetics , Glycoproteins/chemistry , Cell Line , Cell Line, TumorABSTRACT
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.
Subject(s)
Glycomics , Glycopeptides , Formates , Glycomics/methods , Glycopeptides/chemistry , Mass Spectrometry , Polysaccharides/chemistryABSTRACT
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.
Subject(s)
Glycopeptides , Polysaccharides , Glycopeptides/chemistry , Chromatography, Liquid/methods , Glycosylation , Hydrophobic and Hydrophilic InteractionsABSTRACT
Myocardial infarction (MI) is one of the leading causes of deaths worldwide. Because of the incapability of regeneration, the cardiomyocyte loss with MI is replaced by fibrotic scar tissue, which eventually leads to heart failure. Reconstructing regeneration of an adult human heart has been recognized as a promising strategy for cardiac therapeutics. A neonatal mouse heart, which possesses transient regenerative capacity at the first week after birth, represents an ideal model to investigate processes associated with cardiac regeneration. In this work, an integrated glycoproteomic and proteomic analysis was performed to investigate the differences in glycoprotein abundances and site-specific glycosylation between postneonatal day 1 (P1) and day 7 (P7) of mouse hearts. By large-scale profiling and quantifying more than 2900 intact N-glycopeptides in neonatal mouse hearts, we identified 227 altered N-glycopeptides between P1 and P7 hearts. By extracting protein changes from the global proteome data, the normalized glycosylation changes for site-specific glycans were obtained, which showed heterogeneity on glycosites and glycoproteins. Systematic analysis of the glycosylation changes demonstrated an overall upregulation of sialylation and core fucosylation in P7 mice. Notably, the upregulated sialylation was a comprehensive result of increased sialylated glycans with Neu5Gc, with both Neu5Gc and core fucose, and decreased sialylated glycans with Neu5Ac. The upregulated core fucosylation resulted from the increase of glycans containing both core fucose and Neu5Gc but not glycans containing sole core fucose. These data provide a valuable resource for future functional and mechanism studies on heart regeneration and discovery of novel therapeutic targets. All mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD017139.
Subject(s)
Glycopeptides , Proteomics , Animals , Animals, Newborn , Glycosylation , Mice , RegenerationABSTRACT
Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.
Subject(s)
Glycopeptides/analysis , Influenza A Virus, H1N1 Subtype/enzymology , Influenza, Human/virology , Neuraminidase/chemistry , Polysaccharides/analysis , Viral Proteins/chemistry , Animals , Cell Line , Glycosylation , Humans , Influenza A Virus, H1N1 Subtype/chemistry , Insecta , Orthomyxoviridae Infections/virologyABSTRACT
Bisecting N-glycan represents one of the most important modifications to the N-glycan core, and it is involved in various biological processes. Despite many studies on the biological roles of bisecting N-glycans, current approaches for bisecting N-glycan analysis mainly rely on the use of the lectin PHA-E, which are of low specificity and sensitivity. Here, we describe a straightforward method for the recognition of bisecting N-glycans on intact glycopeptides using two characteristic Y ions [peptide+HexNAc3Hex1] and [peptide+HexNAc3Hex1Fuc1] in low energy fragmented MS/MS spectra under higher energy collisional dissociation (HCD) mode. The critical aspect of the method is the combination use of low energy HCD fragmentation and intact glycopeptide analysis. With samples from rat renal tissues, we determined the optimal fragmentation energies and analyzed the influence of core fucosylation on the intensity of the [peptide+HexNAc3Hex1] ion. Using the method, we identified 183 intact glycopeptides with bisecting N-glycans and investigated the primary bisecting N-glycan structures and the possible biological roles of these identified proteins.
Subject(s)
Glycopeptides/chemistry , Polysaccharides/chemistry , Tandem Mass Spectrometry , Amino Acid Sequence , Animals , RatsABSTRACT
Bisecting N-glycan is known to be a metastasis suppressor and plays a regulatory role in the biosynthesis of N-glycans. Previous studies have shown that bisecting N-glycans are capable of modulating both the branching and terminal modifications of glycans. However, these effects have been investigated mainly by glycomic approaches and it remains unclear how they alter when glycans are attached to different glycosites of proteins. Here, we systematically investigated the regulatory roles of bisecting N-glycans in human HK-2 cells using StrucGP, a strategy we developed for structural interpretation of site-specific N-glycans on glycoproteins. The glycoproteomics analysis showed that most of bisecting N-glycans are complex type and often occur in company with core fucosylation. With the overexpression and knockdown of MGAT3, the only enzyme responsible for bisecting N-glycan synthesis, we found that bisecting N-glycans can impact the biosynthesis of N-glycans from multiple aspects, including glycan types, branching, sialylation, fucosylation (different effects for core and terminal fucosylation) as well as the presence of terminal N-acetylglucosamine. Furthermore, gene ontology analysis suggested that most proteins with bisecting N-glycans located in the extracellular region or membrane, where they function mostly in cell adhesion, extracellular matrix regulation and cell signaling. Finally, we showed that overexpression of bisecting N-glycans had a broad impact on the protein expressions of HK-2 cells, involving multiple biological processes. Taken together, our work systematically demonstrated the expression profiles of bisecting N-glycans, and their regulatory effects on the biosynthesis of N-glycans and protein expressions, which provide valuable information for the functional elucidation of bisecting N-glycans.
Subject(s)
Glycoproteins , Polysaccharides , Humans , Glycosylation , Glycoproteins/chemistry , Polysaccharides/chemistryABSTRACT
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.
Subject(s)
B7-H1 Antigen , Polysaccharides , Humans , Glycosylation , Polysaccharides/chemistry , Polysaccharides/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Glycopeptides , N-Acetylneuraminic Acid , Glycopeptides/chemistry , Polysaccharides , Sialic Acids/chemistryABSTRACT
Primary liver cancer, mainly comprising hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), remains a major global health problem. Although ICC is clinically different from HCC, their molecular differences are still largely unclear. In this study, precision N-glycoproteomic analysis was performed on both ICC and HCC tumors as well as paracancer tissues to investigate their aberrant site-specific N-glycosylation. By using our newly developed glycoproteomic methods and novel algorithm, termed 'StrucGP', a total of 486 N-glycan structures attached on 1235 glycosites were identified from 894 glycoproteins in ICC and HCC tumors. Notably, glycans with uncommon LacdiNAc (GalNAcß1-4GlcNAc) structures were distinguished from their isomeric glycans. In addition to several bi-antennary and/or bisecting glycans that were commonly elevated in ICC and HCC, a number of LacdiNAc-containing, tri-antennary, and core-fucosylated glycans were uniquely increased in ICC. More interestingly, almost all LacdiNAc-containing N-glycopeptides were enhanced in ICC tumor but not in HCC tumor, and this phenomenon was further confirmed by lectin histochemistry and the high expression of ß1-4 GalNAc transferases in ICC at both mRNA and protein expression levels. The novel N-glycan alterations uniquely detected in ICC provide a valuable resource for future studies regarding to the discovery of ICC diagnostic biomarkers, therapeutic targets, and mechanism investigations.