Differential expression of N-glycopeptides derived from serum glycoproteins in mild cognitive impairment (MCI) patients.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N-glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N-glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N-glycopeptides showed outstanding AUC values, among them the antithrombin-III Asn224 + 4-5-0-2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N-sites play an important role in neurodegeneration.

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

Multi Omics Applications in Biological Systems.

Traditional methodologies often fall short in addressing the complexity of biological systems. In this regard, system biology omics have brought invaluable tools for conducting comprehensive analysis. Current sequencing capabilities have revolutionized genetics and genomics studies, as well as the characterization of transcriptional profiling and dynamics of several species and sample types. Biological systems experience complex biochemical processes involving thousands of molecules. These processes occur at different levels that can be studied using mass spectrometry-based (MS-based) analysis, enabling high-throughput proteomics, glycoproteomics, glycomics, metabolomics, and lipidomics analysis. Here, we present the most up-to-date techniques utilized in the completion of omics analysis. Additionally, we include some interesting examples of the applicability of multi omics to a variety of biological systems.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S. Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S. Typhimurium genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during S. Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S. Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient S. Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S. Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S. Typhimurium, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S. Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S. Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S. Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.

MS-based glycomics and glycoproteomics methods enabling isomeric characterization.

Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's disease, inflammation, immune deficiencies, congenital disorders, and cancers. The alterations in the distributions of glycan and glycopeptide isomers are involved in the development and progression of several human diseases. However, the microheterogeneity of glycosylation brings a great challenge to glycomic and glycoproteomic analysis, including the characterization of isomers. Over several decades, different methods and approaches have been developed to facilitate the characterization of glycan and glycopeptide isomers. Mass spectrometry (MS) has been a powerful tool utilized for glycomic and glycoproteomic isomeric analysis due to its high sensitivity and rich structural information using different fragmentation techniques. However, a comprehensive characterization of glycan and glycopeptide isomers remains a challenge when utilizing MS alone. Therefore, various separation methods, including liquid chromatography, capillary electrophoresis, and ion mobility, were developed to resolve glycan and glycopeptide isomers before MS. These separation techniques were coupled to MS for a better identification and quantitation of glycan and glycopeptide isomers. Additionally, bioinformatic tools are essential for the automated processing of glycan and glycopeptide isomeric data to facilitate isomeric studies in biological cohorts. Here in this review, we discuss commonly employed MS-based techniques, separation hyphenated MS methods, and software, facilitating the separation, identification, and quantitation of glycan and glycopeptide isomers.

Suppressing the background of LC-ESI-MS analysis of permethylated glycans using the active background ion reduction device.

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

15N metabolic labeling-TMT multiplexing approach to facilitate the quantitation of glycopeptides derived from cell lines.

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles.

Glycosylation of a key cubilin Asn residue results in reduced binding to albumin.

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin's albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.

Isomeric Separation of α2,3/α2,6-Linked 2-Aminobenzamide (2AB)-Labeled Sialoglycopeptides by C18-LC-MS/MS.

Determination of the relative expression levels of the α2,3/α2,6-sialic acid linkage isomers on glycoproteins is critical to the analysis of various human diseases such as cancer, inflammation, and viral infection. However, it remains a challenge to separate and differentiate site-specific linkage isomers at the glycopeptide level. Some derivatization methods on the carboxyl group of sialic acid have been developed to generate mass differences between linkage isomers. In this study, we utilized chemical derivatization that occurred on the vicinal diol of sialic acid to separate linkage isomers on a reverse-phase column using a relatively short time. 2-Aminobenzamide (2AB) labeling derivatization, including periodate oxidation and reductive amination, took only ∼3 h and achieved high labeling efficiency (>90%). Within a 66 min gradient, the sialic acid linkage isomers of 2AB-labeled glycopeptides from model glycoproteins can be efficiently resolved compared to native glycopeptides. Two different methods, neuraminidase digestion and higher-energy collision dissociation tandem mass spectrometry (HCD-MS2) fragmentation, were utilized to differentiate those isomeric peaks. By calculating the diagnostic oxonium ion ratio of Gal2ABNeuAc and 2ABNeuAc fragments, significant differences in chromatographic retention times and in mass spectral peak abundances were observed between linkage isomers. Their corresponding MS2 PCA plots also helped to elucidate the linkage information. This method was successfully applied to human blood serum. A total of 514 2AB-labeled glycopeptide structures, including 152 sets of isomers, were identified, proving the applicability of this method in linkage-specific structural characterization and relative quantification of sialic acid isomers.

Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics.

The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.

The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

A Reciprocal Best-hit Approach to Characterize Isomeric N-Glycans Using Tandem Mass Spectrometry.

Glycosylation is a post-translational modification involved in many important biological functions. The aberrant alteration of glycan structure is implicit with malfunction of cells and possess potential significance in medical diagnosis of complex diseases such as cancer. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been commonly applied to the analysis of complex glycomic samples. However, the characterization of isomeric glycans from their MS/MS spectra in complex biological samples remains challenging. In this paper, we present a novel reciprocal best-hit glycan-spectrum matching (RB-GSM) approach toward characterizing N-glycans. In this method, the MS/MS spectra in the input data set are evaluated against all glycans with the matched precursor mass using customized scoring functions, where a glycan-spectrum matching (GSM) is considered to be true if it is a reciprocal best-hit, that is, it receives the highest score among not only the GSMs between the respective spectrum and all matched glycans, but also the GSMs between the respective glycan and all matched MS/MS spectra in the input data set. We evaluated this RB-GSM approach on N-glycan identification using MS/MS spectra acquired from glycan standards as well as those released from the model glycoprotein fetuin, immunoglobulin G, and human serum samples, which showed the RB-GSM is capable of distinguishing isomeric glycans.

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer.

Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM's advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.

Advances in mass spectrometry-based glycomics-An update covering the period 2017-2021.

The wide variety of chemical properties and biological functions found in proteins is attained via post-translational modifications like glycosylation. Covalently bonded to proteins, glycans play a critical role in cell activity. Complex structures with microheterogeneity, the glycan structures that are associated with proteins are difficult to analyze comprehensively. Recent advances in sample preparation methods, separation techniques, and MS have facilitated the quantitation and structural elucidation of glycans. This review focuses on highlighting advances in MS-based techniques for glycomic analysis that occurred over the last 5 years (2017-2021) as an update to the previous review on the subject. The topics of discussion will include progress in glycomic workflow such as glycan release, purification, derivatization, and separation as well as the topics of ionization, tandem MS, and separation techniques that can be coupled with MS. Additionally, bioinformatics tools used for the analysis of glycans will be described.

Advances in mass spectrometry-based glycoproteomics: An update covering the period 2017-2021.

Protein glycosylation is one of the most common posttranslational modifications, and plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, host-pathogen interaction, and protein stability. Glycoproteomics is a proteomics subfield dedicated to identifying and characterizing the glycans and glycoproteins in a given cell or tissue. Aberrant glycosylation has been associated with various diseases such as Alzheimer's disease, viral infections, inflammation, immune deficiencies, congenital disorders, and cancers. However, glycoproteomic analysis remains challenging because of the low abundance, site-specific heterogeneity, and poor ionization efficiency of glycopeptides during LC-MS analyses. Therefore, the development of sensitive and accurate approaches to efficiently characterize protein glycosylation is crucial. Methods such as metabolic labeling, enrichment, and derivatization of glycopeptides, coupled with different mass spectrometry techniques and bioinformatics tools, have been developed to achieve sophisticated levels of quantitative and qualitative analyses of glycoproteins. This review attempts to update the recent developments in the field of glycoproteomics reported between 2017 and 2021.

Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS.

Glycosylation is known as a critical biological process that can largely affect the properties and the functions of proteins. Glycan isomers have been shown to be involved in a variety of disease progressions. However, the separation and identification of glycan isomers has been a challenge for years due to the microheterogeneity of glycan isomeric structures. Therefore, effective and stable techniques have been investigated over the last few decades to improve isomeric separations of glycans. RPLC has been widely used in biomolecule analysis because of its extraordinary reproducibility and reliability in retention time and separation resolution. However, so far, no studies have achieved high resolution of glycan isomers using this technique. In this study, we focused on further boosting the isomeric separation of permethylated glycans using a 500 mm reversed-phase LC column. To achieve better resolutions on permethylated glycans, different LC conditions were optimized using glycan standards, including core- and branch-fucosylated N-glycan isomers and sialic acid linked isomers, which were both successfully separated. Then, the optimal separation strategy was applied to achieve separations of N- and O-glycan isomers derived from model glycoproteins, including bovine fetuin, ribonuclease B and κ-casein. Baseline separations were observed on multiple sialylated linkage isomers. However, the separation performance of high-mannose isomers needs further improvement. The reproducibility and stability of this long C18 column was also tested by doing run-to-run, day-to-day and month-to-month comparisons of retention times on multiple glycans and the %RSD was found less than 0.92%. Finally, we applied this approach to separate glycan isomers derived from complex biological samples, including blood serum and cell lines, where baseline separations were attained on several isomeric structures. Compared to the separation efficiency of PGC and MGC columns, the RPLC C18 column provides lower resolution but more robust reproducibility, which makes it a good complementary alternative for isomeric separations of glycans.

Analysis of NIST Monoclonal Antibody Reference Material Glycosylation Using the LC-MS/MS-Based Glycoproteomic Approach.

Protein-based therapeutics such as mAbs have become emerging drugs in modern medicine. Most of the approved therapeutic proteins are glycoproteins. Glycosylation is an essential critical quality attribute (CQA) due to the influence that glycoforms have on the safety, efficacy, and pharmacokinetics/pharmacodynamics (PK/PD) of biotherapeutics. Here, we applied an LC-MS/MS-based glycoproteomics approach to characterize Fc glycans of an NISTmAb reference material (RM) 8671 (sample B) and a ß-1,4-galactosidase-treated NISTmAb (sample A). Overall, 48 glycan compositions were identified and quantified. The glycan structure with the highest abundance was FA2, with a relative abundance of 52% in sample A and 38% in sample B. Over 50% of the identified glycans presented at levels smaller than 0.1%. Important glycan attributes were further derived using the quantitative results. The galactosylation level of modified NISTmAb was found to decrease by ∼10% when compared to the galactosylation level of NISTmAb. There was no significant difference between the two samples in the levels of sialylation, fucosylation, and high mannose. Moreover, unglycosylated peptides were also observed at a level of 1-2%.

Direct Comparison of N-Glycans and Their Isomers Derived from Spike Glycoprotein 1 of MERS-CoV, SARS-CoV-1, and SARS-CoV-2.

The emergence of COVID-19 pandemic has engaged the scientific community around the globe in the rapid development of effective therapeutics and vaccines. Owing to its crucial role in the invasion of the host cell, spike (S) glycoprotein is one of the major targets in these studies. The S1 subunit of the S protein (S1 protein) accommodates the receptor-binding domain, which enables the initial binding of the virus to the host cell. Being a heavily glycosylated protein, numerous studies have investigated its glycan composition. However, none of the studies have explored the isomeric glycan distribution of this protein. Furthermore, this isomeric glycan distribution has never been compared to that in S1 proteins of other coronaviruses, severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, which were responsible for past epidemics. This study explores the uncharted territory of the isomeric glycan distribution in the coronaviruses' S1 protein using liquid chromatography coupled to tandem mass spectrometry. We believe that our data would facilitate future investigations to study the role of isomeric glycans in coronavirus viral pathogenesis.

Variability in the Glycosylation Patterns of gp120 Proteins from Different Human Immunodeficiency Virus Type 1 Isolates Expressed in Different Host Cells.

The mature HIV-1 envelope (Env) glycoprotein is composed of gp120, the exterior subunit, and gp41, the transmembrane subunit assembled as trimer by noncovalent interaction. There is a great body of literature to prove that gp120 binds to CD4 first, then to the coreceptor. Binding experiments and functional assays have demonstrated that CD4 binding induces conformational changes in gp120 that enable or enhance its interaction with a coreceptor. Previous studies provided different glycomic maps for the HIV-1 gp120. Here, we build on previous work to report that the use of LC-MS/MS, in conjunction with hydrophilic interaction liquid chromatography (HILIC) enrichment to glycosylation sites, associated with the assorted neutralizing or binding events of glycosylation targeted antibodies from different clades or strains. In this study, the microheterogeneity of the glycosylation from 4 different clades of gp120s is deeply investigated. Aberrant glycosylation patterns were detected on gp120 that originated from different clades, viral sequences, and host cells. The results of this study may help provide a better understanding of the mechanism of how the glycans participate in the antibody neutralizing process that targets glycosylation sites.