Site-specific Glycan Research Articles

Variation of Site-Specific Glycosylation Profiles of Recombinant Influenza Glycoproteins

This work presents a detailed determination of site-specific N-glycan distributions of the recombinant influenza glycoproteins hemagglutinin (HA) and neuraminidase. Variation in glycosylation among recombinant glycoproteins is not predictable and can depend on details of the biomanufacturing process as well as details of protein structure. In this study, recombinant influenza proteins were analyzed from eight strains of four different suppliers. These include five HA and three neuraminidase proteins, each produced from a HEK293 cell line. Digestion was conducted using a series of complex multienzymatic methods designed to isolate glycopeptides containing single N-glycosylated sites. Site-specific glycosylation profiles of intact glycopeptides were produced using a recently developed method and comparisons were made using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 999), whereas digestion replicates and injection replicates showed relatively little variation (similarity score = 957). Notably, glycan distributions for homologous regions of influenza glycoprotein variants showed low variability. Due to the multiple possible sources of variation and inherent analytical difficulties in site-specific glycan determinations, variations were individually examined for multiple factors, including differences in supplier, production batch, protease digestion, and replicate measurement. After comparing all glycosylation distributions, four distinguishable classes could be identified for the majority of sites. Finally, attempts to identify glycosylation distributions on adjacent potential N-glycosylated sites of one HA variant were made. Only the second site (NnST) was found to be occupied using two rarely used proteases in proteomics, subtilisin and esperase, both of which did selectively cleave these adjacent sites.

Rational development of functional hydrophilic polymer to characterize site-specific glycan differences between bovine milk and colostrum

As vastly modified on secreted proteins, N-glycosylation is found on milk proteins and undergo dynamic changes during lactation, characterizing milk protein glycosylation would benefit the elucidation of glycosylation pattern differences between samples. However, their low abundance required specific enrichment. Herein, through rational design and controllable synthesis, we developed a novel multi-functional polymer for the isolation of protein glycosylation. It efficiently separated glycopeptides from complex background inferences with mutual efforts of hydrophilic interaction chromatography (HILIC), metal ion affinity and ion exchange. By fine-tuning Ca2+ as regulators of aldehyde hyaluronic acid (HA) conformation, the grafting density of HA was remarkably improved. Moreover, grafting Ti4+ further enhanced the enrichment performance. Application of this material to characterize bovine milk and colostrum proteins yields 479 and 611 intact glycopeptides, respectively. Comparative analysis unraveled the distinct glycosylation pattern as well the different distribution of glycoprotein abundances between the two samples, offering insights for functional food development.

Site-specific N-glycoproteomic analysis reveals up-regulated fucosylation in seminal plasma of asthenozoospermia.

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.

Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Abstract 3149: Development of a novel site-specific ADC glycan platform with potential for improved in vivo efficacy and stability of the ADC in animal studies

Abstract Antibody-drug conjugates (ADCs) represent a promising modality for delivering cytotoxic drugs to targeted tumor cells while avoiding off-tumor toxicities. Despite successes in the past 20 years, development of effective ADCs with broad therapeutic window remains challenging due to the complexity of conjugation technologies and the instability of the linkers. Most platforms currently in the market as well as under clinical development may face limitations ascribed to the heterogeneity of ADCs with various drug-to-antibody ratio (DAR). This likely resulted from random conjugation and poor overall biophysical characteristics caused by linker hydrophobicity. The limitations may impact the efficacy, safety, bioavailability, and the robustness of manufacturing process of these therapeutic agents. OBI developed a unique glycan ADC platform (GlycOBI) to enable a site-specific conjugation, which is in a 'Plug and Play' format and compatible with any antibodies, linkers, and payloads in various DAR. This is an efficient and scalable process to generate homogenous ADCs. This platform overcomes the limitations of traditional ADCs, resulting in the improvement of efficacy and stability. We developed a non-genetic, engineering-free approach to generate site-specific homogenous ADCs. This was achieved by utilizing OBI proprietary enzymatic technology (EndoSymeOBI), followed by the click chemistry to conjugate the hydrophilic linker-payload via the glycan site that naturally occurs on the antibody’s Fc region. The conjugation process avoids disrupting the antibody structure, ensuring the related ADC has similar biophysical characteristics compared to native antibody. Furthermore, OBI linker technology improves the conjugation efficiency of the payload, as well as reduces the aggregation propensity, and expands the half-life of the ADC products. Anti-TROP2 and anti-HER2 ADCs are developed based on OBI glycan ADC platform (GlycOBI) using a topoisomerase I inhibitor, exatecan, as the payload. Both ADCs demonstrated greater chemical stability in serum stability studies compared to competitor ADCs. Additionally, they showed broad activity against different cancer cell lines in vitro as well as in mouse xenograft models. The preclinical data presented here suggest that GlycOBI is an outstanding ADC platform to improve the performance of ADCs. Further investigations, including studies in the clinical settings, are warranted. Citation Format: Teng-Yi Huang, Yin-Cheng Hsieh, Ka-Shu Fung, Yu-Chao Huang, Chi-Sheng Shia, Ming-Feng Chiang, Nan-Hsuan Wang, Wan-Fen Li, Ming-Tain Lai. Development of a novel site-specific ADC glycan platform with potential for improved in vivo efficacy and stability of the ADC in animal studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3149.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Robust Glycoproteomics Platform Reveals a Tetra-Antennary Site-Specific Glycan Capping with Sialyl-Lewis Antigen for Early Detection of Gastric Cancer.

The lack of efficient biomarkers for the early detection of gastric cancer (GC) contributes to its high mortality rate, so it is crucial to discover novel diagnostic targets for GC. Recent studies have implicated the potential of site-specific glycans in cancer diagnosis, yet it is challenging to perform highly reproducible and sensitive glycoproteomics analysis on large cohorts of samples. Here, a highly robust N-glycoproteomics (HRN) platform comprising an automated enrichment method, a stable microflow LC-MS/MS system, and a sensitive glycopeptide-spectra-deciphering tool is developed for large-scale quantitative N-glycoproteome analysis. The HRN platform is applied to analyze serum N-glycoproteomes of 278 subjects from three cohorts to investigate glycosylation changes of GC. It identifies over 20000 unique site-specific glycans from discovery and validation cohorts, and determines four site-specific glycans as biomarker candidates. One candidate has branched tetra-antennary structure capping with sialyl-Lewis antigen, and it significantly outperforms serum CEA with AUC values > 0.89 compared against < 0.67 for diagnosing early-stage GC. The four-marker panel can provide improved diagnostic performances. Besides, discrimination powers of four candidates are also testified with a verification cohort using PRM strategy. This findings highlight the value of this strong tool in analyzing aberrant site-specific glycans for cancer detection.

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources.

Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing, and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of the recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation followed by ion trap confirmation. This enables a detailed examination of the reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production. All mass spectrometry data files have been deposited in the MassIVE repository under the identifier MSV000091776.

Site-Specific and Multiple Fluorogenic Metabolic Glycan Labeling and Glycoproteomic Profiling in Live Cells.

Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates.

Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a “glycan shield”). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

Analysis of Protein Glycosylation after Rapid Digestion Using Protease-Containing Membranes in Spin Columns.

Glycosylation is an important protein post-translational modification that plays a pivotal role in the bioactivity of therapeutic proteins and in the infectivity of viral proteins. Liquid chromatography with tandem mass spectrometry readily identifies protein glycans with site specificity. However, the overnight incubation used in conventional in-solution proteolysis leads to high turnaround times for glycosylation analysis, particularly when sequential in-solution digestions are needed for site-specific glycan identification. Using bovine fetuin as a model glycoprotein, this work first shows that in-membrane digestion in ∼3 min yields similar glycan identification and quantitation when compared to overnight in-solution digestion. Protease-containing membranes in a spin column enable digestion of therapeutic proteins (trastuzumab and erythropoietin) and a viral protein (SARS-CoV-2 receptor binding domain) in ∼30 s. Glycan identification is similar after in-solution and in-membrane digestion, and limited in-membrane digestion enhances the identification of high-mannose glycans in trastuzumab. Finally, stacked membranes containing trypsin and chymotrypsin allow fast sequential proteolytic digestion to site-specifically identify the glycans of SARS-CoV-2 receptor binding domain. One can easily assemble the protease-containing membranes in commercial spin columns, and spinning multiple columns simultaneously will facilitate parallel analyses.

In situ architecture and membrane fusion of SARS-CoV-2 Delta variant

Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the insitu structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC–HRMS

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood’s capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.

Glycopeptide characterization of Sf9-derived SARS-CoV-2 spike protein recombinant vaccine candidates expedited by the use of glycopeptide libraries.

We report the N-glycosylation pattern of Sf9 insect cell-derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19) (Sanofi). The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing datasets from individual batches from first principles. Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data-dependent acquisition mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification were performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time. Two different spike proteins from six manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to those of earlier reports. Our data clearly show that the overall glycosylation pattern of both spike protein variants was highly similar from batch to batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell-derived spike protein and the results were comparable to published findings.

Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens.

Mass spectrometry-driven glycomics and glycoproteomics, the system-wide profiling of detached glycans and intact glycopeptides from biological samples, respectively, are powerful approaches to interrogate the heterogenous glycoproteome. Efforts to develop integrated workflows employing both glycomics and glycoproteomics have been invested since the concerted application of these complementary approaches enables a deeper exploration of the glycoproteome. This protocol paper outlines, step-by-step, an integrated -omics technology, the "glycomics-assisted glycoproteomics" method, that first establishes the N-glycan fine structures and their quantitative distribution pattern of protein extracts via porous graphitized carbon-LC-MS/MS. The N-glycome information is then used to augment and guide the challenging reversed-phase LC-MS/MS-based profiling of intact N-glycopeptides from the same protein samples. Experimental details and considerations relating to the sample preparation and the N-glycomics and N-glycoproteomics data collection, analysis, and integration are discussed. Benefits of the glycomics-assisted glycoproteomics method, which can be readily applied to both simple and complex biological specimens such as protein extracts from cells, tissues, and bodily fluids (e.g., serum), include quantitative information of the protein carriers and site(s) of glycosylation, site occupancy, and the site-specific glycan structures directly from biological samples. The glycomics-assisted glycoproteomics method therefore facilitates a comprehensive view of the complexity and dynamics of the heterogenous glycoproteome.

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.

Glycosylation network mapping and site-specific glycan maturation in vivo

Glycoprotein processing along a complex highly compartmentalized pathway is a hallmark of eukaryotic cells. We followed the kinetics of intracellular, site-specific glycan processing of a model protein with five distinct N-glycosylation sites and deduced a mathematical model of the secretory pathway that describes a complex set of processing reactions localized in defined intracellular compartments such as the endoplasmic reticulum the Golgi, or the lysosome. The model was able to accommodate site-specific N-glycan processing and we identified phosphorylated glycan structures of the mannose-6-phosphate pathway responsible for the lysosomal sorting of the glycoprotein. Importantly, our model protein can take different routes of the cellular secretory pathway, resulting in an increased glycan complexity of the secreted protein.