N-glycan Compositions Research Articles

An N-glycome tissue atlas of 15 human normal and cancer tissue types determined by MALDI-imaging mass spectrometry

N-glycosylation is an abundant post-translational modification of most cell-surface proteins. N-glycans play a crucial role in cellular functions like protein folding, protein localization, cell–cell signaling, and immune detection. As different tissue types display different N-glycan profiles, changes in N-glycan compositions occur in tissue-specific ways with development of disease, like cancer. However, no comparative atlas resource exists for documenting N-glycome alterations across various human tissue types, particularly comparing normal and cancerous tissues. In order to study a broad range of human tissue N-glycomes, N-glycan targeted MALDI imaging mass spectrometry was applied to custom formalin-fixed paraffin-embedded tissue microarrays. These encompassed fifteen human tissue types including bladder, breast, cervix, colon, esophagus, gastric, kidney, liver, lung, pancreas, prostate, sarcoma, skin, thyroid, and uterus. Each array contained both normal and tumor cores from the same pathology block, selected by a pathologist, allowing more in-depth comparisons of the N-glycome differences between tumor and normal and across tissue types. Using established MALDI-IMS workflows and existing N-glycan databases, the N-glycans present in each tissue core were spatially profiled and peak intensity data compiled for comparative analyses. Further structural information was determined for core fucosylation using endoglycosidase F3, and differentiation of sialic acid linkages through stabilization chemistry. Glycan structural differences across the tissue types were compared for oligomannose levels, branching complexity, presence of bisecting N-acetylglucosamine, fucosylation, and sialylation. Collectively, our research identified the N-glycans that were significantly increased and/or decreased in relative abundance in cancer for each tissue type. This study offers valuable information on a wide scale for both normal and cancerous tissues, serving as a reference for future studies and potential diagnostic applications of MALDI-IMS.

The glycosylation landscape of prostate cancer tissues and biofluids.

An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.

Site-specific glycoproteomic analysis of purified lactoferrin from human and animal milk

Lactoferrin is a highly glycosylated protein, which have important biological functions in the growth and development of neonates. However, the glycoforms and glycosylation sites differed between species. The aim of the study was to identify the glycosylation profile (including glycosites, glycan structures, and glycoforms) of purified lactoferrin from human and animal (cow, goat, sheep) milk by using site-specific glycoproteomics technique. In total, a number of 89 N-glycans were identified in human and animal milk lactoferrin. We identified three N-glycosites with 23 different compositions of N-glycans in cow lactoferrin (CLF), four distinctive N-glycosites with 34 dissimilar N-glycan compositions in goat lactoferrin (GLF), five N-glycosites with 57 different N-glycan compositions in sheep lactoferrin (SLF), while five unique N-glycosites with 50 different N-glycan compositions were ascertained in human lactoferrin (HLF). HLF had the most complex glycan, while animal lactoferrin had the most high-mannose glycoforms. The results of this study further our understanding of lactoferrin differences between human and animal milk, which can provide a perspective on the analysis of differences in functional characteristics.

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.

Elucidation of N-/O-glycosylation and site-specific mapping of sialic acid linkage isomers of SARS-CoV-2 human receptor angiotensin-converting enzyme 2.

Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2-3)Gal (over 80.3%) than that of other N-glycosites (35.6-71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells.

Characterization of site-specific N-glycosylation signatures of isolated uromodulin from human urine.

Uromodulin (Umod, Tamm-Horsfall protein) is the most abundant urinary N-glycoprotein produced exclusively by the kidney. It can form filaments to antagonize the adhesion of uropathogens. However, the site-specific N-glycosylation signatures of Umod in healthy individuals and patients with IgA nephropathy (IgAN) remain poorly understood due to the lack of suitable isolation and analytical methods. In this study, we first presented a simple and fast method based on diatomaceous earth adsorption to isolate Umod. These isolated glycoproteins were digested by trypsin and/or Glu-C. Intact N-glycopeptides with or without HILIC enrichment were analyzed using our developed EThcD-sceHCD-MS/MS. Based on the optimized workflow, we identified a total of 780 unique intact N-glycopeptides (7 N-glycosites and 152 N-glycan compositions) from healthy individuals. As anticipated, these glycosites exhibited glycoform heterogeneity. Almost all N-glycosites were modified completely by the complex type, except for one N-glycosite (N275), which was nearly entirely occupied by the high-mannose type for mediating Umod's antiadhesive activity. Then, we compared the N-glycosylation of Umod between healthy controls (n = 9) and IgAN patients (n = 9). The N-glycosylation of Umod in IgAN patients will drastically decrease and be lost. Finally, we profiled the most comprehensive site-specific N-glycosylation map of Umod and revealed its alterations in IgAN patients. Our method provides a high-throughput workflow for characterizing the N-glycosylation of Umod, which can aid in understanding its roles in physiology and pathology, as well as serving as a potential diagnostic tool for evolution of renal tubular function.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

In-depth profiling of urinary N-glycome in diabetic kidney disease by ultrafast glycoprotein immobilization for glycan extraction (UltraGIG)

Diabetic kidney disease (DKD) is a common and serious kidney-related complication of diabetic mellitus. Although albuminuria and estimated glomerular filtration rate (eGFR) are commonly used diagnostic biomarkers, they have limitations in the diagnosis of DKD due to the lack of specificity and sensitivity. Urinary N-glycans are emerging as novel biomarkers for predicting renal prognosis in DKD. However, most of the current N-glycan profiling methods for DKD were based on lectin affinity enrichment or hydrophilic interaction chromatography (HILIC) with optical detection, which provided limited N-glycome coverage in low throughput. Herein, we developed a novel N-glycan purification method, termed Ultrafast Glycoprotein Immobilization for Glycan extraction (UltraGIG), for in-depth profiling of urinary N-glycome in DKD using mass spectrometry (MS). In UltraGIG, proteins were rapidly (within 40 min) immobilized to resin with high efficiency (over 98%) through NHS-reactive chemistry, and then N-glycans were enzymatically released from the resin by PNGase F. Owing to good efficiency of immobilization of proteins to resin, the subsequent washing steps to remove proteins and other impurities could be easily performed. The UltraGIG showed good selectivity towards glycans (extracting N-glycans from the mixture of IgG and BSA at a 1:100 M ratio) and samples loss was minimized (detection sensitivity at fmol level). A total of 237 N-glycan compositions were identified from urine samples with DKD and healthy controls, which representing the largest data set of N-glycome from DKD. Compared to healthy controls, 3 N-glycans were up-regulated and 14 N-glycans were down-regulated in DKD patients. Collectively, UltraGIG offers a competitive sample purification method for in-depth analysis of urinary N-glycome by MS and provide new insights into biomarker study for the diagnosis of DKD.

Novel Affinity Chromatography Ligand Utilizing the Biological Interaction Between the N‐linked glycans on Monoclonal Antibodies and the FcγIIIA Receptor

Glycosylation is one of the major post‐translational modifications (PTMs) in proteins, with significant effects on protein folding, conformation, stability, and activity. Glycosylation is particularly important for therapeutic protein drugs, such as monoclonal antibodies (mAbs), as it can profoundly impact their pharmacological properties, stability, and safety. Therefore, understanding the inherent heterogeneity of mAb glycoforms is an integral part of their manufacturing processes and quality control.Most marketed mAbs belong to the immunoglobulin G1 (IgG1), IgG2, or IgG4 subclass, carrying N‐glycosylation at the conserved asparagine position 297 (Asn‐297) in the CH2 domain of the Fc region. N‐glycosylation is critical for the Fc receptor binding and consequently affects key antibody effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC). However, assessing ADCC activity using a cell‐based assay remains a complicated, time‐consuming, and expensive task for laboratories.Here, we present a novel workflow leveraging FcγR‐IIIA biology to identify N‐glycan compositions and how to chromatographically determine changes in glycosylation in a time and cost‐effective manner. The TSKgel® FcR‐IIIA analytical and semi‐preparative columns separate mAbs into multiple subsets based on their affinity to the FcγR‐IIIA ligand. Utilizing an increasingly acidic pH gradient, mAb glycoforms are eluted from the column in an order that correlates to their ADCC activity. The analytical TSKgel FcR‐IIIA‐NPR column provides rapid assessment of a mAb's glycoform distribution, whereas the semipreparative TSKgel FcR‐IIIA‐5PW column enables collection of the peaks for more complex orthogonal released N‐glycan analyses and N‐glycosylation profiling using peptide mapping technique.With several mAbs containing various glycosylation patterns, we directly correlated the N‐glycan nature, structure, content, and elution profile on the FcR‐IIIA columns. Furthermore, we present a direct correlation between higher ADCC activity and binding affinity on the FcR‐IIIA columns, indicating that the complexity of the mAb glycosylation pattern is reflected in the observed FcR‐IIIA elution profile. Finally, employing both qualitative and quantitative assessments, we quantified the impact of individual N‐glycan structure and composition on mAb binding to the FcR ligand, providing an additional dimension to our understanding of the FcR‐IIIA columns.This in‐depth correlation analysis provides improved tools to predict, understand, and analyze changes in FcR‐IIIA elution profiles and how changes in the N‐glycan composition influence them. Altogether, this study describes a new framework for utilizing the TSKgel FcR‐IIIA columns for glycosylation profiling, providing a powerful way to simultaneously capture, profile, and characterize the glycosylation state of a given mAb of interest in a comprehensive manner.

Glycomics reveal that ST6GAL1‐mediated sialylation regulates uterine lumen closure during implantation

ObjectivesImplantation failure is a major cause of prenatal mortality. The uterine lumen closure contributes to embryo adhesion to the uterus, but its underlying mechanisms are largely unknown. Our previous study has reported that endometrial fold extension can lead to uterine lumen closure in pigs. The objective of this study was to reveal molecular mechanisms of the uterine lumen closure by characterizing the molecular basis of the endometrial fold extension during implantation in pigs.Materials and methodsUterine and endometrium tissues during implantation were collected in pigs. MALDI‐TOF MS was used to characterize the N‐glycomic profiles. Histochemistry, siRNA transfection, Western blotting, lectin immumoprecipitation, mass spectrometry and assays of wounding healing and cell aggregation were performed to investigate the molecular basis.ResultsWe observed that uterine luminal epithelium (LE) migrated collectively during endometrial fold extension. For the first time, we identified a large number of N‐glycan compositions from endometrium during implantation using MALDI‐TOF MS. Notably, the α2,6‐linked sialic acid and ST6GAL1 were highly expressed in uterine LE when the endometrial folds extended greatly. Subsequently, the role of ST6GAL1‐mediated 2,6‐sialylation in collective epithelial migration was demonstrated. Finally, we found that ST6GAL1‐mediated α2,6‐sialylation of E‐cadherin may participate in collective migration of uterine LE.ConclusionsThe study reveals a mechanism of uterine lumen closure by identifying that ST6GAL1‐mediated α2,6‐sialylation of cell adhesion molecules contributes to endometrial fold extension through regulating collective migration of uterine LE.

Isomer-Specific Monitoring of Sialylated N-Glycans Reveals Association of α2,3-Linked Sialic Acid Epitope With Behcet's Disease.

Behcet’s disease (BD) is an immune disease characterized by chronic and relapsing systemic vasculitis of unknown etiology, which can lead to blindness and even death. Despite continuous efforts to discover biomarkers for accurate and rapid diagnosis and optimal treatment of BD, there is still no signature marker with high sensitivity and high specificity. As the link between glycosylation and the immune system has been revealed, research on the immunological function of glycans is being actively conducted. In particular, sialic acids at the terminus of glycoconjugates are directly implicated in immune responses, cell–cell/pathogen interactions, and tumor progression. Therefore, changes in sialic acid epitope in the human body are spotlighted as a new indicator to monitor the onset and progression of immune diseases. Here, we performed global profiling of N-glycan compositions derived from the sera of 47 healthy donors and 47 BD patients using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to preferentially determine BD target glycans. Then, three sialylated biantennary N-glycans were further subjected to the separation of linkage isomers and quantification using porous graphitized carbon-liquid chromatography (PGC-LC)/multiple reaction monitoring (MRM)-MS. We were able to successfully identify 11 isomers with sialic acid epitopes from the three glycan compositions consisting of Hex5HexNAc4NeuAc1, Hex5HexNAc4Fuc1NeuAc1, and Hex5HexNAc4NeuAc2. Among them, three isomers almost completely distinguished BD from control with high sensitivity and specificity with an area under the curve (AUC) of 0.945, suggesting the potential as novel BD biomarkers. In particular, it was confirmed that α2,3-sialic acid at the terminus of biantennary N-glycan was the epitope associated with BD. In this study, we present a novel approach to elucidating the association between BD and glycosylation by tracing isomeric structures containing sialic acid epitopes. Isomer-specific glycan profiling is suitable for analysis of large clinical cohorts and may facilitate the introduction of diagnostic assays for other immune diseases.

Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles.

Expressed prostatic secretions (EPS), also called post digital rectal exam urines, are proximal fluids of the prostate that are widely used for diagnostic and prognostic assays for prostate cancer. These fluids contain an abundant number of glycoproteins and extracellular vesicles secreted by the prostate gland, and the ability to detect changes in their N-glycans composition as a reflection of disease state represents potential new biomarker candidates. Methods to characterize these N-glycan constituents directly from clinical samples in a timely manner and with minimal sample processing requirements are not currently available. In this report, an approach is described to directly profile the N-glycan constituents of EPS urine samples, prostatic fluids and urine using imaging mass spectrometry for detection. An amine reactive slide is used to immobilize glycoproteins from a few microliters of spotted samples, followed by peptide N-glycosidase digestion. Over 100 N-glycan compositions can be detected with this method, and it works with urine, urine EPS, prostatic fluids, and urine EPS-derived extracellular vesicles. A comparison of the N-glycans detected from the fluids with tissue N-glycans from prostate cancer tissues was done, indicating a subset of N-glycans present in fluids derived from the gland lumens. The developed N-glycan profiling is amenable to analysis of larger clinical cohorts and adaptable to other biofluids.

P–591 Mass spectrometry-based glycomic profiling of the follicular fluid total immunoglobulin G and proteome N-glycomes reveals deregulated inflammatory processes associated with specific controlled ovarian stimulation protocols

Abstract Study question Is there a significant inflammatory-related difference between analyzed follicular fluid (FF) glycome profiles regarding the ovarian stimulation protocol used in patients? Summary answer Observed differences between analyzed glycome profiles from patients that underwent different controlled ovarian stimulation (COS) protocols point to deregulated inflammatory processes associated with specific COS. What is known already Successful physiological folliculogenesis and ovulation require an adequate inflammatory response. On the other hand, COS application in ART relies on induced hormonal activation of systemic inflammatory processes. Several studies have confirmed a rise in inflammatory cytokines, CRP, and other markers of inflammation in patients subjected to different COS protocols, pointing to an enhanced inflammatory response during ovulation stimulation. Glycoproteins and glycans have an indisputable role in immune response modulation: proper glycosylation of glycoproteins plays a pivotal role in the regulation of normal physiological processes, and aberrant glycosylation of glycoproteins has been associated with various pathological states, including inflammation. Study design, size, duration Study design: Cross sectional – FFs from patients that underwent ART in modified natural cycle (MNC group) versus FFs from patients that underwent ART under GnRH antagonist COS (COS group). Size: 20 FFs from 20 patients undergoing ART. Duration: One year. Sampling procedure: Each FF was aspirated from the dominant follicle. In the COS group, only the fluid from the first aspirated follicle of each patient was collected. Participants/materials, setting, methods Study included 20 FF samples from 20 patients divided into two groups according to the applied ovarian stimulation protocol: MNC group (n = 10) and COS group (n = 10). The immunoglobulin G (IgG) was isolated from FF samples by immunoaffinity chromatography. The N-linked glycans derived from IgG molecule and the remaining FF total proteomes were enzymatically cleaved and subjected to derivatization procedure. N-glycomes of FF-isolated IgG and total proteomes were analyzed separately by MALDI-TOF-MS. Main results and the role of chance FF IgG N-glycome profiling The MALDI-TOF-MS based comparative analysis of the individual glycan relative abundances, revealed several significantly deregulated glycoforms between analyzed groups whose levels were significantly elevated (p˂0.05) in the COS vs. MNC group. Furthermore, additional low abundant N-glycan species were also found to be deregulated between the analyzed groups: two monogalactolysed and monosialylated N-glycan compositions were only identified in the COS group. The comparative analysis of FF IgG N-glycome features revealed statistically relevant differences in the levels of two derived traits: galactosylation and bigalactosylation levels of the FF IgG N-glycome, both significantly downregulated (p˂0.05) in the MNC vs. COS profile. Comparative analysis of FF total proteome N-glycome The majority of identified glycan compositions were complex type N-glycans representing more than 98% of the total N-glycome profiles in both analyzed groups. The comparative analysis of individual glycan relative abundances revealed relevant differences in regulation of ten N-glycan species between the two analyzed profiles. In the MNC group, six N-glycan species showed significantly increased abundances (p˂0.05) compared with the COS group. Moreover, two compositions were exclusively identified in the MNC group, while two compositions were identified only in the COS group. Limitations, reasons for caution Since this preliminary study was conducted on relatively small sample size, all results should be verified on a larger sample set. Moreover, focused glycosylation analysis of a panel of individual FF acute phase blood serum derived proteins and immunoglobulins, might additionally clarify the inflammatory mechanisms underlying different ART stimulation protocols. Wider implications of the findings: While glycome profiling of human FFs was conducted for the first time, previous evidence supports the shown association of aberrant inflammation in diverse ART stimulation protocols and in development of various pathological states (i.e. OHSS). Obtained results are in line with previous similar studies performed in the human plasma. Trial registration number uniri-biomed–18–161 1310

Characterization of serum N-glycome alterations in seasonal allergic rhinitis using MALDI-TOF-MS: A pilot study

Seasonal allergic rhinitis (SAR) is an inflammatory process. In this pilot study, matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS)-based analyses were conducted to investigate the effects of SAR on serum N-glycome in a small dataset (n = 10 for both SAR patients and controls). It was detected that two N-glycan compositions (H6N5E2L1 and H6N5E1L2) were down-regulated in SAR patients. Additionally, five tri-antennary N-glycan traits, including both galactosylated and sialylated (A3GS and A3GL) and non-fucosylated (A3F0GE, A3F0GL, A3F0GS) ones, were decreased. Furthermore, three high-antennary and fucosylated N-glycan traits (A3F, A3EF and A4F) were increased in SAR patients compared to controls.

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry.

Simple SummaryIncidence of colorectal cancer (CRC) has been rising in Brazil. To date, no reliable biomarker has been described in CRC for diagnosis and prognosis. Modifications in the N-glycosylation profile are usually associated with many cancers, as CRC. In turn, mass spectrometry (MS)-based methods are the most accurate technology in quantification of N-glycans. Therefore, we described a unique pattern of compositions altered in serum and tissues of stages II and III colon cancer patients, identified by MALDI-TOF/MS and LC-MS technology. N-glycans were mostly found decreased in serum whilst oligomannosidic, hypogalactosylated, and tetra-antennary forms were overexpressed in tumor tissues. Total N-glycome in serum of cancer patients was different from the profile found in serum of healthy individuals. Strikingly, no correlation between tissue N-glycosylation profile and serum profile was observed in cancer patients, posing the question where these compositions are originated from.Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.

Comparative Analysis of Different N-glycan Preparation Approaches and Development of Optimized Solid-Phase Permethylation Using Mass Spectrometry.

Protein N-glycosylation characterization is challenging due to structural micro- and macro-heterogeneity. Although various N-glycan preparation strategies, including purification and derivatization, have been previously developed prior to mass spectrometric analysis, systematic evaluation still needs to be performed. This study compared the different N-glycan purification strategies, including filter-aided sample preparation, de-N-glycosylated protein precipitation, and trypsin digestion followed by reversed phase-based solid-phase extraction, and derivatization approaches, such as solid-phase permethylation, reductive amination, and reduction. With the comparative analysis, an optimized solid-phase permethylation (OSPP) workflow was developed for mass spectrometric N-glycomics, showing simplified analysis for N-glycan compositions and high yields using etanercept. The N-glycan samples released from trastuzumab and adalimumab were utilized to test OSPP to obtain their N-glycan profiles using mass spectrometry. Based on different standard procedures across laboratories, this study provides the reference for analysts to select an appropriate N-glycan preparation method with their research purposes.

Uromodulin Isolation and Its N-Glycosylation Analysis by NanoLC-MS/MS.

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.

Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry.

With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].

Region-Specific Cell Membrane N-Glycome of Functional Mouse Brain Areas Revealed by nanoLC-MS Analysis

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell–cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in “brain-type” glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease.