Glycosylation Analysis Research Articles

Altered Pattern of Serum N-Glycome in Subarachnoid Hemorrhage and Cerebral Vasospasm

Background: Subarachnoid hemorrhage is a serious condition caused by ruptured intracranial aneurysms, resulting in severe disability mainly in young adults. Cerebral vasospasm is one of the most common complication of subarachnoid hemorrhage; thus, active prevention is key to improve the prognosis. The glycosylation of proteins is a critical quality attribute which is reportedly altered in patients diagnosed with acute ischemic stroke. In this study, we examined the N-glycosylation profile of serum glycoproteins in patients with subarachnoid hemorrhage without vasospasm compared to patients with vasospasm. Methods: The serum N-glycans were released by PNGase F (Peptide: N-glycosidase F) digestion and subsequently labeled by procainamide via reductive amination. The samples were analyzed by hydrophilic-interaction liquid chromatography after solid-phase extraction-based sample purification. Results: Besides the glycosylation pattern, we also investigated the biomarkers following subarachnoid hemorrhage. Multiple statistical analyses were performed in order to find significant differences and identify potential prediction factors of cerebral vasospasm. Significant differences were identified such as higher sialylation on bi-, tri-, and tetra-antennary structures in patients with subarachnoid hemorrhage and cerebral vasospasm. Conclusions: Our results suggest that glycosylation analysis can improve the identification of patients with cerebral vasospasm in combination with laboratory parameters.

Preparation of a titanium-functionalized polymeric material rich in hydrophilic groups for phosphoproteome and glycoproteome analyses in serum.

The analysis of protein phosphorylation and glycosylation is critical for investigating disease development. In this work, 1,2-epoxy-5-hexene and N,N-methylenebisacrylamide were polymerized with vinyl phosphate to produce a polymer (denoted as PVME), which contained a variety of hydrophilic groups. The material's hydrophilicity was further enhanced by a ring-opening reaction with cysteine (the product was denoted as Cys-PVEM). Finally, titanium sulfate was combined with Cys-PVME to form titanium-rich polymers (Cys-PVME-Ti4+) for the enrichment of phosphopeptides and glycopeptides. Cys-PVME-Ti4+ has a good sensitivity (0.02 fmol) and selectivity (1 : 1000) with a loading capacity of 62 mg g-1, recyclability (9 cycles), and a good recovery rate (101.6 ± 0.60%) for phosphopeptides, and good sensitivity (0.01 fmol μL-1), selectivity (1 : 2000), a loading capacity of 62.5 mg g-1, recyclability (9 cycles), and a good recovery rate (98.7 ± 1.2%) for glycopeptides. In addition, after enrichment with this material, 27 phosphopeptides with 14 phosphoproteins and 223 glycopeptides associated with 88 glycoproteins were captured from the serum of colorectal cancer patients, while 27 phosphopeptides associated with 14 phosphoproteins and 210 glycopeptides associated with 111 glycoproteins were also captured from the serum of a normal control. Gene ontology (GO) analysis revealed that complement activation, extracellular region, extracellular space, blood coagulation, the IgG immunoglobulin complex, and heparin binding were different between normal control and colorectal cancer, implying that related pathways are likely involved in colorectal cancer pathogenesis.

SLC35A2 loss of function variants affect glycomic signatures, neuronal fate, and network dynamics.

SLC35A2 encodes a UDP-galactose transporter essential for glycosylation of proteins and galactosylation of lipids and glycosaminoglycans. Germline genetic SLC35A2 variants have been identified in congenital disorders of glycosylation and somatic SLC35A2 variants have been linked to intractable epilepsy associated with malformations of cortical development. However, the functional consequences of these pathogenic variants on brain development and network integrity remain elusive. In this study, we use an isogenic human induced pluripotent stem cell-derived neuron model to comprehensively interrogate the functional impact of loss of function variants in SLC35A2 through the integration of cellular and molecular biology, protein glycosylation analysis, neural network dynamics, and single cell electrophysiology. We show that loss of function variants in SLC35A2 result in disrupted glycomic signatures and precocious neurodevelopment, yielding hypoactive, asynchronous neural networks. This aberrant network activity is attributed to an inhibitory/excitatory imbalance as characterization of neural composition revealed preferential differentiation of SLC35A2 loss of function variants towards the GABAergic fate. Additionally, electrophysiological recordings of synaptic activity reveal a shift in excitatory/inhibitory balance towards increased inhibitory drive, indicating changes occurring specifically at the pre-synaptic terminal. Our study is the first to provide mechanistic insight regarding the early development and functional connectivity of SLC35A2 loss of function variant harboring human neurons, providing important groundwork for future exploration of potential therapeutic interventions.

Advances in the development of N-glycopeptide enrichment materials based on hydrophilic interaction chromatography.

Protein glycosylation is one of the most important post-translational modifications, implicated in the development of various diseases, including neurodegenerative diseases, diabetes, and cancers. However, the low content of glycoproteins in biological samples, the diversity and heterogeneity of glycan structures, and insensitive detection methods make glycosylation analysis challenging. As a result, efficient enrichment of glycopeptides from complex samples is a critical step. Efficient enrichment technology can increase the abundance of intact N-glycopeptides in complex biological samples, thereby improving the sensitivity and coverage of glycosylation analysis, which is of great significance for the accurate identification of biomarkers and the development of glycopeptide-based drugs. Among various separation methods for N-glycopeptides, hydrophilic interaction chromatography has received increasing attention, and a variety of enrichment materials have been developed. This article classifies and describes the relevant hydrophilic interaction chromatography materials and provides a comprehensive review of their applications in N-glycopeptide enrichment regarding selectivity, sensitivity, and enrichment performance. Future development trends of ideal glycopeptide enrichment materials are also discussed.

Phylogenetic and phylodynamic analysis of respiratory syncytial virus strains circulating in children less than five years of age in Karachi-Pakistan

BackgroundRespiratory syncytial virus (RSV) is one of the leading causes of infant morbidity and mortality worldwide, especially in Pakistan. To date, few studies have explored RSV epidemiology in different areas of Pakistan. However, none have performed comprehensive phylogenetic and phylodynamic analyses of RSV strains. This study presents a comprehensive genetic and phylodynamic analysis of RSV strains in children under five years in Karachi, Pakistan. MethodsThis study used retrospectively collected nasopharyngeal (swab) samples from 155 children with qPCR-confirmed RSV infection. The samples were used to perform RSV genotyping using PCR employing RSV glycoprotein gene-specific primers. The RSVA and RSVB genotyping was performed using BLAST and Maximum-likelihood (ML) phylogenetic methods. Similarly, the relationship with other RSV strains was analyzed using ML phylogenetic cluster analysis. The RSVA and RSVB mean genetic diversity and coefficient of differentiation were calculated using MEGA7 software. Furthermore, the time to the most common recent ancestor (tMRCA) and effective population size of RSV genotypes A and B were estimated using a Bayesian MCMC analysis. Finally, site selection pressure and glycosylation analyses were performed using FUBAR and NetNGlyc/NetOGlyc tools. ResultsOut of 155, 98 and 57 sequences were RSVA and RSVB, respectively. The tMRCA was estimated to be around 2002 and 2005 for RSVA and RSVB, respectively. RSVA sequences formed two NA1 genotype clusters, comprising 95 and three sequences, respectively. RSVB formed three clusters, where 24 and two sequences clustered with BA9 and BA12 genotypes, respectively, while 31 sequences formed a unique cluster. The RSVA and RSVB glycoprotein gene sequences exhibited N- and O- glycosylation and selection pressure at several sites. RSV B exhibited slightly higher (0.042) nucleotide diversity per site (π) as compared to RSVA (0.019). ConclusionsOur results suggest that RSVA and RSVB strains in Pakistan exhibit distinct genotypic clusters and differ in their estimated tMRCA. Additionally, both genotypes showed glycosylation and selection pressure at specific sites, with RSVB exhibiting higher nucleotide divergence per site (π), indicating its potential to undergo further evolutionary changes and adaptation. Overall, this study provides unique insights into RSV molecular epidemiology. The study may also help improve our understanding of RSV evolutionary changes and the emergence of new genotypes in different regions worldwide and within Pakistan.

Glycosylation analysis of transcription factor TFIIB using bioinformatics and experimental methods

Transcription is a fundamental process involving the interaction of RNA polymerase II and related transcription factors. TFIIB is a transcription factor that plays a significant role in the formation and stability of the preinitiation complex in a precise orientation, as well as in the control of initiation and pre-elongation steps. At the initiation step, TFIIB interacts with three structures: the end of the TATA-binding protein, a GC-rich DNA sequence followed by the TATA box, and the C-terminal domain of RNA polymerase II. It is known that RNA polymerase II is a glycoprotein and contains O-GlcNAc sugar at the C-terminal domain during the initiation stage of transcription. However, it is unclear whether the transcription factors interacting with RNA polymerase II are glycoproteins or not. The study aims to determine the glycosylation (N- and/or O-linked glycosylations) of TFIIB by using bioinformatics in one invertebrate and seven vertebrate species and experimental methods in the sea urchin Paracentrotus lividus oocyte. Both bioinformatics and experimental analysis have shown that TFIIB is a glycoprotein. In addition, PNGase-F enzyme treatment, lectin blotting, and colloidal-gold conjugated lectin labeling results revealed that TFIIB contains O-linked GalNAc, mannose, GlcNAc, and α-2,3-linked sialic acid. Based on our results, we suggest that glycosylation modification may be involved in the transcription mechanism of the TFIIB protein.

Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies.

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions.

Role of N-Glycosylation in Gastrointestinal Cancers.

Gastrointestinal cancers pose a significant global health challenge. N-glycosylation modulates various cellular processes, including key cancer-related mechanisms. Elucidating its involvement in the onset and advancement of these cancers can offer critical insights for enhancing diagnostic and therapeutic approaches. This review outlines the core process of protein N-glycosylation and highlights its contribution to the progression of gastrointestinal cancers, encompassing cell proliferation, survival, invasion, metastasis, and immune evasion, mainly through its impact on critical signaling pathways. Notably, aberrant N-glycosylation patterns have emerged as crucial biomarkers for the diagnosis and prognosis of various gastrointestinal cancers, providing the foundation for more personalized therapeutic approaches. Therapeutic strategies targeting N-glycosylation, such as glycosyltransferase inhibitors and glycoengineering, show significant promise in mitigating tumor aggressiveness and enhancing immune recognition. However, the clinical implementation of N-glycosylation biomarkers requires the standardization of glycosylation analysis techniques and solutions to challenges in sample processing and data interpretation. Future research efforts should concentrate on overcoming these obstacles to unlock the full potential of N-glycosylation in enhancing cancer management and advancing patient outcomes.

Mapping and functional analysis of S1 subunit glycosylation of avian infectious bronchitis virus

Glycosylation of the S1 subunit is a critical post-translational modification in coronavirus antigen proteins and plays a key role in vaccine development. The biological role of highly mutable glycosylation sites in virus evolution, however, remains underexplored. In this study, we identified nine glycosylation sites with high mutation rates on the S1 subunit of Avian Infectious Bronchitis Virus (IBV) through evolutionary analysis. Functional analysis of mutant strains revealed that specific mutations, particularly at position 76 (N → V), significantly enhance the strain's protective efficacy and limit viral replication. These findings provide important insights into the role of glycosylation in viral evolution and offer valuable guidance for the development of more effective IBV vaccines.

Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc., which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC-MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage but also preserved the glycan structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38, and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38, and N83 sites were 82.7%, 100%, and 100% respectively, and 15, 10, and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid, and O-acetylation on sialic acid were 7.28, 4.21, and 0.66 at the intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference of the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC-MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.

5105 Characterization Of Novel PHEX Variants In X-Linked Hypophosphatemic Rickets And Genotype-PHEX Activity Correlation

Abstract Disclosure: H. Wu: None. W. Zhao: None. X. Chen: None. L. Gao: None. C. Xu: None. J. Zhao: None. X-linked hypophosphatemia (XLHR) is the most common genetic form of hypophosphatemic rickets (HR), which is caused by mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Till now, the genotype-phenotype relationship of XLHR and the pathogenic role of PHEX have not been fully determined. Here we summarized clinical features of 49 HR patients and detected 17 novel PHEX variants and 5 novel non-PHEX variants. Then we explored the pathogenesis of new-found variants from protein expression, glycosylation analysis, subcellular localization and endopeptidase activity. The results showed that missense variants (Q189H and X750R) slightly reduced protein expression without obviously altering the length and localization of the protein, while truncating variants significantly impaired the synthesis of PHEX and produced a shorter immature protein retained in cells. No evident correlation was observed between mutation types and clinical phenotypes. However, for the first time ever, we analyzed the relationship between PHEX activity and serum phosphorus level and found that patients with low PHEX activity tended to have severe hypophosphatemia and high rickets severity score (RSS). Then we established two new knock-in XLHR mouse models by introducing into mice two novel Phex variants (c.T1349C and c.C426G) found in patients using CRISPR/Cas9 technology. Both demonstrated the clinical manifestations of XLHR seen in the patients and PhexC426G mice showed more severe phenotype than PhexT[1]349C mice, which further confirmed the rationality of genotype-PHEX enzymatic activity correlation analysis. Therefore, our findings demonstrated that novel PHEX variants could disrupt protein function via affecting protein synthesis, post-translational modification, cellular trafficking and catalytic activity. Our study promoted the understanding of XLHR pathogenic mechanism and PHEX activity-phenotype correlation provided scientific support for precise diagnosis and treatment of XLHR. Presentation: 6/3/2024

An explore method for quick screening biomarkers based on effective enrichment capacity and data mining

Protein glycosylation acts as a crucial role in regulating protein function and maintaining cellular homeostasis. Efficient peptide enrichment can be utilized to effectively solve the inherent challenges of protein glycosylation analysis to search unknown cancer biomarkers. In this research, a low dimensional porous hydrophilic nanosheets with a multi-level porous structure (Co-MOF-SiO2@HA) was synthetized via an easy one-pot method for the efficient enrichment of the N-glycopeptides in the digests of complex biosamples. The synthetized nanosheets Co-MOF-SiO2@HA demonstrated excellent enriching performances including a high enrichment capacity (300 mg g-1 calculated), a spectacular selectivity (IgG digests and BSA digests at the molar ratio of 1/1200), and an excellent spatial confinement ability (IgG digests, IgG and BSA at the molar ratio of 1/1000/1000). As an explore result, after the enrichment of human colorectal cancer tissue and human healthy tissue by the nanosheets, several proteins related to cancers and one protein directly related to well-known human colorectal cancer were identified by detecting the corresponding glycopeptides. It presented the potential value of the feasibility of this analysis mode by nanosheets Co-MOF-SiO2@HA in proteomic analysis.

GlyCompute: towards the automated analysis of protein N-linked glycosylation kinetics via an open-source computational framework.

Understanding the complex biosynthetic pathways of glycosylation is crucial for the expanding field of glycosciences. Computer-aided glycosylation analysis has greatly benefited in recent years from the development of tools found in web-based portals and open-source libraries. However, the in silico analysis of cellular glycosylation kinetics is underrepresented in current glycoscience-related tools and databases. This could be partly attributed to the limited accessibility of kinetic models developed using proprietary software and the difficulty in reliably parameterising such models. This work aims to address these challenges by proposing GlyCompute, an open-source framework demonstrating a novel, streamlined approach for the assembly, simulation, and parameterisation of kinetic models of protein N-linked glycosylation. Specifically, given one or more sets of experimentally observed N-glycan structures and their relative abundances, minimum representations of a glycosylation reaction network are generated. The topology of the resulting networks is then used to automatically assemble the material balances and kinetic mechanisms underpinning the mathematical model. To match the experimentally observed relative abundances, a sequential parameter estimation strategy using Bayesian inference is proposed, with stages determined automatically based on the underlying network topology. The proposed framework was tested on a case study involving the simultaneous fitting of the kinetic model to two protein N-linked glycoprofiles produced by the same CHO cell culture, showing good agreement with experimental observations. We envision that GlyCompute could help glycoscientists gain quantitative insights into the effect of enzyme kinetics and their perturbations on experimentally observed glycoprofiles in biomanufacturing and clinical settings.

Uncovering missing glycans and unexpected fragments with pGlycoNovo for site-specific glycosylation analysis across species

Precision mapping of site-specific glycans using mass spectrometry is vital in glycoproteomics. However, the diversity of glycan compositions across species often exceeds database capacity, hindering the identification of rare glycans. Here, we introduce pGlycoNovo, a software within the pGlyco3 software environment, which employs a glycan first-based full-range Y-ion dynamic searching strategy. pGlycoNovo enables de novo identification of intact glycopeptides with rare glycans by considering all possible monosaccharide combinations, expanding the glycan search space to 16~1000 times compared to non-open search methods, while maintaining accuracy, sensitivity and speed. Reanalysis of SARS Covid-2 spike protein glycosylation data revealed 230 additional site-specific N-glycans and 30 previously unreported O-glycans. pGlycoNovo demonstrated high complementarity to six other tools and superior search speed. It enables characterization of site-specific N-glycosylation across five evolutionarily distant species, contributing to a dataset of 32,549 site-specific glycans on 4602 proteins, including 2409 site-specific rare glycans, and uncovering unexpected glycan fragments.

Direct glycosylation analysis of intact monoclonal antibodies combining ESI MS of glycoforms and MALDI-in source decay MS of glycan fragments

Monoclonal antibody (mAb) glycoengineering has the potential to improve the efficacy of biopharmaceuticals by fine-tuning specific biological properties. Glycosylation analysis is key to monitoring the glycoengineering process. Various mass spectrometry (MS)-based methods are available to characterize mAb glycosylation at different structural levels, but comprehensive analysis is typically time-consuming and costly. Here, we present an approach that combines conventional intact mass measurement of glycoforms by direct infusion ESI-MS with an advanced MALDI-in-source decay FT-ICR MS method for direct analysis of glycans in intact mAbs, without the need for enzymatic release and separation. Using a sodium-doped MALDI matrix, glycans were directly released as ISD fragment ions from the intact mAbs during the ionization process. Measurement of 0,2A fragment signals yielded reproducible glycan profiles that were consistent with conventional methods, yet was achieved with unprecedented speed, providing complementary information to that obtained through intact mass measurement. The methods were applied to standard and glycoengineered trastuzumab and rituximab, allowing rapid glycosylation profiling and structural analysis of glycans by tandem MS of selected ISD fragment ions. This fast approach can facilitate the early-phase development of glycoengineering processes by constraining further in-depth analyses. We envision a broader applicability in studies focused on glycosylation changes in mAbs.

Investigation of the distribution and origin of porcine reproductive and respiratory syndrome virus 1 in the swine production chain: A retrospective study of three farms in Thailand.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a global issue that affects Thai swine as well. In Thailand, PRRSV-2 predominates over PRRSV-1. The origin of PRRSV-1 transmission remains undiscovered. This study traced the source of infected pigs responsible for disease transmission among three pig-fattening farms and analyzed the spread of PRRSV-1. A total of 696 swine samples from breeding and pig-fattening farms in Thailand were screened for PRRSV using open reading frames (ORF7) reverse transcription polymerase chain reaction (RT-PCR). Positive samples were identified as PRRSV-1 using ORF5 RT-PCR. The analysis included the study of nucleotide homology, GP5 amino acid sequences, and N-linked glycosylation patterns to assess the spread of PRRSV-1 across these farms. Genetic examination identified 28 PRRSV-1-positive samples, of which 13 were chosen as representatives. These strains were categorized into three groups based on breeding farm pig houses and showed distinct distribution patterns across pig-fattening farms. Group 1 included piglets transferred from pig house A to Nakhon Pathom, Chonburi, and Sa Kaeo. Groups 2 and 3 showed transfers from pig houses F and H to Chonburi and Sa Kaeo farms. All 13 PRRSV-1 strains were categorized into PRRSV-1 subtype 1/clade H. N-linked glycosylation analysis revealed that nearly all PRRSV-1 strains exhibited a conserved glycosylation pattern at amino acid positions N37, N46, and N53. This pattern is consistent with the glycosylation profile of the previous Thai PRRSV-1 subtype 1/clade H. The present study highlights the persistent presence of PRRSV-1 in Thai swine, which leads to sporadic outbreaks. The molecular genetic analysis identified three primary strain groups dispersed throughout the pig production system, emphasizing the importance of regular monitoring for new PRRSV strains in this herd. Understanding the PRRSV-1 distribution in swine farms is vital for veterinarians. This knowledge supports strategies for eradicating the virus and managing swine health effectively in Thailand.

Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions.

Glycans are oligosaccharides attached to proteins or lipids and affect their functions, such as drug efficacy, structural contribution, metabolism, immunogenicity, and molecular recognition. Conventional glycosylation analysis has relied on destructive, slow, system-sensitive methods, including enzymatic reactions, chromatography, fluorescence labeling, and mass spectrometry. Herein, we propose quantum cascade laser (QCL) infrared (IR) spectroscopy as a rapid, nondestructive method to quantify glycans and their monosaccharide composition. Previously, we demonstrated high-sensitivity IR spectroscopy of protein solution using solvent absorption compensation (SAC) and double-beam modulation (DBM) techniques. However, the SAC-DBM approach suffered a limited frequency scanning range (<400 cm-1) due to the light dispersion by acousto-optic modulators (AOMs). Here, we implemented a mirror-based double-pass AOM in the SAC-DBM scheme and successfully extended the frequency range to (970 to 1840 cm-1), which encompasses the vibrational fingerprint of biomolecules. The extended frequency range allowed the simultaneous observation of monosaccharide ring bands (1000 to 1200 cm-1) and protein amide bands (1500 to 1700 cm-1). We compared the IR spectra of six glycoproteins and two nonglycosylated proteins with the results from intact mass spectrometry. The IR absorbance ratios of the ring band to the amide band of glycoproteins in solutions showed a linear correlation with the ratios of glycan to protein backbone masses. Furthermore, a multivariate analysis produced monosaccharide compositions consistent with the reported database for the glycoproteins, and the monosaccharide compositions were used to improve the predictability of the glycan-protein mass ratio from the IR-absorbance ratio. This nondestructive, high-sensitivity QCL-IR spectroscopy could be used as a standard method to monitor batch-to-batch comparability during drug manufacturing and quantify the glycosylation and monosaccharide composition of new glycoproteins and other glycosylated biosystems.

The role of antibody glycosylation in autoimmune and alloimmune kidney diseases.

Immunoglobulin glycosylation is a pivotal mechanism that drives the diversification of antibody functions. The composition of the IgG glycome is influenced by environmental factors, genetic traits and inflammatory contexts. Differential IgG glycosylation has been shown to intricately modulate IgG effector functions and has a role in the initiation and progression of various diseases. Analysis of IgG glycosylation is therefore a promising tool for predicting disease severity. Several autoimmune and alloimmune disorders, including critical and potentially life-threatening conditions such as systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and antibody-mediated kidney graft rejection, are driven by immunoglobulin. In certain IgG-driven kidney diseases, including primary membranous nephropathy, IgA nephropathy and lupus nephritis, particular glycome characteristics can enhance in situ complement activation and the recruitment of innate immune cells, resulting in more severe kidney damage. Hypofucosylation, hypogalactosylation and hyposialylation are the most common IgG glycosylation traits identified in these diseases. Modulating IgG glycosylation could therefore be a promising therapeutic strategy for regulating the immune mechanisms that underlie IgG-driven kidney diseases and potentially reduce the burden of immunosuppressive drugs in affected patients.

LC-MS/MS of isomeric N-and O-glycopeptides on mesoporous graphitized carbon column

BackgroundThe study of glycopeptides is associated with challenges regarding the microheterogeneity of different isomeric glycans occupying the same glycosylation sites in glycoproteins. It is immensely valuable to perform both qualitative and quantitative site-specific glycosylation analysis of glycopeptide isomers due to their link to several diseases. Achieving isomeric separation of glycopeptides is particularly challenging due to the low abundance of glycopeptides as well as inefficient ionization. Although some methods have demonstrated the isomeric separation of glycopeptides, a more efficient nanoflow-based stationary phase is needed for the isomeric separation of both N- and O-glycopeptides. ResultsIn this study, the separation of N- and O-glycopeptide isomers at 75 °C was achieved with an in-house packed 1 cm long mesoporous graphitized carbon (MGC) column. Different gradient compositions of the optimized mobile phase for separating permethylated glycans on MGC column were tested, and we observed efficient separation of N- and O-glycopeptide isomers at a gradient elution time of 120 min. After achieving the isomeric separation of sialylated glycopeptides from model glycoproteins derived from bovine fetuin, the separation of isomeric glycopeptides derived from asialofetuin, α-1 glycoprotein and human blood serum were also demonstrated. Furthermore, the developed method for the separation of isomeric N- and O-glycopeptide on MGC column showed high reproducibility over three months. We observed an average retention time shift of 1 min and consistent resolution of separated peaks throughout three months. Significance and noveltyMGC column can serve as an efficient tool for obtaining the isomeric separation of N- and O-glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric N- and O-glycopeptide in important biological processes and their correlations to various disease progressions.

The Impact of Protein Glycosylation on the Identification of Patients with Pediatric Appendicitis.

The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as abdominal pain, ultrasonography and altered laboratory parameters (C reactive protein, absolute neutrophil cell number and white blood cell number). The glycosylation pattern of serum N-glycome was analyzed in this study of 38 controls and 40 patients with pediatric appendicitis. The glycans were released by enzymatic deglycosylation followed by fluorescent labeling and solid-phase extraction. The prepared samples were analyzed by hydrophilic interaction liquid chromatography with fluorescence and mass-spectrometric detection. The generated data were analyzed by multiple statistical tests involving the most important laboratory parameters as well. Significant differences associated with the examined patient groups were revealed suggesting the potential use of glycosylation analysis supporting the detection of pediatric appendicitis.