Characterization Of Glycans Research Articles

Assessment of monoclonal antibody glycosylation: a comparative study using HRMS, NMR, and HILIC-FLD

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.Graphical

Computational tools and algorithms for ion mobility spectrometry-mass spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS or IM-MS) is a powerful analytical technique that combines the gas-phase separation capabilities of IM with the identification and quantification capabilities of MS. IM-MS can differentiate molecules with indistinguishable masses but different structures (e.g., isomers, isobars, molecular classes, and contaminant ions). The importance of this analytical technique is reflected by a staged increase in the number of applications for molecular characterization across a variety of fields, from different MS-based omics (proteomics, metabolomics, lipidomics, etc.) to the structural characterization of glycans, organic matter, proteins, and macromolecular complexes. With the increasing application of IM-MS there is a pressing need for effective and accessible computational tools. This article presents an overview of the most recent free and open-source software tools specifically tailored for the analysis and interpretation of data derived from IM-MS instrumentation. This review enumerates these tools and outlines their main algorithmic approaches, while highlighting representative applications across different fields. Finally, a discussion of current limitations and expectable improvements is presented.

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.

Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study.

Lung cancer is the leading cause of cancer-related death, with high morbidity and mortality rates due to the lack of reliable methods for diagnosing lung cancer at an early stage. Low-dose computed tomography can help detect abnormal areas in the lungs, but only 16% of cases are diagnosed early. Tests for lung cancer markers are often employed to determine genetic expression or mutations in lung carcinogenesis. Serum glycome analysis is a promising new method for early lung cancer diagnosis as glycopatterns exhibit significant differences in lung cancer patients. In this study, we employed a solid-phase chemoenzymatic method to systematically compare glycopatterns in benign cases, adenocarcinoma before and after surgery, and advanced stages of adenocarcinoma. Our findings indicate that serum high-mannose levels are elevated in both benign cases and adenocarcinoma, while complex N-glycans, including fucose and 2,6-linked sialic acid, are downregulated in the serum. Subsequently, we developed an algorithm that utilizes 16 altered N-glycans, 7 upregulated and 9 downregulated, to generate a score based on their intensity. This score can predict the stages of cancer progression in patients through glycan characterization. This methodology offers a potential means of diagnosing lung cancer through serum glycome analysis.

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

Ultraviolet Photodissociation Permits Comprehensive Characterization of O-Glycopeptides Cleaved with O-Glycoprotease IMPa.

Complete O-glycosite characterization, including identification of the peptides, localization of the glycosites, and mapping of the glycans, has been a persistent challenge in O-glycoproteomics owing to the technical challenges surrounding O-glycan analysis. Multi-glycosylated peptides pose an even greater challenge owing to their potential heterogeneity. Ultraviolet photodissociation (UVPD) can localize multiple post-translational modifications and is well-suited for the characterization of glycans. Three glycoproteins were assessed based on a strategy combining the use of O-glycoprotease IMPa and HCD-triggered UVPD for the complete characterization of O-glycopeptides. This approach localized multiple adjacent or proximal O-glycosites on individual glycopeptides and identified a previously unknown glycosite on etanercept at S218. Nine different glycoforms were characterized as a multi-glycosylated peptide from etanercept. The performance of UVPD was compared to that of HCD and EThcD for the localization of O-glycosites and the characterization of the constituent peptides and glycans.

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

Recent advances of data‐independent acquisition mass spectrometry‐based proteomics

Fucose Migration Pathways Identified Using Infrared Spectroscopy**

AbstractFucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry.

Fucose Migration Pathways Identified Using Infrared Spectroscopy.

Fucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry.

A rapid and convenient enzyme digestion method for the analysis of N-glycans using exoglycosidase-impregnated polyacrylamide gels fabricated in an automatic pipette tip.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.

Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Mechanistic Study into Free Radical-Activated Glycan Dissociations through Isotope-Labeled Cellobioses.

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radical-induced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13C and/or 18O isotopes into cellobiose to study the mechanisms for free radical-induced dissociation of glycans. The eight isotope-labeled cellobioses include 1-13C, 3-13C, 1'-13C, 2'-13C, 3'-13C, 4'-13C, 5'-13C, and 1'-13C-4-18O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y1 + 0,4X0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as 1,5X0 + H, 1,4X0 + H, 2,4X0 + H-OH, Y1 + 0,4X0, 2,5X1-H, 3,5X0-H, 0,3X0-H, 1,4X0-H, and B2-3H, are confidently assigned. The mechanisms for the formations of these ions are investigated and supported by quantum chemical calculations. These ions are generally initiated by hydrogen abstraction followed by sequential β-elimination and/or radical migration. Here, the mechanistic study for free radical-induced glycan dissociation allows us to interpret all of the free radical-induced fragment ions accurately and, therefore, enables the differentiation of stereochemical isomers. Moreover, it provides fundamental knowledge for the subsequent development of bioinformatics tools to interpret the complex free radical-induced glycan spectra.

Mass spectrometry-based glycoprofiling of biopharmaceuticals by using an automated data processing tool: SimGlycan®.

The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography-tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography-tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography-mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography-tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan® , etc., can display pivotal analytical capabilities in automated liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.

Lectin Analysis of SARS-CoV-2-Positive Nasopharyngeal Samples Using GLYcoPROFILE® Technology Platform.

Nasopharyngeal samples are currently accepted as the standard diagnostic samples for nucleic acid amplification testing and antigenic testing for the SARS-CoV-2 virus. In addition to the diagnostic capacity of SARS-CoV-2-positive crude nasopharyngeal samples, their qualitative potential for direct glycan-specific analysis, in order to uncover unique glycol profiles, was assessed. In this study we provide glycan characterization of SARS-CoV-2-positive and -negative nasopharyngeal samples directly from lectin interactions. Although with limited throughput, this study evaluated the clinical sensitivity and specificity of the GLYcoPROFILE® technology platformon45crude nasopharyngeal samples collected between November 2020 and April 2022. Each GLYcoPROFILE® of 39 SARS-CoV-2-positive samples was compared toglycoprofiling on a panel of 10 selected lectins and the results were paralleled with SARS-CoV-2-negative samples' results. The GLYcoPROFILE® showed a clear distinction between positive and negative samples with WFA, GSL-II, PHA-L (GlcNAc-specific) and BPA (GalNAc-specific) highlighted as relevant lectins in SARS-CoV-2-positive samples. In addition, a significant, positive statistical correlation was found for these lectins (p < 0.01).

Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Modification of Morphology and Glycan Pattern of the Oviductal Epithelium of Baboon Papio hamadryas during the Menstrual Cycle.

Simple SummaryThe oviduct or Fallopian tube is the anatomical region where fertilization and early embryonic development take place. The morphology and activity of the epithelial cells are under the control of the sex hormone levels. The non-ciliated cells secrete oviduct-specific estrogen-dependent glycoproteins whose glycan component, despite its key role in the oviductal function, has not yet been studied in baboon species. This study examined the morphology and the glycan composition of the oviductal epithelium of the baboon Papio hamadryas during the menstrual cycle. Different segments of the laparoscopically removed oviducts from healthy adult females during the follicular, preovulatory, and luteal phases were processed. The stage of the menstrual cycle was based on the sex hormone levels and the vaginal cytology features. The morphological and glycopattern analyses revealed that in the epithelium lining, all the oviductal segments were taller, more differentiated, and richer in glycoconjugates during the preovulatory phase than in the follicular and luteal phases. A region-specific glycosylation pattern was also detected. These results provide an insight into the molecular differences occurring in the oviductal regions during the phases of the menstrual cycle of the baboon oviduct, which is a primate phylogenetically close to humans and whose reproductive tract is similar to that of women and influenced by the same hormonal events.The mammalian oviduct is a highly specialized structure where fertilization and early embryonic development occur. Its mucosal epithelium is involved in maintaining and modulating a dynamic intraluminal fluid. The oviductal epithelium consists of ciliated and non-ciliated (secretory) cells whose differentiation and activity are sex hormone-dependent. In this study, we investigated for the first time both the morphology and the glycan composition of baboon oviductal epithelium during the menstrual cycle. Oviducts were laparoscopically removed from 14 healthy adult female Papio hamadryas whose menstrual cycle phase was assessed based on the sex hormone levels and the vaginal cytology features. Histological investigations were carried out on fimbriae, infundibulum, ampulla, and isthmus separately fixed in 4% (v/v) paraformaldehyde, embedded in paraffin wax, and stained with hematoxylin-eosin for morphological analyses and using a panel of nine fluorescent lectins for glycoconjugate characterization. The histomorphological analysis revealed that in the entire oviduct (i) the ciliated and non-ciliated cells were indistinguishable during the follicular and luteal phases, whereas they were highly differentiated during the preovulatory phase when the non-ciliated cells exhibited apical protrusions, (ii) the epithelium height was significantly higher in the preovulatory phase compared to other menstrual phases, and (iii) the number of ciliated cells significantly (p ≤ 0.05) increased from the fimbriae to the infundibulum and progressively reduced in the other oviductal segments with the lower presence of ciliated cells in the isthmus. The glycan characterization revealed a complex and region-specific composition during the different phases of the menstrual cycle. It can be summarized as follows: (i) high-mannosylated N-linked glycans (Con A reactivity) were present throughout the oviductal epithelium during the entire menstrual cycle and characteristically in the apical protrusions of non-ciliated cells of the ampulla during the preovulatory phase; (ii) sialoglycans with α2,3-linked sialic acids (MAL II binding) were expressed along the entire oviductal surface only during the preovulatory phase, whereas α2,6-linked ones (SNA affinity) were also detected in the surface of the luteal phase, although during the preovulatory phase they were characteristically found in the glycocalyx of the isthmus cilia, and O-linked sialoglycans with sialic acids linked to Galβl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA) were found in the entire oviductal surface during all phases of the menstrual cycle; (iii) GalNAc terminating O-linked glycans (HPA staining) were mainly expressed in the entire oviducts of the luteal and preovulatory phases, and characteristically in the apical protrusions of the isthmus non-ciliated cells of the preovulatory phase; and (iv) fucosylated glycans with α1,2-linked fucose (LTA reactivity) occurred in the apical surface of fimbriae during the luteal phase, whereas α1,3/4-linked fucose (UEA I binders) were present in the apical protrusions of the ampulla non-ciliated cells and in the apical surface of isthmus during the preovulatory phase as well as in the isthmus apical surface of follicular-phase oviducts. These results demonstrate for the first time that morphological and glycan changes occur in the baboon oviductal epithelium during the menstrual cycle. Particularly, the sex hormone fluctuation affects the glycan pattern in a region-specific manner, probably related to the function of the oviductal segments. The findings add new data concerning baboons which, due to their anatomical similarity to humans, make an excellent model for female reproduction studies.

Linkage-specific identification and quantification of sialylated glycans by TIMS-TOF MS through conjugation with metal complexes

Mass spectrometry is an indispensable technology for the characterization of glycans. However, specific identification of isomeric glycans especially sialylated glycan isomers using mass spectrometry alone is challenging, which is why orthogonal techniques are needed. Aiming to achieve simple, rapid, and specific identification of sialyl-linkage isomers, we reported herein a trapped ion mobility spectrometry time of flight mass spectrometry (TIMS-TOF MS) method for linkage-specific identification of sialylated glycans through conjugation with metal complexes. Two pairs of sialyl-linkage isomers including 3'/6′-sialyllactose (3'/6′-SL) and 3'/6′-sialyl-N-acetyllactosamine (3'/6′-SLN) conjugated with the diethylenetriamine (DETA) or 2,2′; 6′,2″-terpyridine (Terpy) ligand and transition metal ion (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, or Zn2+) were studied by TIMS-TOF MS. The two pairs of sialylated isomers were successfully separated with a metal-ligand system, and relative quantification of sialyl-linkage isomers was demonstrated. In addition, the linkage of the sialic acid moiety can also be distinguished with MS/MS in combination with the metal-ligand system.

Development of mass spectrometric glycan characterization tags using acid-base chemistry and/or free radical chemistry.

Despite recent advances in glycomics, glycan characterization still remains an analytical challenge. Accordingly, numerous glycan-tagging reagents with different chemistries were developed, including those involving acid-base chemistry and/or free radical chemistry. Acid-base chemistry excels at dissociating glycans into their constituent components in a systematic and predictable manner to generate cleavages at glycosidic bonds. Glycans are also highly susceptible to depolymerization by free radical processes, which is supported by results observed from electron-activated dissociation techniques. Therefore, the free radical activated glycan sequencing (FRAGS) reagent was developed so as to possess the characteristics of both acid-base and free radical chemistry, thus generating information-rich glycosidic bond and cross-ring cleavages. Alternatively, the free radical processes can be induced via photodissociation of the specific carbon-iodine bond which gives birth to similar fragmentation patterns as the FRAGS reagent. Furthermore, the methylated-FRAGS (Me-FRAGS) reagent was developed to eliminate glycan rearrangements by way of a fixed charged as opposed to a labile proton, which would otherwise yield additional, yet unpredictable, fragmentations including internal residue lossesor multiple external residue losses. Lastly, to further enhance glycan enrichment and characterization, solid-support FRAGS was developed.

Isolation and characterization of high-mannose type glycans containing five or six mannose residues from hen egg yolk

High-mannose type glycans play important roles in biosynthesis of glycoproteins including glycoprotein quality control system. In the endoplasmic reticulum (ER), α1,2-mannosidases cleave several mannose (Man) residues to give small high-mannose type glycans, such as glycans containing five or six mannose residues (M5-glycan or M6-glycan). These glycans are reported to act as a signal for degradation processes of glycoproteins in the ER. In this work, we isolated the M5-glycan and the M6-glycan from delipidated egg yolk and confirmed that their structures were identical to human type glycans based on rigorous NMR experiments, suggesting the potential use for semisynthesis of glycoconjugates and glycan analysis.

Characterizing M-protein light chain glycosylation via mass spectrometry

ObjectivesMonoclonal gammopathy of undetermined significance (MGUS) patients with M-proteins containing n-glycosylated light chains (GLC) have an increased risk for progression to symptomatic plasma cell disorders (PCD). Large-scale research involving the determination of glycan specific moieties is understudied due to the lack of clinically viable methods. This report documents a proof-of-concept glycan characterization method for patients with M-protein GLCs. Design and methodsTwenty-three previously characterized MGUS patients with glycosylated light chains identified by MASS-FIX were used for this study. Glycosylated light chains were enriched from patient serum using light chain (LC) specific Sepharose nanobody beads (NB), followed by glycan digestion via PNGase F. Glycan moieties were derivatized on-target using Girard’s reagent T for MALDI-TOF analysis and confirmed with top-down GLC LC-ESI-Q-TOF-MS analysis. ResultsIntact GLC LC-ESI-Q-TOF-MS and cleaved glycan MALDI-TOF MS analysis had 100% agreement for the top three intensity glycans between spectra and 88 percent agreement for all reported glycan moieties. GLC moieties among patients were similar with fucosylation being the only notable difference. Additionally, doubly glycosylated light chains were observed in two patients. ConclusionsThe MALDI-TOF method provides the tools to characterize and evaluate GLCs in a clinical setting as it is adaptable to our clinical MASS-Fix assay, relatively cheap, and accurate in glycan moiety assignments as confirmed by top-down GLC LC-ESI-Q-TOF-MS.