Characterization Of N-glycosylation Research Articles

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.

High-throughput N-glycoproteomics with fast liquid chromatographic separation

N-glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N-glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N-glycosylation at the intact N-glycopeptide level, providing comprehensive information of peptide backbones, N-glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N-glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N-glycopeptides were identified, where 422 site- and structure-specific differential N-glycosylation on 145 N-glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N-glycoproteomes at large scale.

Integration of high-pH fractionation and cotton-HILIC enrichment approaches for analyzing N-glycopeptides

Glycosylation, a prevalent post-translational modification, plays a significant role in diverse biological processes and exhibits associations with various diseases, including cancer. Among the different types of glycosylation, N-glycosylation is particularly prominent. Glycosylation analysis also plays a crucial role in understanding the efficacy of protein biopharmaceuticals, as the majority of these drugs are glycoproteins. The precise detection and characterization of N-glycosylation require a series of enrichment steps. On the other hand, peptide fragmentation methods constitute a crucial step in reducing sample complexity. Moreover, enrichment strategies coupled with mass spectrometry are essential for the analysis and identification of N-glycopeptides. This research aims to enhance the detection methods for N-glycopeptides and N-glycoproteins by employing strategies that involve peptide fractionation and glycopeptide enrichment. This study targeted to assess and compare the efficacy of an integration method and a direct enrichment approach in terms of identifying glycopeptides from the glycoproteome of human plasma. The results demonstrate that the integration method detects 212 N-glycopeptides and 88 N-glycoproteins, while Cotton-HILIC direct enrichment detects 88 N-glycopeptides and 41 N-glycoproteins. In conclusion, the employment of Cotton-HILIC enrichment with high pH fractionation exhibits greater qualitative abilities in characterizing the structures and functions of glycopeptides.

In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein

COVID-19 is caused by SARS-CoV-2 infection and remains one of the biggest pandemics around the world since 2019. Vaccination has proved to be an effective way of preventing SARS-CoV-2 infection and alleviating the hospitalization burden. Among different forms of COVID-19 vaccine design, the spike protein of SARS-CoV-2 virus is widely used as a candidate vaccine antigen. As a surface protein on the virus envelop, the spike was reported to be heavily N-glycosylated and glycosylation had a great impact on its immunogenicity and efficacy. Besides, N-glycosylation might vary greatly on different expression systems and sequence variant designs. Therefore, comprehensive analysis of spike N-glycosylation is of great significance for better vaccine understanding and quality control. In this study, full characterization of N-glycosylation was performed for a Chinese Hamster Ovary (CHO) cell expressed variant-designed spike protein. The spike protein featured the latest six-proline substitution design together with the incorporation of a combination of mutation sites. Trypsin and Glu-C digestion coupled with PNGase F strategies were adopted, and effective LC–MS/MS methods were applied to analyze samples. As a result, a total of 19 N-glycosites were identified in the recombinant pike protein at intact N-glycopeptide level. Quantitative analysis of released glycan by LC–MS/MS was also performed, and 31 high-abundance N-glycans were identified. Sequencing analysis of glycan was further provided to assist glycan structure confirmation. Moreover, all of the analyses were performed on three consecutive manufactured batches and the glycosylation results on both glycosite and glycans showed good batch-to-batch consistency. Thus, the reported analytical strategy and N-glycosylation information may well facilitate studies on SARS-CoV-2 spike protein analysis and quality studies.Graphical

Sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex TMT-labeling

Protein sialylation participates many biological processes in a linkage-specific manner, and aberrant sialylation has been associated with many malignant diseases. Mass spectrometry-based quantitative N-glycoproteomics has been widely adopted for quantitative analysis of aberrant sialylation, yet multiplexing method at intact N-glycopeptides level is still lacking. Here we report our study of sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex tandem mass tags (TMT)-labeling. With lung cancer as a model system, differential sialylation in cancer tissues relative to adjacent non-tumor tissues was characterized at the intact N-glycopeptide level with N-glycosite information. TMT-labeled intact N-glycopeptides with and without sialic acid alkylamidation were subject to reversed-phase liquid chromatography-nano-electron spray ionization-tandem mass spectrometry (RPLC-nanoESI-MS/MS) analysis to provide comprehensive characterization of N-glycosylation with and without sialic acid at the intact N-glycopeptide level with structure and N-glycosite.In this study, 6384 intact N-glycopeptides without sialylation were identified and 521 differentially expressed intact N-glycopeptides from 254 intact N-glycoproteins were quantified. Eight intact N-glycoproteins responsible for N-glycan biosynthesis were identified as glycosyltransferases. In total, 307 sialylated intact N-glycopeptides with linkage-specific sialic acid residues were identified together with 29 N-glycans with α2,6-linked sialic acids and 55 N-glycans with α2,3-linked sialic acids. Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-(COVID)-19. Additionally, many types of N-glycosylation including terminal N-galactosylation, core and/or branch fucosylation, α2,6-sialylation and terminal bisecting N-acetylglucosamine were identified and quantified in intact N-glycoproteins from immunoglobulin family.

Comprehensive site- and structure-specific characterization of N-glycosylation in model plant Arabidopsis using mass-spectrometry-based N-glycoproteomics

The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR ≤ 1%, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge. Wtih the experimental evidence support of structure-diagnostic fragment ions, 81 glycan structures from 54 glcan compostions were unambiguouly distinguished. The comprehensive experimental site- and structure-specific N-glycosylation data reported in this study will serve as a fundamental valuable reference for the coming functional studies of this widely adopted model organism of plant.

Structure-Specific N-Glycoproteomics Characterization of NIST Monoclonal Antibody Reference Material 8671.

The characteristics of monoclonal antibodies (mAbs) cohering various function effectors show great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There has been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentation of N-glycan moieties. Here, we report our structure-specific N-glycoproteomics characterization of NIST monoclonal antibody reference material 8671 using GPSeeker, and 59 N-glycan structures (including 16 pairs of isomers) are characterized.

Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis.

The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.

Post-natal developmental changes in the composition of the rat neocortical N-glycome.

Asparagine-linked glycosylation (N-glycosylation) plays a key role in many neurodevelopmental processes, including neural cell adhesion, neurite outgrowth and axon targeting. However, little is known about the dynamics of N-glycosylation during brain development and, in particular, how the N-glycome of the developing neocortex differs from that of the adult. The aim of this study, therefore, was to perform a thorough characterization of N-glycosylation in both the adult and neonatal rat neocortex in order to gain insights into the types of changes occurring in the N-glycome during neurodevelopment. To this end, we used hydrophilic interaction ultraperformance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry to compare the adult neocortical N-glycome with that of 24- and 48-h neonates. We report that the abundance of complex N-glycans is significantly lower in adults compared with neonates. Furthermore, the proportion of charged complex N-glycans is also greatly reduced. This decrease in the abundance of complex N-glycans is offset by a corresponding increase in the proportion of truncated and, to a lesser extent, hybrid N-glycans. Lastly, we report that although the proportion of oligomannose N-glycans remains constant at around 24%, the distribution of high-mannose subtypes shifts from predominantly large subtypes in neonates to smaller subtypes in the adult. In summary, our findings indicate that N-glycan synthesis in the rat neocortex is fundamentally different in neonates compared with adults with a general shift occurring from large, sialylated N-glycans towards smaller, neutral structures as neonates develop into adults, coupled with a parallel shift towards smaller oligomannose structures.

GPSeeker Enables Quantitative Structural N-Glycoproteomics for Site- and Structure-Specific Characterization of Differentially Expressed N-Glycosylation in Hepatocellular Carcinoma.

N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with the presence of different N-glycan structures at a single specific glycosite. These different structures may have exactly the same monosaccharide composition but totally different differential expressions and pathological relevance. Mass spectrometry-based N-glycoproteomics has so far been successful in large-scale characterization of these N-glycans at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of the intact N-glycopeptide search engine GPSeeker and the GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In benchmark characterization of differentially expressed N-glycosylation in hepatocellular carcinoma HepG2 cells relative to LO2 cells, 5 405 and 1 081 intact N-glycopeptides with putative linkage structures were identified and quantified with isotopic dimethyl labeling and 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among the5 405 IDs, 837 were identified with no less than onestructure diagnostic fragment ion for the N-glycan moieties. Besides double isomers of sialic acid linkages and fucose sequences, quadruple isomers from combination of two linages and two sequences were chromatographically separated and confidently identified; microheterogeneity with different differentially expressions were observed on 183 out of the 231 quantifiedN-glycosites. This GPSeeker-centered quantitative structural N-glycoproteomics pipeline can be widely applied to precise qualitative and quantitative characterization of N-glycosylation with physiological and pathological relevance.

Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Multiplexing N-glycan analysis by DNA analyzer.

Analysis of N-glycan structures has been gaining attentions over the years due to their critical importance to biopharma-based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N-glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio-safety and bio-activity. The ability to comprehensively characterize highly complex mixtures of N-glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time-consuming sample pretreatment procedures. CE-LIF is one of the typical techniques for N-glycan analysis due to its high separation efficiency. In this paper, a 16-capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N-glycan assay throughput. Routinely, the labeling dye used for CE-LIF is 8-aminopyrene-1,3,6-trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross-validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross-validation. The optimized method combines the parallel separation capacity of multiple-capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N-glycans. These new methods provided enough useful structural information to permit N-glycan structure elucidation with only one sample injection.

Quantitative MS analysis of therapeutic mAbs and their glycosylation for pharmacokinetics study

Therapeutic mAbs play an important role in the treatment of a wide range of diseases. Due to their complexity, comprehensive evaluation of their pharmacokinetics has yet to be fully achieved. It is crucial to develop sensitive, accurate, reliable, and reproducible methods for quantitation of mAbs in complex samples. In addition, it is also important to evaluate the PTMs which can affect their safety and/or effectiveness. MS-based methods provide an emerging approach for quantitation of proteins and their modification forms. In this review, we give a brief overview of quantification analysis of mAbs in complex biological samples and the characterization of N-glycosylation by biological MS.

Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine.

The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.

N-Glycosylation and plant cell growth.

N-linked glycosylation is one of the most prevalent cotranslational protein modifications in plants. It is initiated by a conserved process in the endoplasmic reticulum and subsequently involves a series of different N-glycan maturation steps that take place in the ER and Golgi apparatus. Despite our vast knowledge on the different processing steps we still understand very little about the role of characteristic glycoforms present on individual plant glycoproteins. Here, we describe convenient tools that allow the fast and reliable characterization of N-glycosylation on plant glycoproteins. The presented protocols can be adopted to other plant species and to the characterization of N-glycans from different glycoproteins.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

Differential N‐glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.

Different routes to the same ending: comparing the N‐glycosylation processes of Haloferax volcanii and Haloarcula marismortui, two halophilic archaea from the Dead Sea

Recent insight into the N-glycosylation pathway of the haloarchaeon, Haloferax volcanii, is helping to bridge the gap between our limited understanding of the archaeal version of this universal post-translational modification and the better-described eukaryal and bacterial processes. To delineate as yet undefined steps of the Hfx. volcanii N-glycosylation pathway, a comparative approach was taken with the initial characterization of N-glycosylation in Haloarcula marismortui, a second haloarchaeon also originating from the Dead Sea. While both species decorate the reporter glycoprotein, the S-layer glycoprotein, with the same N-linked pentasaccharide and employ dolichol phosphate as lipid glycan carrier, species-specific differences in the two N-glycosylation pathways exist. Specifically, Har. marismortui first assembles the complete pentasaccharide on dolichol phosphate and only then transfers the glycan to the target protein, as in the bacterial N-glycosylation pathway. In contrast, Hfx. volcanii initially transfers the first four pentasaccharide subunits from a common dolichol phosphate carrier to the target protein and only then delivers the final pentasaccharide subunit from a distinct dolichol phosphate to the N-linked tetrasaccharide, reminiscent of what occurs in eukaryal N-glycosylation. This study further indicates the extraordinary diversity of N-glycosylation pathways in Archaea, as compared with the relatively conserved parallel processes in Eukarya and Bacteria.