Structural Analysis Of Glycans Research Articles

Direct Identification of Complex Glycans via a Highly Sensitive Engineered Nanopore.

The crucial roles that glycans play in biological systems are determined by their structures. However, the analysis of glycan structures still has numerous bottlenecks due to their inherent complexities. The nanopore technology has emerged as a powerful sensor for DNA sequencing and peptide detection. This has a significant impact on the development of a related research area. Currently, nanopores are beginning to be applied for the detection of simple glycans, but the analysis of complex glycans by this technology is still challenging. Here, we designed an engineered α-hemolysin nanopore M113R/T115A to achieve the sensing of complex glycans at micromolar concentrations and under label-free conditions. By extracting characteristic features to depict a three-dimensional (3D) scatter plot, glycans with different numbers of functional groups, various chain lengths ranging from disaccharide to decasaccharide, and distinct glycosidic linkages could be distinguished. Molecular dynamics (MD) simulations show different behaviors of glycans with β1,3- or β1,4-glycosidic bonds in nanopores. More importantly, the designed nanopore system permitted the discrimination of each glycan isomer with different lengths in a mixture with a separation ratio of over 0.9. This work represents a proof-of-concept demonstration that complex glycans can be analyzed using nanopore sequencing technology.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.

Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple.

The Golgi apparatus is one of the major sites of protein and lipid glycosylation and processing. Protein N-glycosylation plays critical roles in protein folding, transport, stability, and activity. Various glycosyltransferases and glycoside hydrolases are localized at each cisterna in the Golgi apparatus and synthesize a large variety of N-glycan structures. The biosynthetic pathways of N-glycans are complicated, which hiders the rational design of glycan metabolic pathways. In addition, the analysis of glycan structure requires specialized instruments for analyses such as mass spectrometry, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, which are not familiar to all laboratories. Here, we introduce relatively simple methods for N-glycan analysis, including disruption of genes encoding glycosyltransferases or glycoside hydrolases, glycan structural analysis using lectins and mass spectrometry, and visualization of glycan metabolic pathways in silico.

Fast and Ultrasensitive Glycoform Analysis by Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

The glycoform of a therapeutic monoclonal antibody (mAb) has a significant impact on its effector function as well as its safety and pharmacokinetics. Glycoform heterogeneity is influenced by various factors, including the producing cells and cell culture processes. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) and applied the technique to the glycan structural analysis of mAbs. To achieve both the highest sensitivity and the most comprehensive glycan profiling, we integrated our energy-resolved oxonium ion monitoring (Erexim) method with SFC-MS to construct a new SFC-Erexim technology. An 8 min analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-Erexim detected a total of 102 glycoforms, with a detection limit of 5 attomoles. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-Erexim compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. The SFC-MS-based glycoform profiling method will provide an ideal platform for the in-depth analysis of precise glycan structure and abundance.

Synergistic use of glycomics and single-molecule molecular inversion probes for identification of congenital disorders of glycosylation type-1.

Congenital disorders of glycosylation type 1 (CDG‐I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG‐I is a partial absence of complete N‐glycans on transferrin. However, recent findings of a diagnostic N‐tetrasaccharide for ALG1‐CDG and increased high‐mannose N‐glycans for a few other CDG suggested the potential of glycan structural analysis for CDG‐I gene discovery. We analyzed the relative abundance of total plasma N‐glycans by high resolution quadrupole time‐of‐flight mass spectrometry in a large cohort of 111 CDG‐I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single‐molecule molecular inversion probes (smMIPs) for sequencing of CDG‐I candidate genes on the basis of specific N‐glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N‐tetrasaccharide in ALG2‐CDG patients and a novel fucosylated N‐pentasaccharide as specific glycomarker for ALG1‐CDG. Moreover, group‐specific high‐mannose N‐glycan signatures were found in ALG3‐, ALG9‐, ALG11‐, ALG12‐, RFT1‐, SRD5A3‐, DOLK‐, DPM1‐, DPM3‐, MPDU1‐, ALG13‐CDG, and hereditary fructose intolerance. Further differential analysis revealed high‐mannose profiles, characteristic for ALG12‐ and ALG9‐CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG‐I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG‐I patient cohorts.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Exposure of silver nanocolloids causes glycosylation disorders and embryonic deformities in medaka

Silver nanomaterials such as silver nanocolloids (SNC) contribute to environmental pollution and have adverse ecological effects on aquatic organisms. In particular, chemical exposure of fish during embryogenesis leads to deformities and puts the population at risk. Although glycans and glycosylation are known to be important for proper morphology in embryogenesis, little glycobiology-based research has examined morphological disorders caused by environmental pollutants. This study addressed the glycobiological effects of SNC exposure on medaka embryogenesis. After exposure of medaka embryos to SNC, deformities such as small heads and deformed eyes were observed. The expression of five glycan-related genes (alg2, gnsb, b4galt2, b3gat1a, and b3gat2) was significantly altered, with changes depending on the embryonic stage at exposure, with more severe deformities with exposure at earlier stages. In situ hybridization analyses indicated that the five genes were expressed mainly in the head region; exposure of SNC suppressed alg2 and gnsb and enhanced b4galt2 and b3gat1a expression relative to controls on day 7. Loss (siRNA)- and gain (RNA overexpression)-of-function experiments confirmed that alg2, gnsb, and b4galt2 are essential for embryogenesis. The effects of SNC exposure on glycan synthesis were estimated by glycan structure analysis. In the medaka embryo, high mannose–type glycans were dominant, and SNC exposure altered glycan synthesis. The alteration was more significant when exposure occurred at an early stage of medaka embryogenesis. Thus, SNC exposure causes embryonic deformities in medaka embryos through disordered glycosylation.

Practical Use of Analytical Tools for Glycan Structures Related to Functionality and Safety of Biopharmaceuticals

It is known that glycosylation of biopharmaceuticals relates to drug potency and half-life in blood. Since glycan modification is essential for the effector effect in antibody drugs, biopharmaceuticals are produced by using biosynthesis of mammalian cells but not microorganisms like as Escherichia coli. In particular, a biantennary N-glycan attached on Fc region of antibody is an important factor for interaction with its receptor FcRIIIa, and lack of a core-fucose residue attached to innermost GlcNAc at reducing end of N-glycan dramatically enhances interaction between FcRIIIa and IgG. Recently, it is reported that a particular galactose residue at non-reducing end of biantennary N-glycan also promotes the interaction with FcRIIIa receptor. On the other hand, glycan structure of post-translational modification by the use of mammalian-derived cells for production of biopharmaceuticals is different from that of human in some cases, and non-human type glycans is observed sometimes. For instance, high-mannose N-glycan is observed in unpreferable process step of manufacturing production and non-human type glycans are found depend on host cell type. Antigenicity of α1,3galactose (α1,3Gal) antigen and N-glycolylneuraminic acid (NeuGc) are observed at non-human type glycans well-known as xenoantigen. Because human lacks the enzyme activities of α1,3Gal and NeuGc synthesis for during the process of evolution with acquired gene mutations, immune response may act as a exogeneous antigen when they are injected to human patients. Therefore, contamination of antigenic glycosylation is considered to affect the safety of biopharmaceuticals. Currently, general standards have not been established regarding methods for glycan analysis from the viewpoint of the functionality and safety of biopharmaceuticals. Furthermore, it seems that the comprehensive supply system of glycan standards and recognition probes is still partial. We will report our developed tools regarding glycan standards and recognition probes. The glycan standards are available for quantitative analysis of reducing terminal core fucose and non-reducing terminal galactose (G2 and 6-G1 glycans) to evaluate the effector function of antibody drugs. The recognition probes derived from chicken are expected to be applied for qualitative analysis of an allergy risk factor caused by xenoantigen (α1,3Gal and NeuGc epitopes). The tools developed in this study would contribute to the analysis of glycan structures that affect the quality of biopharmaceuticals.

Cell engineering for the production of hybrid-type N-glycans in HEK293 cells.

Glycoprotein therapeutics are among the leading products in the biopharmaceutical industry. The heterogeneity of glycans in therapeutic proteins is an issue for maintaining quality, activity and safety during bioprocessing. In this study, we knocked out genes encoding Golgi α-mannosidase-II, MAN2A1 and MAN2A2 in human embryonic kidney 293 (HEK293) cells, establishing an M2D-KO cell line that can produce recombinant proteins mainly with hybrid-type N-glycans. Furthermore, FUT8, which encodes α1,6-fucosyltransferase, was knocked out in the M2D-KO cell line, establishing a DF-KO cell line that can express noncore fucosylated hybrid-type N-glycans. Two recombinant proteins, lysosomal acid lipase and constant fragment of human IgG1, were expressed in the M2D-KO and DF-KO cell lines. Glycan structural analysis revealed that complex-type N-glycans were removed in both M2D-KO and DF-KO cells. Our results suggest that these cell lines are suitable for the production of therapeutic proteins with hybrid-type N-glycans. Moreover, KO cell lines would be useful as models for researching the mechanism of antimetastatic effects in human tumours by swainsonine treatment.

Advances in Structural Analysis of Glycans

Advances in Structural Analysis of Glycans

Advances in Structural Analysis of Glycans

Advances in Structural Analysis of Glycans

Increased yield of enzymatic synthesis by chromatographic selection of different N-glycoforms of yeast invertase.

Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl β-d-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.

Altered glycosylation of glycodelin in endometrial carcinoma

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

Flow Chemistry System for Carbohydrate Analysis by Rapid Labeling of Saccharides after Glycan Hydrolysis.

This study demonstrates the utilization of a flow chemistry system for continuous glycan hydrolysis and saccharide labeling to assist with the existing methods in glycan structural analysis. Acidic hydrolysis of glycans could be accelerated in a flow system. Aldoses and α-ketoacid-type saccharides were effectively labeled with naphthalene-2,3-diamine (NADA) at 60 °C for 10 min to form the fluorescent naphthimidazole (NAIM) and quinoxalinone (QXO) derivatives, respectively. The NADA-labeled derivatives improved the structural determination and composition analysis for their parent saccharides by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Furthermore, this protocol was applied to determine the SA–Gal–Glc sequence of GM3-sugar out of six possible permutations.

Multistage mass spectrometry with intelligent precursor selection for N-glycan branching pattern analysis

Biological functions of N-glycans are frequently related to their unique branching patterns. Multistage mass spectrometry (MSn) has become the primary method for glycan structural analysis. However, selection of the best fragment as the precursor for the next round of product-ion scanning is important but difficult. We have previously proposed the concept and designed the approach of glycan intelligent precursor selection (GIPS) to guide MSn experiments, but its use in N-glycans is not straightforward as some N-glycans are of high similarity in branching patterns. In the present work we introduced new elements to GIPS to improve its performance in N-glycan branching pattern analysis. These include a hypothesis and significance test, based on Bayes factor, and DPbiased as a new precursor selection strategy. The improved GIPS was successfully applied to identification of individual N-glycans, and incorporated into MALDI-MS N-glycan profiling for assignment of N-glycans obtained from glycoproteins and complex human serum.

Toward Automatic and Comprehensive Glycan Characterization by Online PGC-LC-EED MS/MS.

Despite the recent advances in mass spectrometry (MS)-based methods for glycan structural analysis, characterization of glycomes remains a significant analytical challenge, in part due to the widespread presence of isomeric structures and the need to define the many structural variables for each glycan. Interpretation of the complex tandem mass spectra of glycans is often laborious and requires substantial expertise. Broad adoption of MS methods for glycomics, within and outside the glycoscience community, has been hindered by the shortage of bioinformatics tools for rapid and accurate glycan sequencing. Here, we developed an online porous graphitic carbon liquid chromatography (PGC-LC)-electronic excitation dissociation (EED) MS/MS method that takes advantage of the superior isomer resolving power of PGC and the structural details provided by EED MS/MS for characterization of glycan mixtures. We also made improvements to GlycoDeNovo, our de novo glycan sequencing algorithm, so that it can automatically and accurately identify glycan topologies from EED tandem mass spectra acquired online. The majority of linkages can also be determined de novo, although in some cases, biological insight may be needed to fully define the glycan structure. Application of this method to the analysis of N-glycans released from ribonuclease B not only revealed the presence of 18 high-mannose structures, including new isomers not previously reported, but also provided relative quantification for each isomeric structure. With fully automated data acquisition and topology analysis, the approach presented here holds great potential for automated and comprehensive glycan characterization.

Surface Effects of Iron Oxide Nanoparticles on the MALDI In-Source Decay Analysis of Glycans and Peptides

Inorganic nanoparticles as MALDI matrices have recently been explored to study the molecular mass determination and structural analysis of glycans and peptides. However, the specific factors contributing to the success of the analysis are not well elucidated. In this paper, we investigated the roles of nanoparticle surface coatings and additive ions in MALDI in-source decay (ISD) analysis of model glycans and peptides. Specifically, iron oxide nanoparticles with four defined capping molecules (gluconic acid, citric acid, lactobionic acid, or glutathione) were tested, and the roles of additives (NH4OH, NaOH, LiOH, NaCl, or trifluoroacetic acid) were examined. For a model glycan, maltoheptaose, and a model peptide, substance P acid, nanoparticle capping molecules, additive cations, and additive anions altogether influenced the molecular ion sensitivity and ISD fragmentation efficiency.

Glycoengineering of HEK293 cells to produce high-mannose-type N-glycan structures.

Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1, MAN1A2 and MAN1B1, in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.

GENETICS OF RESPONSE TO COGNITIVE BEHAVIOR THERAPY IN ADULTS WITH MAJOR DEPRESSION

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

High-Throughput Automated Micro-permethylation for Glycan Structure Analysis.

Permethylation is an essential technique for the detailed structural characterization of glycans by mass spectrometry. However, it requires specialized skills and equipment and is labor-intensive. While this limits glycan analysis to a handful of experts, the increasing awareness of the tremendous importance of glycans in physiological processes of health and disease has drastically raised the demand for detailed structural characterization of glycans. We have developed a simple microplate permethylation method that relies upon solid phase extraction using C18 tips to purify the permethylated glycans. The procedure is easy to perform, making it accessible to nonexperts, and fast, promising to accelerate glycan research. A 96-well plate of released glycan samples can be permethylated in less than an hour. The procedure can be carried out without discontinuous steps in an automatic pipet and integrated seamlessly with glycan release and mass spectrometry.