Publications by authors named "David J Harvey"

157 Publications

FGDB: Database of follicle stimulating hormone glycans.

Comput Struct Biotechnol J 2021 22;19:1635-1640. Epub 2021 Mar 22.

Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, United States.

Glycomics, the study of the entire complement of sugars of an organism has received significant attention in the recent past due to the advances made in high throughput mass spectrometry technologies. These analytical advancements have facilitated the characterization of glycans associated with the follicle-stimulating hormones (FSH), which play a central role in the human reproductive system both in males and females utilizing regulating gonadal (testicular and ovarian) functions. The irregularities in FSH activity are also directly linked with osteoporosis. The glycoanalytical studies have been tremendously helpful in understanding the biological roles of FSH. Subsequently, the increasing number of characterized FSH glycan structures and related glycoform data has thrown a challenge to the glycoinformatics community in terms of data organization, storage and access. Also, a user-friendly platform is needed for providing easy access to the database and performing integrated analysis using a high volume of experimental data to accelerate FSH-focused research. FSH Glycans DataBase (FGDB) serves as a comprehensive and unique repository of structures, features, and related information of glycans associated with FSH. Apart from providing multiple search options, the database also facilitates an integrated user-friendly interface to perform the glycan abundance and comparative analyses using experimental data. The automated integrated pipelines present the possible structures of glycans and variants of FSH based on the input data, and allow the user to perform various analyses. The potential application of FGDB will significantly help both glycoinformaticians as well as wet-lab researchers to stimulate the research in this area. FGDB web access: https://fgdb.unmc.edu/.
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http://dx.doi.org/10.1016/j.csbj.2021.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050109PMC
March 2021

ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2015-2016.

Authors:
David J Harvey

Mass Spectrom Rev 2021 Jul 16;40(4):408-565. Epub 2021 Mar 16.

Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom.

This review is the ninth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/mas.21651DOI Listing
July 2021

In vivo modification of the goat mammary gland glycosylation pathway.

N Biotechnol 2021 Mar 4;61:11-21. Epub 2020 Nov 4.

Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile; Biotechnology and Biomedicine Center SpA, Granada 168, Villumanque, Concepcion, Chile. Electronic address:

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.
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http://dx.doi.org/10.1016/j.nbt.2020.11.001DOI Listing
March 2021

Networks of HIV-1 Envelope Glycans Maintain Antibody Epitopes in the Face of Glycan Additions and Deletions.

Structure 2020 08 19;28(8):897-909.e6. Epub 2020 May 19.

School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK; Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. Electronic address:

Numerous broadly neutralizing antibodies (bnAbs) have been identified that target the glycans of the HIV-1 envelope spike. Neutralization breadth is notable given that glycan processing can be substantially influenced by the presence or absence of neighboring glycans. Here, using a stabilized recombinant envelope trimer, we investigate the degree to which mutations in the glycan network surrounding an epitope impact the fine glycan processing of antibody targets. Using cryo-electron microscopy and site-specific glycan analysis, we reveal the importance of glycans in the formation of the 2G12 bnAb epitope and show that the epitope is only subtly impacted by variations in the glycan network. In contrast, we show that the PG9 and PG16 glycan-based epitopes at the trimer apex are dependent on the presence of the highly conserved surrounding glycans. Glycan networks underpin the conservation of bnAb epitopes and are an important parameter in immunogen design.
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http://dx.doi.org/10.1016/j.str.2020.04.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416112PMC
August 2020

NEGATIVE ION MASS SPECTROMETRY FOR THE ANALYSIS OF N-LINKED GLYCANS.

Authors:
David J Harvey

Mass Spectrom Rev 2020 09 24;39(5-6):586-679. Epub 2020 Apr 24.

Nuffield Department of Medicine, Target Discovery Institute, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom.

N-glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure-revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N-linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix-assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.
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http://dx.doi.org/10.1002/mas.21622DOI Listing
September 2020

MASS SPECTROMETRIC FRAGMENTATION OF TRIMETHYLSILYL AND RELATED ALKYLSILYL DERIVATIVES.

Mass Spectrom Rev 2020 03 5;39(1-2):105-211. Epub 2019 Dec 5.

Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, Massachusetts, 02115.

This review describes the mass spectral fragmentation of trimethylsilyl (TMS) and related alkylsilyl derivatives used for preparing samples for analysis, mainly by combined gas chromatography and mass spectrometry (GC/MS). The review is divided into three sections. The first section is concerned with the TMS derivatives themselves and describes fragmentation of derivatized alcohols, thiols, amines, ketones, carboxylic acids and bifunctional compounds such as hydroxy- and amino-acids, halo acids and hydroxy ethers. More complex compounds such as glycerides, sphingolipids, carbohydrates, organic phosphates, phosphonates, steroids, vitamin D, cannabinoids, and prostaglandins are discussed next. The second section describes intermolecular reactions of siliconium ions such as the TMS cation and the third section discusses other alkylsilyl derivatives. Among these latter compounds are di- and trialkyl-silyl derivatives, various substituted-alkyldimethylsilyl derivatives such as the tert-butyldimethylsilyl ethers, cyclic silyl derivatives, alkoxysilyl derivatives, and 3-pyridylmethyldimethylsilyl esters used for double bond location in fatty acid spectra. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 0000:1-107, 2019.
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http://dx.doi.org/10.1002/mas.21590DOI Listing
March 2020

Ion Mobility-Mass Spectrometry of Glycoconjugates.

Methods Mol Biol 2020 ;2084:203-219

Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.

Glycoconjugates are diverse biomolecules that are dynamically assembled to regulate and fine-tune numerous cellular processes. Their biosynthesis is nontemplate-driven, achieved stepwise in discrete locations within the cell, giving rise to a range of complex branched structures that pose a significant challenge in structural biology. Mass spectrometry is the leading method for analysis of glycoconjugates, and the addition of ion mobility has proven valuable for improving structural assignments of individual glycans in complex biological mixtures. In this chapter, we briefly discuss recent applications of IM for glycomics and describe how to acquire, interpret, and analyze IM-MS data for the analysis of glycans.
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http://dx.doi.org/10.1007/978-1-0716-0030-6_13DOI Listing
December 2020

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Mol Cell Proteomics 2020 01 7;19(1):11-30. Epub 2019 Oct 7.

Graduate School of Analytical Science and Technology, Chungnam National University, Gung-dong 220, Yuseong-Gu, Daejeon 305-764, Korea (South).

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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http://dx.doi.org/10.1074/mcp.RA119.001677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944243PMC
January 2020

Separation of Isomeric Glycans by Ion Mobility and Liquid Chromatography-Mass Spectrometry.

Anal Chem 2019 08 1;91(16):10604-10613. Epub 2019 Aug 1.

Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy , University of Gothenburg , Gothenburg , Sweden.

Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (LC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and -glycans. Here, we present a direct comparison of the IM- and LC-separation of -glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by LC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.
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http://dx.doi.org/10.1021/acs.analchem.9b01772DOI Listing
August 2019

Follicle-Stimulating Hormone Glycobiology.

Endocrinology 2019 06;160(6):1515-1535

Target Discovery Institute, Nuffield Department of Medicine, Oxford University, Oxford, United Kingdom.

FSH glycosylation varies in two functionally important aspects: microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity, arising from partial FSHβ subunit glycosylation. Although advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate, and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, which exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHβ glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all of the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. To resolve these alternatives, well-characterized FSH glycoform preparations are necessary.
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http://dx.doi.org/10.1210/en.2019-00001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534497PMC
June 2019

Glycosylation profiling of dog serum reveals differences compared to human serum.

Glycobiology 2018 11;28(11):825-831

New England Biolabs Inc., 240 County Road, Ipswich, MA, USA.

Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG - the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.
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http://dx.doi.org/10.1093/glycob/cwy070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192460PMC
November 2018

Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen.

Cell Rep 2018 08;24(8):1958-1966.e5

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; Biological Sciences and the Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK. Electronic address:

Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-associated envelope glycoprotein (Env) spikes. The same bnAbs also bind to recombinant, soluble trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clinical grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native versus recombinant Env glycosylation will guide vaccine design and manufacturing programs.
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http://dx.doi.org/10.1016/j.celrep.2018.07.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113929PMC
August 2018

N-glycan microheterogeneity regulates interactions of plasma proteins.

Proc Natl Acad Sci U S A 2018 08 15;115(35):8763-8768. Epub 2018 Aug 15.

Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom;

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.
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http://dx.doi.org/10.1073/pnas.1807439115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126716PMC
August 2018

Structural Studies of Fucosylated N-Glycans by Ion Mobility Mass Spectrometry and Collision-Induced Fragmentation of Negative Ions.

J Am Soc Mass Spectrom 2018 06 22;29(6):1179-1193. Epub 2018 May 22.

Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewis and Lewis epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1950-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003995PMC
June 2018

Mass spectrometric analysis of glycosylated viral proteins.

Authors:
David J Harvey

Expert Rev Proteomics 2018 05 23;15(5):391-412. Epub 2018 May 23.

a Target Discovery Institute, Nuffield Department of Medicine , University of Oxford , Oxford , UK.

Introduction: Viral diseases contribute much to human and animal suffering and enormous efforts are directed at developing appropriate vaccines for protection. Glycoproteins constitute much of the viral surfaces and are obvious targets for such vaccine development. This review describes mass spectrometric methods used for the structural determination of these compounds. Areas covered: The review describes the structures of the N- and O-linked glycans found on glycoproteins and mass spectrometric methods for their ionization and fragmentation. The steps, such as determination of glycan attachment sites and the structures of the attached glycans following their release from the glycoproteins are described and examples are given of the uses of the various analytical methods using mainly influenza, Ebola and HIV as representative examples. Also included are tables listing work on many other viruses. Expert commentary: Recent technological advances, such as the introduction of ion mobility techniques, have greatly improved analyses in this area and have enabled larger amounts of information to be gathered in shorter time periods on ever smaller amounts of material. Such techniques should greatly accelerate the discovery of vaccine targets and lead to the production of vaccines for diseases not currently available.
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http://dx.doi.org/10.1080/14789450.2018.1468756DOI Listing
May 2018

Fucose Migration in Intact Protonated Glycan Ions: A Universal Phenomenon in Mass Spectrometry.

Angew Chem Int Ed Engl 2018 06 25;57(25):7440-7443. Epub 2018 May 25.

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195, Berlin, Germany.

Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision-induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold-ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low-energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment.
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http://dx.doi.org/10.1002/anie.201801418DOI Listing
June 2018

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2013-2014.

Authors:
David J Harvey

Mass Spectrom Rev 2018 07 17;37(4):353-491. Epub 2018 Apr 17.

Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom.

This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.
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http://dx.doi.org/10.1002/mas.21530DOI Listing
July 2018

Collision Cross Sections and Ion Mobility Separation of Fragment Ions from Complex N-Glycans.

J Am Soc Mass Spectrom 2018 06 19;29(6):1250-1261. Epub 2018 Apr 19.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1930-1DOI Listing
June 2018

Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments.

J Am Soc Mass Spectrom 2018 05 5;29(5):972-988. Epub 2018 Mar 5.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on ManGlcNAc isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1890-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940726PMC
May 2018

Integrity of Glycosylation Processing of a Glycan-Depleted Trimeric HIV-1 Immunogen Targeting Key B-Cell Lineages.

J Proteome Res 2018 03 8;17(3):987-999. Epub 2018 Feb 8.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, United Kingdom.

Broadly neutralizing antibodies (bNAbs) that target the trimeric HIV-1 envelope glycoprotein spike (Env) are tools that can guide the design of recombinant Env proteins intended to engage the predicted human germline precursors of bNAbs (gl-bNAbs). The protein components of gl-bNAb epitopes are often masked by glycans, while mature bNAbs can evolve to accommodate or bypass these shielding glycans. The design of germline-targeting Env immunogens therefore includes the targeted deletion of specific glycan sites. However, the processing of glycans on Env trimers can be influenced by the density with which they are packed together, a highly relevant point given the essential contributions under-processed glycans make to multiple bNAb epitopes. We sought to determine the impact of the removal of 15 potential N-glycan sites (5 per protomer) from the germline-targeting soluble trimer, BG505 SOSIP.v4.1-GT1, using quantitative, site-specific N-glycan mass spectrometry analysis. We find that, compared with SOSIP.664, there was little overall change in the glycan profile but only subtle increases in the extent of processing at sites immediately adjacent to where glycans had been deleted. We conclude that multiple glycans can be deleted from BG505 SOSIP trimers without perturbing the overall integrity of the glycan shield.
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http://dx.doi.org/10.1021/acs.jproteome.7b00639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846105PMC
March 2018

Identification of Lewis and Blood Group Carbohydrate Epitopes by Ion Mobility-Tandem-Mass Spectrometry Fingerprinting.

Anal Chem 2017 02 1;89(4):2318-2325. Epub 2017 Feb 1.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , OX1 3QU Oxford, United Kingdom.

Glycans have several elements that contribute to their structural complexity, involving a range of monosaccharide building blocks, configuration of linkages between residues and various degrees of branching on a given structure. Their analysis remains challenging and resolving minor isomeric variants can be difficult, in particular terminal fucosylated Lewis and blood group antigens present on N- and O-glycans. Accurately characterizing these isomeric structures by current techniques is not straightforward and typically requires a combination of methods and/or sample derivatization. Yet the ability to monitor the occurrence of these epitopes is important as structural changes are associated with several human diseases. The use of ion mobility-mass spectrometry (IM-MS), which separates ions in the gas phase based on their size, charge and shape, offers a new potential tool for glycan analysis and recent reports have demonstrated its potential for glycomics. Here we show that Lewis and blood group isomers, which have identical fragmentation spectra, exhibit very distinctive IM drift times and collision cross sections (CCS). We show that IM-MS/MS analysis can rapidly and accurately differentiate epitopes from parotid gland N-glycans and milk oligosaccharides based on fucosylated fragment ions with characteristic CCSs.
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http://dx.doi.org/10.1021/acs.analchem.6b03853DOI Listing
February 2017

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer.

J Virol 2017 Jan 3;91(2). Epub 2017 Jan 3.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom

The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage induces a compact structure which not only renders the spike capable of fusion but also limits further maturation of its extensive glycosylation. The redirection of the glycosylation pathway to preserve underprocessed oligomannose-type glycans is an important feature in immunogen design, as glycans contribute to or influence the epitopes of numerous broadly neutralizing antibodies. Here we present a quantitative site-specific analysis of a recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) compared to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a detailed molecular map of a trimer-associated glycan remodeling that forms a localized subdomain of the native mannose patch. The formation of native trimers is a critical design feature in shaping the glycan epitopes presented on recombinant vaccine candidates.

Importance: The envelope spike of human immunodeficiency virus type 1 (HIV-1) is a target for antibody-based neutralization. For some patients infected with HIV-1, highly potent antibodies have been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 vaccine research to elicit these antibodies by immunization with recombinant mimics of the viral spike. These antibodies have evolved to recognize the dense array of glycans that coat the surface of the viral molecule. We show how the structure of these glycans is shaped by steric constraints imposed upon them by the native folding of the viral spike. This information is important in guiding the development of vaccine candidates.
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http://dx.doi.org/10.1128/JVI.01894-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215339PMC
January 2017

Travelling-wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N-glycans.

J Mass Spectrom 2016 Nov;51(11):1064-1079

Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1-acid glycoprotein and thyroglobulin were measured with a travelling-wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man GlcNAc from chicken ovalbumin and Man GlcNAc Fuc from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision-induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high-mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high-mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility-m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jms.3828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150983PMC
November 2016

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity.

MAbs 2016 Nov/Dec;8(8):1498-1511. Epub 2016 Sep 3.

b Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford , Oxford , UK.

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.
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http://dx.doi.org/10.1080/19420862.2016.1228504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098438PMC
November 2017

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein.

Cell Rep 2016 Mar 10;14(11):2695-706. Epub 2016 Mar 10.

Oxford Glycobiology Institute and Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.
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http://dx.doi.org/10.1016/j.celrep.2016.02.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805854PMC
March 2016

Travelling-wave ion mobility and negative ion fragmentation of high-mannose N-glycans.

J Mass Spectrom 2016 Mar;51(3):219-35

Department of Biological Sciences, University of Warwick, Coventry, CV47AL, UK.

The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed.
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http://dx.doi.org/10.1002/jms.3738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821469PMC
March 2016

Fragmentation and ion mobility properties of negative ions from N-linked carbohydrates: Part 7. Reduced glycans.

Rapid Commun Mass Spectrom 2016 Mar;30(5):627-34

Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia.

Rationale: Negative ion collision-induced dissociation (CID) spectra of released N-glycans provide very informative structural information relating to branching patterns and location of residues such as fucose. For some structural studies, particularly those involving chromatography, glycans are often reduced to avoid production of multiple peaks from α- and β-anomers. We examined the effect of reduction on the production of diagnostic fragment ions and on the ion mobility properties of N-glycans.

Methods: Released N-glycans from the glycoproteins bovine fetuin, ribonuclease B, chicken ovalbumin, and porcine thyroglobulin were reduced with sodium cyanoborohydride and both negative ion CID spectra and ion mobility properties of their phosphate adducts were examined with a Waters Synapt G2Si travelling-wave ion mobility mass spectrometer with electrospray sample introduction. Estimated collisional cross sections were measured with dextran as the calibrant,

Results: Fragment ions were similar to those from the unreduced glycans with the exception that the prominent (2,4) A cleavage ion from the reducing terminus was replaced by a prominent [M-H3 PO4](-) ion. Other ions arising from the chitobiose core were of lower relative abundance than those from the unreduced glycans. Estimated collisional cross sections were similar to those of the unreduced compounds but with symmetrical arrival time distribution (ATD) profiles, unlike those of the unreduced glycans whose peaks often contained prominent asymmetry. This observation showed that this asymmetry was due to anomer separation.

Conclusions: Reduction of the reducing terminal GlcNAc residue resulted in fewer diagnostic ions from the chitobiose core but fragmentation of the remainder of the molecules generally paralleled that of the unreduced glycans. Thus, most structural information, with the exception of the linkage position of fucose on the core GlcNAc, was available. ATD peaks were symmetrical with the result that cross sections were more appropriate for data-base searching than those from the non-reduced compounds where asymmetry produced lower precision in the measurement.
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http://dx.doi.org/10.1002/rcm.7467DOI Listing
March 2016

Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).

Glycobiology 2016 Mar 3;26(3):230-50. Epub 2015 Nov 3.

Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.
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http://dx.doi.org/10.1093/glycob/cwv096DOI Listing
March 2016

GlycoMob: an ion mobility-mass spectrometry collision cross section database for glycomics.

Glycoconj J 2016 06 28;33(3):399-404. Epub 2015 Aug 28.

Biomolecular Frontiers Research Centre, Macquarie University, Sydney, NSW 2109, Australia.

Ion mobility mass spectrometry (IM-MS) is a promising analytical technique for glycomics that separates glycan ions based on their collision cross section (CCS) and provides glycan precursor and fragment masses. It has been shown that isomeric oligosaccharide species can be separated by IM and identified on basis of their CCS and fragmentation. These results indicate that adding CCSs information for glycans and glycan fragments to searchable databases and analysis pipelines will increase identification confidence and accuracy. We have developed a freely accessible database, GlycoMob ( http://www.glycomob.org ), containing over 900 CCSs values of glycans, oligosaccharide standards and their fragments that will be continually updated. We have measured the absolute CCSs of calibration standards, biologically derived and synthetic N-glycans ionized with various adducts in positive and negative mode or as protonated (positive ion) and deprotonated (negative ion) ions.
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http://dx.doi.org/10.1007/s10719-015-9613-7DOI Listing
June 2016

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2011-2012.

Authors:
David J Harvey

Mass Spectrom Rev 2017 05 13;36(3):255-422. Epub 2015 Aug 13.

Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, OX1 3QU, UK.

This review is the seventh update of the original article published in 1999 on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2012. General aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, and fragmentation are covered in the first part of the review and applications to various structural types constitute the remainder. The main groups of compound are oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:255-422, 2017.
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http://dx.doi.org/10.1002/mas.21471DOI Listing
May 2017
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