Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2021-2022.

The use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates is a well-established technique and this review is the 12th update of the original article published in 1999 and brings coverage of the literature to the end of 2022. As with previous review, this review also includes a few papers that describe methods appropriate to analysis by MALDI, such as sample preparation, even though the ionization method is not MALDI. The review follows the same format as previous reviews. It is divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of computer software for structural identification. (2) Applications to various structural types such as oligo- and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other general areas such as medicine, industrial processes, natural products and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. MALDI is still an ideal technique for carbohydrate analysis, particularly in its ability to produce single ions from each analyte and advancements in the technique and range of applications show little sign of diminishing.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2017-2018.

This review is the tenth 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 2018. Also included are papers that describe methods appropriate to glycan and glycoprotein 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, new methods, matrices, derivatization, MALDI imaging, fragmentation and the use of 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. Most of the applications are 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 highlights the impact that MALDI imaging is having across a range of diciplines. MALDI is still an ideal technique for carbohydrate analysis and advancements in the technique and the range of applications continue steady progress.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2019-2020.

This review is the tenth 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 2020. 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. The review is basically divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of arrays. (2) Applications to various structural types such as oligo- and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other areas such as medicine, industrial processes and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. The reported work shows increasing use of incorporation of new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented nearly 40 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show little sign of diminishing.

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

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.

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

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.

MASS SPECTROMETRIC FRAGMENTATION OF TRIMETHYLSILYL AND RELATED ALKYLSILYL DERIVATIVES.

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.

Identification of N-glycans with GalNAc-containing antennae from recombinant HIV trimers by ion mobility and negative ion fragmentation.

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

Formation and fragmentation of doubly and triply charged ions in the negative ion spectra of neutral N-glycans from viral and other glycoproteins.

Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M - H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans.

N-glycan microheterogeneity regulates interactions of plasma proteins.

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.

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

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 (PGCLC-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 N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC 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.

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

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.

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

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.

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

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.

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

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.

Mass spectrometric analysis of glycosylated viral proteins.

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.

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

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.

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

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.

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

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.

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

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.