Amplification and Preparation of Cellular O-Glycomes for Functional Glycomics.

Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSn). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.

Structures of N-Glycans of Bothrops Venoms Revealed as Molecular Signatures that Contribute to Venom Phenotype in Viperid Snakes.

The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.

Distinct human α(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (LeX; CD15) and/or sialyl Lewis-X (sLeX; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human LeX/sLeX biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLeX is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize LeX, and, among all FTs, FT6 holds a unique capacity in creating sLeX and LeX determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLeX on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes.

Next-Generation Glycan Microarray Enabled by DNA-Coded Glycan Library and Next-Generation Sequencing Technology.

Interactions of glycans with proteins, cells, and microorganisms play important roles in cell-cell adhesion and host-pathogen interaction. Glycan microarray technology, in which multiple glycan structures are immobilized on a single glass slide and interrogated with glycan-binding proteins (GBPs), has become an indispensable tool in the study of protein-glycan interactions. Despite its great success, the current format of the glycan microarray requires expensive, specialized instrumentation and labor-intensive assay and image processing procedures, which limit automation and possibilities for high-throughput analyses. Furthermore, the current microarray is not suitable for assaying interaction with intact cells due to their large size compared to the two-dimensional microarray surface. To address these limitations, we developed the next-generation glycan microarray (NGGM) based on artificial DNA coding of glycan structures. In this novel approach, a glycan library is presented as a mixture of glycans and glycoconjugates, each of which is coded with a unique oligonucleotide sequence (code). The glycan mixture is interrogated by GBPs followed by the separation of unbound coded glycans. The DNA sequences that identify individual bound glycans are quantitatively sequenced (decoded) by powerful next-generation sequencing (NGS) technology, and copied numbers of the DNA codes represent relative binding specificities of corresponding glycan structures to GBPs. We demonstrate that NGGM generates glycan-GBP binding data that are consistent with that generated in a slide-based glycan microarray. More importantly, the solution phase binding assay is directly applicable to identifying glycan binding to intact cells, which is often challenging using glass slide-based glycan microarrays.

Advancing MSn spatial resolution and documentation for glycosaminoglycans by sulfate-isotope exchange.

Glycosaminoglycans (GAGs) are carbohydrate polyionic polymers that participate in a host of critically important biological processes. A significant difficulty in the comprehensive structural characterization of GAGs is the determination of specific sulfation position isomers. We chose to circumvent sulfate lability by its liberation followed by specific isotope exchange that makes it amenable to methylation, collisional induced dissociation, and MSn disassembly for a detailed structural characterization. A set of chemistries that include sulfate release, isotopic (CD3- and CD3-CO-) replacement, and methylation have been modified to yield a stable product ideal for sequencing by MSn. Disassembly of these samples provides a detailed read-out of sequence inclusive of all sulfation sites. As documenting steps, we applied these chemical modifications to a series of disaccharides and a synthetic GAG pentamer, Arixtra®. Upon disassembly, glycosidic and cross-ring cleavages define the monomer composition including individual sulfation positions. The N- and O-sulfates are differentiated by deuterium-containing mass compositions. The uronic methylesters do not significantly alter the fragmentation patterns. A fragment library of these products is being assembled as an adjunct to our larger fragment library, some 15 years in the making.

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(ß3)GlcNAc, Gal(ß4)GlcNAc and Gal(ß3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and ß4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(ß4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.

Isomeric complexity of glycosylation documented by MSn.

Re-analysis of two breast cancer cell lines, MCF-7 and MDA-MB-231, has shown multiple isomeric structures exposed by sequential mass spectrometry, MS n . Several released glycan compositions were re-evaluated, which indicated variations in polylactosamine and fucosylation structures. Probable isomer numbers, when considering both stereo and structural entities, are significant and the varying types are mentioned. The structural isomers of linkage position are most frequently considered, while stereo isomers are usually assumed from biosynthetic data. Evaluation of any new sample should be cautious and merits careful attention to empirical data. While isomers are usually considered a chromatographic problem (e.g., LCMS, IMMS) and most frequently considered a separations problem, such results will always be challenged by identification and documentation. MSn data provide a direct spatial solution that includes spectral data for characterization (mass and abundance) supported by a universal library match feature.

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses.

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.

Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses.

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MS(n) analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MS(n) are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.

The glycosylation-dependent interaction of perlecan core protein with LDL: implications for atherosclerosis.

Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI-DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.

Platelets support extracellular sialylation by supplying the sugar donor substrate.

Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators. Cargos of sugar donor substrates for glycosyltransferase activity have also been reported in platelets. Here, we implemented a cell-based system to interrogate platelets for their ability to deliver effectively the sugar donor substrate for extracellular ST6Gal-1 to function. We report that thrombin-activated platelets, at physiologic concentration and pH, can efficiently and effectively substitute for CMP-sialic acid in extracellular ST6Gal-1-mediated sialylation of target cell surfaces. Activated platelets can also supply the sialic acid donor to sialylate the synthetic acceptor, Gal(ß1,4)GlcNAcα-o-benzyl, with the product Sia(α2,6)Gal(ß1,4)GlcNAcα-o-benzyl structurally confirmed by LC/MS. Platelet-secreted donor substrate was recovered in the 100,000 × g sediment, strongly suggesting the association of this otherwise soluble substrate, putatively CMP-sialic acid, within platelet microparticles. Sequestration within microparticles may facilitate delivery of glycosylation substrate at effective dosages to sites of extracellular glycosylation while minimizing excessive dilution.

The structures of glycophorin C N-glycans, a putative component of the GPC receptor site for Plasmodium falciparum EBA-140 ligand.

Glycophorins C and D are highly glycosylated integral sialoglycoproteins of human red blood cell membranes carrying the Gerbich blood group antigens. The O- and N-glycosidic chains of the major erythrocyte glycoprotein (Lisowska E. 2001, Antigenic properties of human glycophorins - an update. Adv Exp Med Biol, 491:155-169; Tomita M and Marchesi VT. 1975, Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci USA, 72:2964-2968.) are well characterized but the structure of GPC N-glycans has remained unknown. This problem became important since it was reported that GPC N-glycans play an essential role in the interaction with Plasmodium falciparum EBA-140 merozoite ligand. The elucidation of these structures seems essential for full characterization of the GPC binding site for the EBA-140 ligand. We have employed detailed structural analysis using sequential mass spectrometry to show that many GPC N-glycans contain H2 antigen structures and several contain polylactosamine structures capped with fucose. The results obtained indicate structural heterogeneity of the GPC N-glycans and show the existence of structural elements not found in glycophorin A N-glycans. Our results also open a possibility of new interpretation of the data concerning the binding of P. falciparum EBA-140 ligand to GPC. We hypothesize that preferable terminal fucosylation of N-glycosidic chains containing repeating lactosamine units of the GPC Gerbich variant could be an explanation for why the EBA-140 ligand does not react with GPC Gerbich and an indication that the EBA-140 interaction with GPC is distinctly dependent on the GPC N-glycan structure.

Toward a platform for comprehensive glycan sequencing.

From a series of recently published reports, an analytical platform has been proposed for a quantitative and qualitative measure of N- and O-glycosylation, complete with peptide-glycan connectivity and detailed structural understanding. As distant as this may appear, a best methods approach will appear that must move us beyond the cartoon stage of structural understanding. Thus, with this unifying goal in mind, we summarize a series of individually promising first phase protocols of sample preparation (release, purification, and quantification) that remain congruent with a concluding phase (methylation and MS(n)) for documented structural detail. Sequential enzymatic N-glycan and chemical O-glycan release from glycopeptides with intervening solid phase extraction and derivatization will provide for a comparative quantification measure of glycosylation. The O-glycan release will be nonreductive and coupled with Michael addition to a pyrazolone analog (1-phenyl-3-methyl-5-pyrazolone) with both the peptide and glycan labeled. The product glycans are stable to methylation and appropriate for sequential disassembly (MS(n)). An application using human serum and cancer samples has been detailed characterizing sLe(x) and comparable valence epitopes. This integrated platform will provide opportunities at variable points to contrast, share, and advance alternative protocols in a collaborative effort that is greatly needed. This integrated platform provides end point opportunities to confirm structural details compiled from synthetic standards and well characterized biologics by MS(n).

Anomalous N-glycan structures with an internal fucose branched to GlcA and GlcN residues isolated from a mollusk shell-forming fluid.

This report describes the structural details of a unique N-linked valence epitope on the major protein within the extrapallial (EP) fluid of the mollusk, Mytilus edulis. Fluids from this area are considered to be responsible for shell expansion by a self-assembly process that provides an organic framework for the growth of CaCO3 crystals. Previous reports from our laboratories have described the purification and amino acid sequence of this EP protein, which was found to be a glycoprotein (EPG) of approximately 28 KDa with 14.3% carbohydrate on a single N-linked consensus site. Described herein is the de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap sequential mass spectrometry (ITMS(n)) resolving structure by tracking precursor-product relationships through successive rounds of collision induced disassociation (CID), thereby spatially resolving linkage and branching details within the confines of the ion trap. Three major glycomers were detected, each possessing a 6-linked fucosylated N-linked core. Two glycans possessed four and five identical antennae, while the third possessed four antennas, but with an additional methylfucose 2-linked to the glucuronic acid moiety, forming a pentasaccharide. The tetrasaccharide structure was: 4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc, while the pentasaccharide was shown to be as follows: mono-O-methyl-Fuc(1-2)-4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc. Samples were differentially deuteriomethylated (CD3/CH3) to localize indigenous methylation, further analyzed by high resolution mass spectrometry (HRMS) to confirm monomer compositions, and finally gas chromatography mass spectrometry (GC-MS) to assign structural and stereoisomers. The interfacial shell surface location of this major extrapallial glycoprotein, its calcium and heavy metal binding properties and unique structure suggests a probable role in shell formation and possibly metal ion detoxification. A closely related terminal tetrasaccharide structure has been reported in spermatozoan glycolipids of freshwater bivalves.

The conserved oligomeric Golgi complex is required for fucosylation of N-glycans in Caenorhabditis elegans.

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.

High Performance IT-MS Sequencing of Glycans (Spatial Resolution of Ovalbumin Isomers).

This report outlines and applies a high performance sequencing technology to evaluate the glycome of a common model glycoprotein, ovalbumin. The targets were the N-linked glycans enzymatically released from the protein, the N-glycoproteome. These product glycans were reduced, methylated and directly infused into the MS using a chip-based nanoelectrospray with the ions structurally characterized by sequential disassembly. Ten major ions were selected for detailed analysis. Isomer topologies (glycan connectivity) were determined from ion pathways of disassembly. Linkage information was revealed by specific cross-ring cleavage fragments within smaller oligomers. Both connectivity and linkage features were assisted with described bioinformatic tools and details confirmed with a standards library of fragments. The number of isomeric structures found within these 10 parent ions were 37, more than double earlier reports, and setting a new goal for developing technology. In this non-chromatographic, high performance spatial approach, the focus has been patterned to be comprehensive, and stay within the bounds of a plausible high throughput strategy consistent with automation. Selective structures are described in the text to appraise readers of the general approach; a more comprehensive coverage has been included in supplemental material.

The N-glycome of human plasma.

N-linked glycans isolated from human plasma proteins have been profiled and sequenced by mass spectrometry using an ion trap instrument (ITMSn). The released glycans were prepared as reduced, methylated analogues and directly infused into a chip-based nanoelectrospray ionization system and analyzed by ITMSn. The resulting mass profiles (MS1) of IgG-depleted and nondepleted plasma samples were contrasted and these results were again compared with recent literature reports. Before depletion, approximately 50 independent glycan ions were detected; this more than doubled to 106 after depletion. The mass range profiled was 1-5 kDa which included many doubly and triply charged ions that were resolved by higher MS resolution. Selected ions in the depleted sample were disassembled to define their detailed structure providing a high-performance sequencing result. The simplicity of this nonchromatographic, direct infusion and gas-phase structural characterization compares most favorably with the latest reports using alternative instrumentation and adjunct techniques.

Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I).

LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAcalpha2-3Galbeta1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAcalpha2-3Galbeta1-3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a, respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside-protein interactions.

Rapid de-O-glycosylation concomitant with peptide labeling using microwave radiation and an alkyl amine base.

Procedures are detailed for a quantitative release of O-linked glycans from peptides that now provide a shorter reaction time, a possible identification of O-linked sites, and a quantification of all reaction products. The release was initiated by a mild base, dimethylamine, and accelerated by microwave radiation. Differential analysis using standard glycoproteins has shown improved release efficiency concurrent with facile incorporation of dimethylamine into the former O-linked sites. In situ glycan reduction insures protection against peeling and is synchronous with subsequent studies by high performance MS(n) sequencing. The protocols were established with a synthetic O-GlcNAc peptide that would mimic the linkage chemistry and applied to a well characterized glycoprotein bovine fetuin with both N- and O-linked glycans and a highly glycosylated swine mucin.