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.

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.

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.

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.

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.

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.

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.

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.

Structural documentation of glycan epitopes: sequential mass spectrometry and spectral matching.

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MS(n) sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N- and O-glycans of proteins. The latter are most frequently pendant through a CID-labile HexNAc glycosidic linkage. The synthetic samples were supplied by collaborating colleagues and commercial sources and usually possessed a readily released reducing-end linker, a by-product of synthesis. All samples were comparably methylated, extracted, and MS(n) disassembled to compare their linkage and branching spectral details. Both sample types provide B-ion type fragments early in a disassembly pathway and their compositions are a suggestion of structure. Further steps of disassembly are necessary to confirm the details of linkage and branching. Included in this study were various Lewis and H antigens, 3- and 6-linked sialyl-lactosamine, NeuAc-2,8-NeuAc dimer, and Galα1,3Gal. Sample infusion provided high quality spectral data whereas disassembly to small fragments generates reproducible high signal/noise spectra for spectral matching. All samples were analyzed as sodium adducted positive ions. This study includes comparability statistics and evaluations on several mass spectrometers.

Tools to MSn Sequence and Document the Structures of Glycan Epitopes.

Sequential disassembly (MSn) has been applied to fully characterise and document native samples containing glycan epitopes with their synthetic analogues. Both sample types were prepared by methylation, solvent phase extracted, directly infused and spatially resolved. Product ions of all samples were compiled and contrasted using management tools prepared for the fragment ion library. Each of the epitopes was further disassembled to confirm the multiple structural isomers probable within component substructures of linkage and branching. All native samples tested proved to be matched with their synthetic analogues and reasonably identical on either linear or cylindrical ion traps. Not surprisingly, spectra of mixed epitopes fragment independently, being uninfluenced by similarities. The approach has been coupled with computational tools for data handling and presentation.

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.

N-glycome profiling of Bothrops jararaca newborn and adult venoms.

Glycosylation is an important post-translational modification of snake venom proteins and contributes to venom proteome complexity. Many snake venom components are known to be glycosylated, however, very little is known about the carbohydrate structures present in venom glycoproteins. Previous studies showed that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and shift in animal size are associated with changes in the venom proteome of the snake Bothrops jararaca. In this study we explored the composition of the N-glycome released from newborn and adult B. jararaca venom proteins. We used an ion trap mass spectrometer (IT-MS) to disassemble glycan structures based on the use of several pathways of MS (MSn) and demonstrate the presence of some structural isomers in both newborn and adult venom B. jararaca N-glycans. The main N-glycans identified in both venoms are of the hybrid/complex type however some mannose-rich type structures were also detected. The N-glycan composition of newborn and adult venoms did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms.

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.

Structural characterization of an oligosaccharide made by Neisseria sicca.

Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization--time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its beta-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) beta-L-rhamnose (1-3) beta-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap MS.

Thirteen high mannose isomers have been structurally characterized within three glycomers, Man(5)GlcNAc(2), Man(7)GlcNAc(2), and Man(8)GlcNAc(2) released from bovine ribonuclease B, six previously unreported. The study was carried out with a single ion trap instrument involving no chromatography. Three previously characterized isomers from Man(7) and Man(8) (three each) have been identified plus one unreported Man(7) isomer. Incomplete alpha-glucosidase activity on the Man(6) and Man(7) glycoproteins appears to account for two additional isomeric structures. The preeminence of ion traps for detail analysis was further demonstrated by resolving three new isomers within the Man(5) glycomer summing to the six previously unreported structures in this glycoprotein. All reported structures represent a distribution of Golgi processing remnants that fall within the Man(9)GlcNAc(2) footprint. Topologies were defined by ion compositions along a disassembly pathway while linkage and branching were aided by spectral identity in a small oligomer fragment library. Isomers from this glycoprotein appear to represent a distribution of Golgi processing remnants, and an alphanumeric classification scheme has been devised to identify all products. Although numerous analytical strategies have been introduced to identify selected components of structure, it has been the continued focus of this and previous reports to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Duplication of these and results from comparable standards could bring an important analytical focus to carbohydrate sequencing that is greatly lacking.

Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc.

It is well established that high doses of monomeric immunoglobulin G (IgG) purified from pooled human plasma [intravenous immunoglobulin (IVIG)] confer anti-inflammatory activity in a variety of autoimmune settings. However, exactly how those effects are mediated is not clear because of the heterogeneity of IVIG. Recent studies have demonstrated that the anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Here we determine the precise glycan requirements for this anti-inflammatory activity, allowing us to engineer an appropriate IgG1 Fc fragment, and thus generate a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency. This therapeutic molecule precisely defines the biologically active component of IVIG and helps guide development of an IVIG replacement with improved activity and availability.

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry.

In an effort to understand the role of molecular glycosylation in cancer a murine model has been used to characterize and fingerprint malignancies in established cell lines that manifest all the hallmarks of metastatic disease: spontaneous development, local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Using astrocyte cell controls, we compared N-linked glycosylation from a nonmetastatic brain tumor cell line and two different metastatic brain tumor cells. Selected ions in each profile were disassembled by ion trap mass spectrometry (MS(n)) which exhibited multiple structural differences between each tissue. These unique structures were identified within isomeric compositions as pendant nonreducing termini of di- and trisaccharide fragments, probably transparent to a tandem MS approach but distinctively not to sequential ion trap MS(n) detection.