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Dauber A.,Boston Childrens Hospital | Dauber A.,Cambridge Broad Institute | Ercan A.,Immunology and Allergy | Lee J.,5 Francis Street | And 17 more authors.
Human Molecular Genetics | Year: 2014

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migrationcausedbymutationsin the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.© The Author 2014. Published by Oxford University Press. All rights reserved.All rights reserved.


Zhou H.,University of New Hampshire | Zhou H.,Harvard University | Hanneman A.J.,University of New Hampshire | Hanneman A.J.,Glycan Connections LLC. | And 3 more authors.
Journal of Proteome Research | Year: 2013

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 (ITMSn) 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 (CD 3/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. © 2013 American Chemical Society.


Ashline D.J.,Glycan Connections LLC | Hanneman A.J.S.,Glycan Connections LLC | Zhang H.,University of New Hampshire | Reinhold V.N.,Glycan Connections LLC | Reinhold V.N.,University of New Hampshire
Journal of the American Society for Mass Spectrometry | Year: 2014

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MSn 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 MSn 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. [Figure not available: see fulltext.] © 2013 American Society for Mass Spectrometry.


PubMed | Glycan Connections LLC
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2014

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.


PubMed | Glycan Connections LLC
Type: Journal Article | Journal: Journal of the American Society for Mass Spectrometry | Year: 2014

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 Gal1,3Gal. 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.


PubMed | Glycan Connections LLC and University of New Hampshire
Type: | Journal: Discovering the subtleties of sugars : proceedings of the 3rd Beilstein Glyco-Bioinformatics Symposium : June 10th - 14th, 2013, Potsdam, Germany. International Beilstein Symposium on Glyco-Bioinformatics (3rd : 2013 : Potsdam, Germany) | Year: 2016

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.

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