Shared Resource Cores

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PROJECT SU MMARY (See instructions): Analysis of glycoconjugate structure/functions requires specialized expertise from various disciplines. Many of these tools and approaches are not only difficult, but also require expensive and complex equipment. We have established our Shared Resource Core to not only contain the components that are most essential for the aims of this PEG, but also that build upon the unique strengths of our participating investigators. Each sub-Core is detailed in their separate sections. Sub-Core 01 - Mass Spectrometry -Drs. van Eyk and Zhang are leaders in mass spectrometric analysis of proteins and glycoproteins. The Core is extraordinarily well equipped. The Core's offerings include: 1) Identification of proteins; 2) global quantification of N-linked or O-linked glycoproteins using solidphase extraction of glycopeptides; 3) Lectin affinity/MS analyses; 4) Monitoring changes in glycosylation by lectin microarrays; 5) The use of "Click" chemistry for analyses of metabolically produced glycoconjugates; 6)Glycan profiling by MALDI-MS" (AXIMA-Resonance) and GlycanAnalyser. 7) Cell surface capture technology for isolation of glycoproteins; 2) Targeted capture of soluble glycoproteins from cytoplasm or extracellular environment; 3) MRM MS/MS assays for targeted quantification ofglycoproteins and glycosylation sites. Sub-Core C2 - NMR - When possible, glycan structure is best determined by NMR. Dr. Allen Bush is a leading expert in the structural analysis of glycans by NMR. His sub-Core supports the PEG by offering structural expertise for oligosaccharides, polysaccharides, glycopeptides, small glycoproteins and glycolipids. State of the art NMR equipment and methods are available in this sub-Core. Sub-Core C3 - Chemistry - Dr. Kevin Yarema is a leading chemical glycobiologist. His sub-Core will support the PEGs by: 1) providing sugar analogs for "metabolic glycoengineering" (eg. SCFA-hexosamines); 2) performing custom synthesis of inhibitors (eg. D-PDMP, TMG), azido sugars and sugar nucleotides, glycan-tagging tools and other small molecules. Sub-Core 04 - 0-GlcNAc Resources - Drs. Hart and Zachara are leaders in the analysis of 0-GlcNAc. In support of the PEGs we offer: 1) Tools for detecting 0-GlcNAc, OGT and OGA; 2) Develop site-specific 0-GlcNAc antibodies; 3) Provide synthetic 0-GlcNAc peptides for antibody production and MS standards; 4) Assays for OGT and OGA activity; 5) Quantitation of sugar nucleotides & nucleotides; 6) Assist with site mapping and quantification on single proteins or globally. This Shared Resource provides key technologies to allow rapid advancement of glycoscience research critical to the mission of the NHLBI.