AN IMPROVED PLATFORM FOR GAG DISACCHARIDE ANALYSIS USING LC/MS

使用 LC/MS 进行 GAG 二糖分析的改进平台

• 批准号: 8365548
• 负责人: JOSEPH ZAIA
• 金额: $ 2.31万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365548
• 关键词: Acetates; American; Biology; Caliber; Carbon; Chromatography; Data; Deacetylation; Disaccharides; Funding; GAG Gene; Glucosamine; Glycobiology; Goals; Grant; Heparan Sulfate Biosynthesis; Heparan Sulfate Proteoglycan; Heparin Lyase; Heparitin Sulfate; Human; Inorganic Sulfates; J-Chain Immunoglobulins; Journals; Laboratories; Length; Mammals; Mass Spectrum Analysis; Medicine; National Center for Research Resources; Nephrology; Organ; Parents; Pattern; Phase; Phenotype; Play; Principal Investigator; Protein Binding; Proteins; Publishing; Regulation; Research; Research Infrastructure; Resolution; Resources; Role; Sampling; Societies; Solid; Source; Structure; Sulfatases; Surface; System; Testing; Time; Tissue Microarray; Tissues; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Update; WT1 gene; Water; base; cost; extracellular; glomerular filtration; improved; interest; liquid chromatography mass spectrometry; stem cell niche; sulfation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The structurally-polydisperse heparan sulfate (HS) chains are responsible for the interactions of HS proteoglycans with a wide variety of proteins, playing critical roles in biology. One HS biosynthesis step, the coordinated N-deacetylation/ N-sulfation on the GlcNAc residues, can be interrupted by the limited supply of 3'-phosphoadenosine 5'-phosphosulfate, and result in the formation of unsubstituted glucosamine residues within HS chains. Although endogenous N-unsubstituted saccharides have only been identified in a handful of studies, they have been implicated to be important in cellular and pathophysiological phenomena. Therefore, there is a clear need to systematically study the occurrence, and determine the structure and distribution patterns of these free-amino-containing HS disaccharides from mammals in a tissue-specific manner. At the present time, all samples received by the MSR for HS analysis are extracted and then digested exhaustively heparin lyases. The disaccharides are then profiled using SEC/MS. The data are used to determine the average chain lengths and average incorporation of sulfate and acetate groups. The data serve to identify the distribution of disaccharides in the internal regions of the parent chain as well as the non-reducing end. It is the non-reducing end that is of particular interest for protein binding studies. Update on progress: The SEC LC/MS system is used for disaccharide analysis of every sample received in the laboratory. It has been used for several core and collaborative studies that have been published (1-5). We have tested new Waters UPLC SEC stationary phases for use with GAG disaccharide analysis. The goal is to be able to scale down the column diameter using the UPLC SEC material, taking advantage of its increased efficiency. In initial studies, a UPLC SEC column appropriate for proteins was not appropriate for GAG disaccharides. We are waiting for Waters to release a smaller pore size UPLC SEC solid phase. It is necessary to increase the chromatographic resolution, sensitivity, and to reduce analysis time for GAG disaccharide analysis. These capabilities are needed in particular for analysis of GAGs from tissue surfaces and from tissue microarrays. Towards these ends, we are testing both UPLC HILIC and Chip-based porous graphitized carbon chromatography. 1. Staples, G. O., Shi, X., and Zaia, J. (2011) Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2, PLoS ONE 6, e16689. 2. Schumacher, V., Schlotzer-Schrehardt, U., Karumanchi, S. A., Shi, X., Zaia, J., Jeruschke, S., Zhang, D., Pavenstaedt, H., Drenckhan, A., Amann, K., Ng, C., Hartwig, S., Ng, K.-H., Ho, J., Kreidberg, J. A., Taglienti, M., Royer-Pokora, B., and Ai, X. (2011) WT1 regulation of Sulf expression is crucial to maintaining the glomerular filtration barrier, Journal of the American Society for Nephrology Accepted 2/22/11. 3. Staples, G. O., Shi, X., and Zaia, J. (2010) Extended NS domains reside at the non-reducing end of heparan sulfate chains, J Biol Chem 285, 18336-18343. 4. Langsdorf, A., Schumacher, V., Shi, X., Tran, T., Zaia, J., Jain, S., Taglienti, M., Kreidberg, J. A., Fine, A., and Ai, X. (2010) Expression regulation and function of Sulfs in the spermatogonial stem cell niche, Glycobiology 21, 152-161. 5. Shi, X., and Zaia, J. (2009) Organ-specific heparan sulfate structural phenotypes, J Biol Chem 284, 11806-11814.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 硫酸乙酰肝素（HS）是一种结构多分散的蛋白多糖，它与多种蛋白质相互作用，在生物学中起着重要的作用。一个HS生物合成步骤，即GlcNAc残基上的协调N-脱乙酰化/N-硫酸化，可以被有限供应的3 '-磷酸腺苷5'-磷酸硫酸盐中断，并导致HS链内未取代的葡糖胺残基的形成。虽然内源性N-未取代的辅酶A只在少数研究中被确定，但它们在细胞和病理生理现象中具有重要意义。因此，有一个明确的需要，系统地研究的发生，并确定这些游离氨基含HS二糖从哺乳动物的结构和分布模式，在组织特异性的方式。 目前，MSR收到的用于HS分析的所有样品都经过提取，然后用肝素裂解酶彻底消化。 然后使用SEC/MS分析二糖。数据用于确定平均链长和硫酸酯和乙酸酯基团的平均掺入。 这些数据用于确定二糖在母链内部区域以及非还原端的分布。 非还原端是蛋白结合研究中特别感兴趣的。 最新进展： SEC LC/MS系统用于实验室收到的每份样品的二糖分析。 它已被用于已发表的几个核心和合作研究（1-5）。 我们测试了用于GAG二糖分析的新型沃茨UPLC SEC固定相。 我们的目标是能够利用UPLC SEC材料的高效率来缩小色谱柱直径。 在初始研究中，适用于蛋白质的UPLC SEC色谱柱不适用于GAG二糖。 我们正在等待沃茨发布更小孔径的UPLC SEC固相。 提高色谱分离度、灵敏度、缩短分析时间是分析GAG二糖的必要条件。 这些能力是需要的，特别是从组织表面和组织微阵列的GAG的分析。 为此，我们正在测试UPLC HILIC和基于芯片的多孔石墨化碳色谱。 1. 斯台普斯，G. O.，施，X.，和Zaia，J.（2011）Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2，PLoS ONE 6，e16689. 2. Schumacher，V.，Schlotzer-Schrehardt，美国，Karumanchi，S.一、施，X.，Zaia，J.，Jeruschke，S.，Zhang，D.，中国农业科学院农业研究所所长，帕文施塔特，H.，Drenckhan，A.，阿曼，K.，Ng，C.，Hartwig，S.，Ng，K.- H、何，J，Kreidberg，J. A.，Taglienti，M.，Royer-Pokora，B.，和Ai，X.（2011）WT 1调节Sulf表达对维持肾小球滤过屏障至关重要，美国肾脏病学会杂志接受2/22/11。 3. 斯台普斯，G. O.，施，X.，和Zaia，J.（2010）Extended NS domains resident at the non-reducing end of heparan sulfate chains，J Biol Chem 285，18336-18343. 4. Langsdorf，A.，Schumacher，V.，施，X.，Tran，T.，Zaia，J.，贾恩，S.，Taglienti，M.，Kreidberg，J. A.，好吧A和Ai，X.等人（2010）精原干细胞龛中硫的表达调节和功能，糖生物学21，152-161。 5. 施，X.，和Zaia，J.（2009）Organ-specific heparan sulfate structural phenotypes，J Biol Chem 284，11806-11814。