AN IMPROVED PLATFORM FOR GAG DISACCHARIDE ANALYSIS USING LC/MS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 结构上多分散的硫酸乙酰肝素(HS)链负责HS蛋白多糖与多种蛋白质的相互作用，在生物学中起着至关重要的作用。HS生物合成的一个步骤，即GlcNAc残基上的N-脱乙酰化/N-硫化，可以被3‘-磷酸腺苷5’-磷酸硫酸盐的有限供应中断，并导致HS链中未被取代的氨基葡萄糖残基的形成。虽然内源性N-未取代糖仅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，显然有必要系统地研究哺乳动物中这些含有游离氨基的HS二糖的存在，并以组织特异性的方式确定其结构和分布模式。 目前，MSR收到的所有HS分析样本都被提取出来，然后彻底消化肝素裂解酶。然后用SEC/MS分析双糖的特征，这些数据被用来确定硫酸盐和醋酸基的平均链长和平均掺入。该数据用于识别双糖在母链内部区域以及非还原末端的分布。对于蛋白质结合研究来说，它的非还原末端是特别有趣的。 最新进展情况： SEC LC/MS系统用于实验室收到的每个样品的双糖分析。它已被用于已发表的几项核心和协作研究(1-5)。我们已经测试了新的Waters UPLC SEC固定相，用于GAG双糖分析。目标是能够使用UPLC SEC材料缩小柱直径，同时利用其提高的效率。在最初的研究中，适合蛋白质的UPLC SEC柱不适合用于GAG二糖。我们正在等待Waters释放一种孔径较小的UPLC SEC固相。 GAG双糖分析需要提高层析分辨率、灵敏度，缩短分析时间。这些能力尤其需要用于分析来自组织表面和来自组织微阵列的GAG。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.Staples，G.O.，Shii，X.，and Zaia，J.(2011)人胞外硫酸酯酶HSulf2修饰的哺乳动物肝素硫酸盐的糖组分分析，PLoS one 6，e16689。 2.Schumacher，V.，Schlotzer-Schrehardt，U.S.，Karumachi，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.和Ai，X.(2011)WT1对Sulf表达的调控对维持肾小球滤过屏障至关重要，2011年2月22日接受《美国肾脏病学会杂志》。 3.Staples，G.O.，Shi.，X.和Zaia，J.(2010年)扩展的NS结构域位于硫酸乙酰肝素链的非还原末端，J Biol Chem285,18336-18343。 4.Langsdorf，A.，Schumacher，V.，Shii，X.，Tran，T.，Zaia，J.，Jain，S.，Taglienti，M.，Kreidberg，J.A.，Fine，A.和Ai，X.(2010)Sulff在精原干细胞生态位中的表达调控和功能，糖生物学21,152-161。 5.施霞和扎亚，J.(2009年)器官特异性硫酸乙酰肝素结构表型，生物化学杂志第284期，11806-11814。

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.