Detection and Histopathology Localization of O-Glycans and Glycosaminoglycans in Tissues

摘要 人们已经对其结构和功能角色进行了大量的研究 四种主要的多糖类型(例如，N-连接、O-连接、糖胺聚糖(GAG)) 和鞘糖脂(GSL))与疾病状态的关系。这些不同的葡聚糖是如何 实际上，组织内的分布几乎没有被研究过，而且经常每一类都是 相互独立地学习。此外，对这些糖类的分析通常是 在组织或细胞提取物中完成，放弃任何本地化机会。任何先前的组织测绘 这依赖于广泛的碳水化合物结合凝集素或碳水化合物。 抗原抗体。这些试剂可用于将结构糖链基序定位在 但它们一般不区分N-糖蛋白或O-糖蛋白，也不区分GSL 结构，因为所有这些都可以共享相同的糖链结构靶点。为了解决这个问题，我们的实验室 最近开发了一种新的方法来直接在组织上描述N-连接的多糖 使用高分辨率的MALDI-FTICR仪器对MALDI-MS进行成像。这 使用福尔马林固定的组织是最有效的方法，提供了前所未有的机会 分析世界各地储存的疾病和正常的福尔马林固定组织块。该方法还包括 非常有效地鉴定了冷冻组织中的N-连接的多糖和主要的GSL物种。 目前该提案的目标是开发相关的MALDI组织成像方法来治疗O- 连接的粘蛋白类型的葡聚糖和软骨素/肝素插嘴类。这将提供 史无前例的能力，不仅可以检查这四种多糖的个体分布 组织中的分类，但也允许比较它们在相同组织中的分布。 该提案直接涉及RFA-RM-15-008开发分析方法和 这些工具能够快速而详细地表征来自 生物来源。

Abstract Extensive amounts of research has been done individually on the structure and functional roles ofthe four major glycan types (e.g., N-linked, O-linked, glycosaminoglycan (GAG) andglycosphingolipids (GSL)) in relation to disease states. How these different glycans are actuallydistributed within tissues has barely been studied, and frequently each class is studiedindependent of one another. Furthermore, analysis of these glycan classes are usually done intissue or cell extracts, forfeiting any localization opportunities. Any prior tissue mapping that hasbeen done has relied on broad class carbohydrate binding lectins, or carbohydrate antigenantibodies. These reagents may be used to localize structural glycans motifs within a giventissue, but they do not generally distinguish N-glycan or O-glycan proteins, nor GSL structures,as these could all share the same glycan structural target. To address this, our lab has recentlydeveloped a novel method to profile N-linked glycans directly on tissue using MALDI-imagingmass spectrometry using a high-resolution MALDI-FTICR instrument. This approach is mosteffective using formalin-fixed tissues, offering an unprecedented opportunity to analyze diseaseand normal formalin-fixed tissue blocks stored world-wide. The method is also very effective atidentification of both N-linked glycans and major GSL species in frozen tissues. The goal of thecurrent proposal is to develop related MALDI tissue imaging methods for O- linked mucin typeglycans and chondroitin/heparin GAG classes. This will provide unprecedented capabilities toexamine not only the individual distributions of these four glycan classes in tissues, but alsoallow comparisons of their distributions together in the same tissues. The proposal directlyaddresses the RFA-RM-15-008 goal of developing analytical methods and tools to enable rapidand detailed characterization of the complete glycan representation from a biological source.