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

摘要 大量的研究已经单独完成的结构和功能的作用 四种主要聚糖类型（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。这些不同的聚糖 几乎没有研究过它们在组织中的实际分布，而且每一类都经常被 相互独立的研究。此外，这些聚糖类的分析通常是 在组织或细胞提取物中完成，丧失任何定位机会。任何既往组织标测 已经完成的研究依赖于广泛的碳水化合物结合凝集素， 抗原抗体这些试剂可用于将结构聚糖基序定位在多核苷酸内。 但它们通常不能区分N-聚糖或O-聚糖蛋白，也不能区分GSL 结构，因为这些都可以共享相同的聚糖结构靶标。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，直接在组织上分析N-连接聚糖， 使用高分辨率MALDI-FTICR仪器的MALDI-imagingmass spectrometry。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， 分析储存在世界各地的疾病和正常福尔马林固定组织块。该方法也 非常有效地鉴定了冷冻组织中的N-连接聚糖和主要GSL种类。 目前的目标是发展相关的MALDI组织成像方法，用于O- 连接的粘蛋白型聚糖和软骨素/肝素GAG类。这将提供 前所未有的能力，不仅检查这四种聚糖的个体分布， 组织中的类别，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开发分析方法的目标， 能够快速和详细地表征完整聚糖表示的工具， 生物源。

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.