基於液相層析質譜平台的醣質體分析方法對成年斑馬魚的器官特異之末端醣表位的深入詮釋

Abstract

隨著質譜儀器與電腦運算技術的進步，對於不同物種、器官、細胞或疾病進行醣質體學與醣蛋白體學分析，進而嘗試闡述其生物功能意義的研究方式，是近年生命科學愈受重視的一新興領域。除了以人類為主的醣質體學研究以外，科學家們也研究其他物種所表現的醣質，試圖建立各種模型來探討並解決人體中與醣相關的疾病、生物途徑與機制，藉此提升人類的醫學知識，增進社會福祉。本碩士論文的研究工作，意旨針對斑馬魚的腦、腸與卵巢，以更高精確度與高感度的液相層析串聯式質譜儀分析法，進一步解析鑑定其蛋白醣質體的特色，建構深度的醣末端分子表位於各組織器官的表現分佈圖譜。其中一重點在於首度探討斑馬魚的硫酸化醣質，利用了固相萃取法，將甲基化衍生後帶負電的硫酸化醣質分離出來，再透過自動化質譜圖數據擷取與後續多面向篩選統整分析，鑑定其主要結構、硫酸化位點與相對表現量。主要具體研究成果除發現並解構了斑馬魚腦部與腸特有的唾液酸及硫酸化醣質體表位，首次在腦中找到O-醣基化的甘露醣鏈，印證了斑馬魚體內具有製造O-醣基化甘露醣鏈的相關酵素表現的文獻報導；並在N-醣基化醣鏈上以質譜的方式證實帶有硫酸化修飾的人類自然殺手-1(HNK-1)末端表位結構，交互驗證了先前其他研究團隊透過抗體染色推論斑馬魚腦內的人類自然殺手-1抗原的表現。由於小鼠與人類的腦中皆有類似的末端表位以及O、N-醣基化的多醣鏈表現，透過全面性地研究斑馬魚的醣質體，以期能幫助學界在未來研究醣生物學相關的生理功能與疾病時，建立一個相對於小鼠或人類而言便利、成本低廉、更符合需求的斑馬魚替代模型。

Driven largely by recent technical advances in mass spectrometry, glycomics is increasingly being pursued. It raises our knowledge and provides evidence for one of the most fascinating post-translational modifications, which adds glycans onto proteins and alters their functions. Danio rerio (zebrafish) is one of model organisms that is well-suited for genetic and developmental studies, serving in many instances as relevant disease models that offer many advantages besides mouse model. Current understanding of the embryos and adult zebrafish glycomes indicates high similarity with, as well as distinctive differences from that of human and mice. Similarities on gene level as paralogous genes are also high, which may be exploited as keys to uncover glycosylation-associated pathologies in human. Previous structural studies on the zebrafish glycome, by ways of MALDI MS and MS/MS and other techniques in organ-specific manner has provided a broad distribution map of a diverse range of N-, O-GalNAc and lipid-linked glycans but many details are still missing. In my thesis work, the settings and applicability of a higher sensitivity and precision nanoLC-MS2/MS3 platform to non-mammals e.g. zebrafish were first investigated and then fine-tuned to home in on delineating the various unique fucosylated, sialylated, and/or sulfated terminal glycotopes, particularly those expressed in adult brain, in comparison with ongoing comprehensive murine brain glycomics currently undertaken in the laboratory by others. Notably, the presence of sulfated HNK-1 glycotope as carried on sialylated N-glycans in brain was unambiguously identified by the first ever sulfoglycomics performed on zebrafish.