- 學位論文
硫酸化醣質體之質譜分析技術的研發和應用
Development and Applications of Mass Spectrometry-based Sulfoglycomics
摘要
硫酸化修飾的醣類在生物免疫系統、生殖系統，和發炎反應裡，皆被認為是重要的生物辨識分子。最廣泛的研究為淋巴細胞回歸至淋巴組織內，兩者之間的作用力是藉由淋巴細胞表面上的受體辨識週邊淋巴結表面的涎黏蛋白上的特定硫酸化醣類結構 6-sulfo sLeX。此外，近年來利用醣晶片的研究暗示不同種類的硫酸化醣類末端結構為一些免疫系統裡的凝集素所選擇辨識的結構。目前這些經硫酸化修飾的末端醣類結構大部分是藉由抗體所解出，被掛在怎樣的醣類主體結構及是否有更多未知的硫酸化醣類結構的資訊仍有限。 目前廣泛應用的醣質體質譜分析平台，因為硫酸化修飾的醣類結構其微量且帶有負電的特性，並不適合應用在分析硫酸化醣質體。因此，此論文的主要目的是建立硫酸化醣質體的分析方法論，搭配高敏感和高精確性的質譜儀，建立分析平台並應用在不同生物樣品上。首先建立適當的樣品前置處理流程，化學衍生法配合兩階段式的萃取和分離，分別為反相層析和弱陰離子交換層析，可成功的將甲基化的硫酸化醣類純化、並分離出不同帶電性的硫酸化醣類。後端質譜分析流程為，確認適當的基質以利於質譜在負離子模式下偵測出硫酸化醣質體以得到輪廓圖譜。接著透過串聯式質譜儀的搭配，在負離子模式下，可判別硫酸化的鍵結和位置。在正離子模式下和不同碰撞能量形式的串聯式質譜分析，得到硫酸化醣類末端結構是否掛在特定的醣鏈上或特定核心結構，和其他岩藻醣，唾液酸的鍵結。藉著搭配不同的質譜分析其互補的斷片模式可有效定序硫酸化醣類的完整結構。 此建立好的硫酸化醣質體分析方法已成功的應用在老鼠淋巴組織比如週邊淋巴結、黏膜相關的淋巴組織派伊爾結(Payer's patch)和硫酸化轉移酵素的轉殖細胞株。在老鼠週邊淋巴組織上鑑定出除O型醣鏈上以外，在N型醣鏈上亦帶有特定的硫酸化醣類結構 6-sulfo sLeX，並發現到縱使剔除掉硫酸化轉移酵素基因的老鼠，其週邊淋巴組織上仍表現硫酸化醣類。此外，成功分析不同位置的淋巴結組織上硫酸化醣類的差異性，並在老鼠派伊爾結上的O型醣鏈發現新奇的硫酸化修飾的醣類結構。最後，分析轉殖硫酸化轉移酵素的細胞株探討酵素在生物體內對受質的專一性，發現到不同號的硫酸化轉移酵素可辨識著不同的O型醣鏈的核心結構。這些成功的例子顯示此硫酸化醣質體質譜分析平台的穩定性，期望能藉此方法論的開發能輔助鑑定硫酸化醣類結構和找出許多原本受限於分析方法的未知新奇硫酸化醣類結構。
並列摘要
Sulfate modification on terminal oligosaccharyl epitopes carried on N-glycans and O-glycans has increasingly been indicated as critical determinant mediating a diverse range of biological functions. For example, lymphocyte homing to lymphoid tissue is critically dependent on recognition between L-selectin on lymphocytes and 6-sulfo sLeX epitope carried on the O-glycans on the high endothelial venule of peripheral lymph nodes. Additionally, many other sulfated glycotopes have recently been identified as preferred ligands for Siglecs and galectins in immune systems via glycan array screening. To define its true physiological relevance, a robust sulfoglycomic analysis is needed to establish the expression pattern, exact structures and the glycan carriers of these sulfated epitopes. Due to a natural low abundance and the negatively charged nature, sulfated glycans are often not easily detected by current approaches in mass spectrometry (MS) based glycomic analysis. This thesis work aims to develop methodology for sulfoglycomic analysis, and to apply the developed methodology to different biological samples. First, the standard permethylation protocol was further developed and optimized for sulfated glycans, allowing efficient recovery of the permethyl derivatives by use of reverse phase C18 cartridge for a high sensitivity first screen by MALDI-MS in the negative ion mode. A better matrix, 3,4-diaminobenzophenone, was identified, which significantly enhances detection sensitivity. Moreover, through systematic MALDI-MS/MS analysis of synthetic sulfated glycan standards in negative ion mode, we identified diagnostic fragment ions to unambiguously determine the location and linkage of the sulfate moiety. Subsequent microscale fractionation based on NH2 beads packed in microtip offers enrichment of sulfated glycans away from highly abundant non-sulfated glycans, so as to enable better chance of detection of sulfated glycans in the positive ion mode. It was found that previously established low and high energy CID MS/MS fragmentation characteristics in the positive ion mode are directly applicable to permethylated sulfated glycans, which enable a complete sequencing including glycosyl linkage and branching determination that are otherwise less amenable in negative ion mode. The developed methodology provided a high sensitivity mapping of the sulfoglycomes derived from mouse lymph nodes and human GlcNAc-6-sulfotransferase transfected cell lines. It was found that 6-sulfo sLeX glycotope can be carried on N-glycans and not restricted in expression to O-glycans of mouse peripheral lymph nodes. Moreover, sulfated N-glycans and O-glycans were still detected in mice with double knockouts of the critical GlcNAc6ST-1 and -2, suggesting the contribution of other sulfotransferases in making the residual sulfated epitopes including those sulfated at Gal. Distinctive sulfoglycomic pattern of mouse Peyer’s patches was observed in comparison with that of peripheral lymph nodes, and a novel sulfated SdA-like glycotope carried on O-glycans was identified by MS/MS sequencing and further linkage analysis. Finally, in the case of colon cancer cells transfected with different human GlcNAc6STs, it was found that only human GlcNAc6ST-2 can add sulfate on both core 2 and extended core 1 based O-glycans, whereas both GlcNAc6ST-1 and GlcNAc6ST-3 will not act on extended core 1 structures. These results are fully consistent with elevated expression of GlcNAc6ST-2 in colonic carcinomas, which contributed to positive MECA79 immunostaining against the sulfated glycotope carried on extended Core 1 structures.