以高階液相層析串聯質譜分析技術研發高通量醣質體末端表位鑑定平台

Advancing a High Throughput Glycotope-Centric Glycomics Workflow Based on NanoLC-MS2-Product Dependent-MS3 Analysis of Permethylated Glycans

蕭正得(Cheng-Te Hsiao)

指導教授 ： 邱繼輝

國立臺灣大學/生命科學院/生化科學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700295

摘要

細胞表面的醣基化修飾存在於不同的醣蛋白及醣脂質上，其多樣性的醣結構組合而成的醣質體已知在生物體中扮演極為重要的功能與角色。例如，醣質體的末端表位參與細胞間的相互辨識，或是生物分子間藉由醣質體與凝集素而連結，以及疾病相關的病原體藉由細胞表面的醣質體所造成感染，除此之外，癌症的發生往往也伴隨著不正常的醣化表現。醣類的結構組成，主要是由固定的核心次單元進行延伸，藉由不同醣鏈骨架的延長、分支，以及末端醣的修飾所組成，其結構的最末端稱之為醣質體末端表位，這也是生物系統中辨識醣質體最重要的決定位置。因此，建立高效率的醣質體分析平台，使之精準地分析其末端表位的結構是極其重要的。隨著儀器的日新月異，新世代的質譜儀可集合不同感測器及碰撞室功能於單一儀器，可在不同的分析組合下提高檢測速度，並提供不同斷片模式於多次質譜分析，進而提高了獲得有用醣斷片資訊的可能性。由於醣質體及其個別結構無模板可供預測，不像蛋白質體的質譜分析，可以利用水解後的胜肽進行質譜斷裂，再以軟體快速比對序列資料，因此醣質體質的質譜分析缺少相對應的軟體及資料庫也將是分析上的另一個挑戰。
本論文的研究工作主旨即在於(一)利用新世代高解析液相層析串聯質譜儀結合蛋白質體研究的層析方法，在不更換溶液及管柱前提下，完成利用不同電性切換，探討不同樣品間的醣質體及其特定(硫酸化)醣末端表位分析。(二)配合質譜儀及方法的演進，同時進行軟體開發，用以處理大量的質譜數據並分析出有用的醣質體資訊；並配合不同質譜檢測及不同儀器參數或不同的醣類樣品，開發其對應的功能及參數，及找出定性及相對定量方法。(三)利用此平台分析不同生物樣品的醣質體變化及發現相對少量的末端表位，在利用胃癌細胞醣質體為受質的研究中，成功發現不同岩藻糖苷酵素對特定端岩藻糖末端表位具有不同專一性，以及分析老鼠紋狀體組織醣質體發現含少見的雙唾液酸末端表位。
本論文已完成醣質體末端表位鑑定平台開發，包含整合高解析醣質體的質譜技術及高通量的數據分析。故此平台將可應用於不同生物樣品的體醣質分析，及利用實驗設計用以分析在不同生理條件、不同基因表現和不同化學作用下的醣質體變化。隨著新世代高解析質譜儀和醣質體生物資訊的快速發展，結合次世代基因定序科技及其他科學研究，可讓疾病的預防與診療能夠更為精準，以期達到精準醫療的最終目標。

關鍵字

醣質體 ； 醣質體末端表位 ； 高解析液相層析串聯質譜儀 ； 醣質生物資訊 ； 精準醫療

並列摘要

Glycans attached on lipid and proteins mediate a variety of structural and functional roles in cell–cell recognition, cell-matrix adhesion and host-pathogen interactions. Aberrant glycosylation has been implicated in the development of diseases such as cancer. N- or O-glycans have common core structures which can be extended either in linear or branched form, and are then modified or terminally capped by sialic acid, fucose, and sulfate at different positions to generate the critical terminal glyco-epitopes, or glycotopes. To better address their biological functions, it is essential to develop a precise glycomic analytical platform that will globally survey the tremendously heterogeneous glycan structures expressed by a cell to seek out, identify and quantify the all-important glycotopes. This is made possible by the advent of Orbitrap Fusion Tribrid mass spectrometer system, which combines quadrupole, Orbitrap, and ion trap mass analyzers, with increasing speed of HCD and CID fragmentation that can be performed in parallel or in successive stages for maximum experimental flexibility. Taking advantages of these latest advances in mass spectrometry (MS), the primary goal of this thesis work is to establish a high throughput glycotope-centric glycomics workflow based on nanoLC-MS2-product dependent-MS3 analysis of permethylated glycans. Such nanoLC-MS2/MS3 data acquisition mode was previously shown to be effective for mapping the sulfated glycotopes in negative ion mode, and now extended in this work to distinguish closely related isomeric fucosylated glycotopes carried on non-sulfated glycans in positive ion mode. The diagnostic MS3 ions were validated via analysis of glycan standards while the reverse phase C18 nanoLC conditions were optimized by running against complex glycomic samples derived from gastric and colon cancer cell lines. With elevated temperature, isomeric constituents of smaller O-glycans were shown to be well resolved but not those of larger size carrying increasing numbers of fucose, sialic acids and sulfates. To facilitate data analysis, a data mining computational tool, GlyPick, was developed and implemented at various levels to filter out true glycan MS2 spectra and associated MS3 triggered on target glycotopes; to search for, identify and quantify their relative amount based on the summed intensity of the diagnostic MS2/MS3 ions to allow comparison across different samples; as well as to assign glycosyl compositions to inferred monoisotopic precursors and to quantify their relative abundance, in a fully automated fashion. Finally, the established glycotope-centric glycomics workflow was applied to 1) map the glycomic changes in gastric fucosylated glycotopes in response to treatments by fucosidases of distinct specificities; 2) search for and identify with confident rare disialylated glycotopes on the N-glycans from mouse brain striatum. The demonstrated practical utilities of this novel analytical platform will allow high sensitivity in depth glycomic mapping that can parallel the progress in applying next-generation sequencing (NGS) and proteomics to precision medicine for disease treatment and prevention that takes into account individual variability.

並列關鍵字

Glycomics ； Glycotopes ； LC?MS ； Orbitrap Fusion ； Glycoinformatics ； Precison medicine

參考文獻

References

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