邏輯演繹序列串聯質譜法應用於N-聚醣的結構鑑定

Abstract

N-連接醣基化是最重要的蛋白質轉譯後修飾之一，它在生物體中有十分重要的作用，因此N-聚醣的結構鑑定非常重要，但由於N-聚醣的異構物眾多、結構十分複雜，目前N-聚醣的結構鑑定仍十分具有挑戰性。我們實驗室過去開發了一種新的質譜方法¬¬¬¬¬¬¬，邏輯演繹序列串聯質譜法（logically derived sequence tandem mass spectrometry, LODES/MSn），用於進行醣的結構鑑定。本研究使用了兩種不同去醣基化的方法，並通過一系列的萃取步驟純化N-聚醣，再使用液相層析儀、質譜儀和邏輯演繹序列串聯質譜法，對萃取出的N-聚醣進行結構鑑定。本研究從豆子中萃取出一些在生合成路徑中不常見的高甘露N-聚醣，透過比較不同樣品的萃取離子層析圖（extracted ion chromatogram），測量了不同樣品中這些不常見的N-聚醣異構物的訊號強度差異，發現在紅豆中這些不常見的N-聚醣最為豐富。為了確認這些N-聚醣的產生，針對紅豆的萃取步驟改變不同的反應條件，確認了在不同反應條件下，在生合成路徑中不常見的高甘露N-聚醣的存在。本研究利用萃取出的的N-聚醣，協助建立部分的N-聚醣資料庫，再透過比對資料庫，快速地鑑定其他生物樣品，包含各種豆子、肉等的N-聚醣結構。

N-linked glycosylation is one of the most important post-translational modifications of proteins, and it plays a very important role in organisms. Therefore, the structural identification of N-glycans is very important. However, the structural identification of N-glycans is still very challenging due to the numerous isomers and complex structures of N-glycans. A new mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn), was developed in our laboratory in the past for the structural identification of sugars. In this study, two different de-glycosylation methods were used, and N-glycans were purified by a series of extraction steps, followed by liquid chromatography, mass spectrometry, and logically derived sequence tandem mass spectrometry, used to identify extracted N-glycans structure. In this study, some high-mannose N-glycans that are not commonly found in biosynthetic pathways were extracted from beans, and these uncommon N-glycans were measured in different samples by comparing the extracted ion chromatograms of different samples. Differences in the signal intensities of glycan isomers found that these uncommon N-glycans were most abundant in red beans. To confirm the production of these N-glycans, different reaction conditions were changed for the red bean extraction step, confirming the presence of high-mannose N-glycans that are not commonly found in biosynthetic pathways under different reaction conditions. In this study, the extracted N-glycans were used to assist in the establishment of a partial N-glycan database, and applied this database for rapid structural determination of the N-glycans in other biological samples, including various beans and meat.