邏輯演繹序列串聯質譜法應用於蛋類N-聚醣的結構鑑定

Abstract

我們實驗室團隊開發出一個創新的質譜分析方法，邏輯演繹序列串聯質譜法（logically derived sequence tandem mass spectrometry, LODES/MSn），有別於傳統方法只能解析部分結構，該方法能將醣的結構完整分析出來。本研究使用酵素萃取蛋類當中的N-聚醣，並利用高效能液相層析法(High-Performance Liquid Chromatography, HPLC)、質譜儀及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。透過比較不同樣品的層析圖（Chromatogram）後發現許多N-聚醣含有多種異構物。本實驗著重於鑑定這些特殊N-聚醣的結構，再搭配少許的酵素反應加以佐證，結果發現出許多目前在生物合成路徑並未見到的N-聚醣，甚至有些不尋常的N-聚醣含量是占大多數。(HexNAc)1[(Man)3(GlcNAc)2]組成的N-聚醣依照生物合成路徑應只存在1種可能，卻在蛋類當中發現共5種異構物，(Hex)1(HexNAc)1[(Man)3(GlcNAc)2]組成的N-聚醣依照生物合成路徑應只存在2種可能，卻在蛋類當中發現共5種異構物，(HexNAc)2[(Man)3(GlcNAc)2]組成的N-聚醣依照生物合成路徑應只存在1種可能，卻在蛋類當中發現共4種異構物，在(Hex)2(HexNAc)1[(Man)3(GlcNAc)2]中發現2種結構，在(Hex)1(HexNAc)2[(Man)3(GlcNAc)2]中發現1種結構、在(HexNAc)3[(Man)3(GlcNAc)2]中發現3種結構，在(Hex)2(HexNAc)2[(Man)3(GlcNAc)2]中發現1種結構、在(HexNAc)4[(Man)3(GlcNAc)2]中發現1種結構，因此我們推測可能存在尚未發現的生物合成路徑。Our laboratory has developed an innovative mass spectrometry-based approach called logically derived sequence tandem mass spectrometry (LODES/MSⁿ). Unlike traditional techniques that can only provide partial structural information, LODES/MSⁿ enables comprehensive structural elucidation of carbohydrates. In this study, N-glycans were enzymatically extracted from egg samples and subjected to structural analysis using high-performance liquid chromatography (HPLC), mass spectrometry, and the LODES/MSⁿ method. By comparing chromatograms from different samples, we found that many N-glycans exist in multiple isomeric forms. This study focuses on the structural identification of these unusual N-glycans, supported by limited enzymatic reactions. Our results revealed numerous N-glycans that have not been observed in known biosynthetic pathways. Remarkably, some of these atypical N-glycans were found to be the predominant forms. Based on known biosynthetic pathways, N-glycans with the composition (HexNAc)₁[(Man)₃(GlcNAc)₂] are expected to exist in only one structure. However, in egg samples, we identified five distinct isomers. Similarly, for N-glycans with the composition (Hex)₁(HexNAc)₁[(Man)₃(GlcNAc)₂], only two structures are theoretically possible, yet we detected five isomers. In the case of (HexNAc)₂[(Man)₃(GlcNAc)₂], only one structure is theoretically possible, but four were observed. For the composition (Hex)₂(HexNAc)₁[(Man)₃(GlcNAc)₂], two isomers were identified.For (Hex)₁(HexNAc)₂[(Man)₃(GlcNAc)₂], one isomer was found. For (HexNAc)₃[(Man)₃(GlcNAc)₂], three isomers were found. For (Hex)₂(HexNAc)₂[(Man)₃(GlcNAc)₂], one isomer was found.For (HexNAc)₄[(Man)₃(GlcNAc)₂], one isomer was found. These findings suggest the potential existence of previously unrecognized biosynthetic pathways.