使用邏輯演繹序列串聯質譜法鑑定碳水化合物的結構

Abstract

碳水化合物在生活中具有多種功能，它與蛋白質和去氧核糖核酸等其他生物大分子一樣重要。但是，我們對碳水化合物的瞭解遠遠落後於我們對蛋白質和核酸的瞭解相比。這主要是因為碳水化合物的結構分析是非常具有挑戰性。質譜法是一種高度靈敏的分析工具，適用於分析生物大分子。大多數常用的質譜法涉及碳水化合物的衍生化或僅識別碳水化合物結構的一部分。為了開發一種通用且方便使用的質譜法以用於確定碳水化合物的一級結構，我們開發了一種新的質譜法來識別未衍生化的寡糖的結構。這種方法，稱爲邏輯演繹序列串聯質譜法（LODES/MSn ），可以提供鎖鏈結構，異頭構型，單醣成分和分支位置。LODES/MSn 使用鈉離子加合物的低能量碰撞誘導解離 (CID)，從而能夠裂解選擇性化學鍵，這是為後續 CID 識別結構決定性碎片離子的合理解離序列，並且特別是製備了雙醣 CID譜的數據庫。這方法首先應用在各種標準寡糖，以證明方法的準確性。然後應用在從牛蛋白質、大豆蛋白、人乳腺上皮細胞中提取的 N-聚醣和高甘露糖 N-聚醣和人類乳腺癌。

Carbohydrates have various functions in life, they are as important as other classes of macrobiomolecules such as protein and DNA. However, our understanding of carbohydrates is far lagged behind compared to what we have learned protein and DNA. This is mainly because the structural analysis of carbohydrates is challenging. Mass spectrometry is highly sensitive and a robust analytical tool for macro-biomolecule analysis. Most of the commonly used mass spectrometry-based methods involve the derivatization of carbohydrates or only identify part of the carbohydrate structure. With the aim to develop a universal yet user friendly mass spectrometry-based method to determine the primary structure of carbohydrates/glycans, we developed a new method for complete structural identification of underivatized oligosaccharides. This method, logically derived sequence tandem mass spectrometry (LODES/MSn), can provide assignments of linkages, anomeric configurations, monosaccharide constituents, and branch locations. LODES/MSn entails low-energy collision induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of various types of standard oligosaccharides as a proof of concept. Then, we applied LODES/MSn to structural assignment of sialylated oligosaccharides, N-glycans, and high mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma.