Characterisation and Comparability Laboratory, The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Co. Dublin, A94 X099, Ireland; School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, D02 R590, Ireland.
Characterisation and Comparability Laboratory, The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Co. Dublin, A94 X099, Ireland.
Anal Chim Acta. 2021 Sep 22;1179:338840. doi: 10.1016/j.aca.2021.338840. Epub 2021 Jul 9.
Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation. The difficulty in comprehensively characterizing the serum N-glycome arises from the absence of a biosynthetic template resulting in great structural heterogeneity and complexity. To help overcome these challenges we developed a 2-dimensional liquid chromatography platform which utilizes offline weak anion exchange (WAX) chromatography in the first dimension and hydrophilic interaction liquid chromatography (HILIC) in the second dimension to separate N-glycans by charge, corresponding to degree of sialylation, and size, respectively. Performing these separations offline enables subsequent derivatization with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) for sialic acid linkage determination and the identification of sialic acid linkage isomers. Subsequent tandem mass spectrometry analysis revealed the identification of 212 complete and partial N-glycan structures including low abundant N-glycans containing acetyl and sulphate modifications. The identifications obtained through this platform were then applied to N-glycans released from a set of stage 3 gastric cancer serum samples obtained from patients before (pre-op) and after (post-op) tumour resection to investigate how the serum N-glycome can facilitate differentiation between the two pathological states.
糖基化是一种重要的翻译后修饰，它参与了多种重要的生物学功能。蛋白质糖基化特征，特别是 N-糖基化，受个体病理状态变化的影响，例如疾病的存在，因此，N-聚糖作为潜在的疾病生物标志物受到了广泛关注。人血清是基于 N-聚糖的生物标志物研究的理想来源，因为循环蛋白代表了个体的生理状态，许多血清蛋白都含有 N-糖基化。全面描述血清 N-糖组的困难源于缺乏生物合成模板，导致结构高度异质性和复杂性。为了克服这些挑战，我们开发了一种二维液相色谱平台，该平台在第一维利用离线弱阴离子交换（WAX）色谱法，在第二维利用亲水相互作用液相色谱法（HILIC），根据电荷、唾液酸化程度和大小分别分离 N-聚糖。这些分离在离线状态下进行，随后用 4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基吗啉氯化物（DMT-MM）进行衍生化，以确定唾液酸连接，并鉴定唾液酸连接异构体。随后的串联质谱分析揭示了 212 种完整和部分 N-聚糖结构的鉴定，包括含有乙酰基和硫酸基修饰的低丰度 N-聚糖。通过该平台获得的鉴定结果随后应用于从一组 3 期胃癌患者术前（术前）和术后（术后）肿瘤切除获得的血清样本中释放的 N-聚糖，以研究血清 N-糖组如何促进两种病理状态的区分。
