In-Depth Site-Specific O‑Glycosylation Analysis of Glycoproteins and Endogenous Peptides in Cerebrospinal Fluid (CSF) from Healthy Individuals, Mild Cognitive Impairment (MCI), and Alzheimer’s Disease (AD) Patients

Site-specific
O-glycoproteome mapping in complex biological systems
provides a molecular basis for understanding the structure–function
relationships of glycoproteins and their roles in physiological and
pathological processes. Previous O-glycoproteome analysis in cerebrospinal
fluid (CSF) focused on sialylated glycoforms, while missing information
on other glycosylation types. In order to achieve an unbiased O-glycosylation
profile, we developed an integrated strategy combining universal boronic
acid enrichment, high-pH fractionation, and electron-transfer and
higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide
analysis. We applied this strategy to analyze the O-glycoproteome
in CSF, resulting in the identification of 308 O-glycopeptides from
110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms.
To our knowledge, this is the largest data set of O-glycoproteins
and O-glycosites reported for CSF to date. We also developed a peptidomics
workflow that utilized the EThcD and a three-step database searching
strategy for comprehensive PTM analysis of endogenous peptides, including
N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly,
among the 1411 endogenous peptides identified, 89 were O-glycosylated,
and only one N-glycosylated peptide was found, indicating that CSF
endogenous peptides were predominantly O-glycosylated. Analyses of
the O-glycoproteome and endogenous peptidome PTMs were also conducted
in the CSF of MCI and AD patients to provide a landscape of glycosylation
patterns in different disease states. Our results showed a decreasing
trend in fucosylation and an increasing trend of endogenous peptide
O-glycosylation, which may play an important role in AD progression.

位点特异性O-糖蛋白质组（O-glycoproteome）图谱绘制在复杂生物体系中可为解析糖蛋白的构效关系及其在生理与病理过程中的作用提供分子层面依据。此前针对脑脊液（cerebrospinal fluid, CSF）的O-糖蛋白质组分析多聚焦于唾液酸化糖型，却未覆盖其他糖基化类型的相关信息。为获得无偏倚的O-糖基化图谱，我们开发了一套整合策略：结合通用硼酸富集、高pH分级分离技术，以及电子转移与高能碰撞解离（electron-transfer and higher-energy collision dissociation, EThcD），以优化完整O-糖肽的分析效果。我们将该策略应用于脑脊液O-糖蛋白质组的分析，最终从110个O-糖蛋白中鉴定出308条O-糖肽，覆盖唾液酸化与非唾液酸化两类糖型。据我们所知，这是目前已报道的脑脊液来源O-糖蛋白及O-糖基化位点规模最大的数据集。我们还开发了一套肽组学分析流程：依托EThcD技术与三步法数据库检索策略，可对内源肽进行全面的翻译后修饰（Post-translational modification, PTM）分析，涵盖N-糖基化、O-糖基化及其他常见肽类翻译后修饰类型。值得注意的是，在鉴定得到的1411条内源肽中，有89条发生了O-糖基化修饰，仅发现1条N-糖基化肽，这表明脑脊液内源肽的糖基化修饰以O-糖基化为主。我们还对轻度认知障碍（Mild Cognitive Impairment, MCI）与阿尔茨海默病（Alzheimer's Disease, AD）患者的脑脊液样本开展了O-糖蛋白质组及内源肽组翻译后修饰分析，以刻画不同疾病状态下的糖基化模式全貌。研究结果显示，岩藻糖基化水平呈下降趋势，而内源肽的O-糖基化水平则呈上升趋势，这一变化或许在阿尔茨海默病的疾病进程中发挥重要作用。