Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA.
Mol Cell Proteomics. 2021;20:100081. doi: 10.1016/j.mcpro.2021.100081. Epub 2021 Apr 20.
As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.
作为与中枢神经系统（CNS）细胞外液直接交换的体液，脑脊液（CSF）是 CNS 相关疾病生物标志物发现的丰富来源。已经对 CSF 进行了广泛的蛋白质组学分析，但缺乏旨在揭示特定部位 CSF N-糖蛋白组的研究。在 CSF 中进行特定部位 N-糖蛋白质组学研究的初步努力得到的覆盖范围有限，阻碍了 CSF 中基于糖基化的疾病生物标志物发现的进一步实验设计。在本研究中，我们开发了一种 N-糖蛋白质组学方法，该方法结合了亲水相互作用色谱（HILIC）增强的 N-糖肽顺序富集和硼酸盐富集与电子转移和更高能量碰撞解离（EThcD），用于大规模完整 N-糖肽分析。将所开发的方法应用于人 CSF 样品的分析，总共从 511 个 N-糖基位点和 285 个 N-糖蛋白中鉴定出 2893 个完整的 N-糖肽。据我们所知，这是迄今为止报道的 CSF 中最大的特定部位 N-糖蛋白组数据集。该数据集为更好地理解糖蛋白的结构-功能关系及其在 CNS 相关生理和病理过程中的作用提供了分子基础。由于越来越多的证据表明糖基化缺陷参与阿尔茨海默病（AD）的发病机制，在本研究中，对来自健康对照和 AD 患者的 CSF 样本进行了比较深入的 N-糖蛋白质组学分析，结果得到了相当的 N-糖蛋白组覆盖范围，但不同类别糖型的表达模式明显不同，例如 AD CSF 样本中的岩藻糖基化减少。检测到一些 N-糖蛋白的糖基化模式发生改变，包括α-1-抗胰蛋白酶、ephrin-A3 和肌肽酶 CN1 等，这些糖蛋白可能是进一步基于糖基化的 AD 研究的潜在有趣靶点，并最终可能导致对糖基化在 AD 进展中的作用的分子阐明。
