Development of complementary enrichment strategies for analysis of N-linked intact glycopeptides and potential site-specific glycoforms in Alzheimer's disease

IF 5.6 1区化学 Q1 CHEMISTRY, ANALYTICAL

Talanta Pub Date : 2025-07-16 DOI:10.1016/j.talanta.2025.128595

Zhenpeng Deng , Yan Wang , Xiaoyan Liu , Xingfa Ren , Weibing Zhang , Mingming Dong , Yusong Ge , Yongliang Yu , Mingliang Ye

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引用次数: 0

Abstract

Protein glycosylation is a critical post-translational modification, and knowledge of site-specific glycoforms is essential for developing biomarkers and therapeutic drugs. Although LC-MS/MS-based glycoproteomics strategies enable the identification of site-specific glycoforms at proteomics scale, their coverage is still low largely because of the poor glycopeptide enrichment performance. HILIC is thought to allow "unbiased" enrichment of intact glycopeptides, and it is broadly used to analyze the site-specific glycoforms at proteomics scale. To maximize glycopeptide capturing, the samples are always loaded onto HILIC with high acetonitrile (ACN) content (typically 80 %). In this study, we found that some HILIC columns could effectively capture glycopeptides at around 70 % ACN. We further demonstrated that the system could identify the highly hydrophilic glycopeptides that can not be identified by conventional methods. The excellent complementarity of 70 % ACN enrichment methods greatly enhances the coverage (>20 %) of N-glycoproteome identification in human blood. The developed methods were further applied to investigate the N-glycosylation changes in the plasma of Alzheimer's disease (AD) and mild cognitive impairment (MCI) patients. It was found that fucosylated glycopeptides were up-regulated and sialylated glycopeptides were down-regulated as the disease progressed. Altered glycosylation patterns were detected for a number of site-specific glycoforms, which serve as potentially interesting targets for further glycosylation-based AD progression. Our results reveal that using complementary strategies offers a comprehensive approach to studying N-glycoproteomics, paving the way for in-depth glycoproteomics analysis.

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用于分析阿尔茨海默病中n -连接完整糖肽和潜在位点特异性糖型的互补富集策略的发展

蛋白质糖基化是一种关键的翻译后修饰，了解位点特异性糖型对于开发生物标志物和治疗药物至关重要。尽管基于LC-MS/ ms的糖蛋白组学策略能够在蛋白质组学尺度上鉴定位点特异性糖型，但由于糖肽富集性能差，其覆盖率仍然很低。HILIC被认为允许完整糖肽的“无偏”富集，并且广泛用于蛋白质组学尺度上的位点特异性糖型分析。为了最大限度地捕获糖肽，样品总是装载在高乙腈（ACN）含量（通常为80%）的HILIC上。在这项研究中，我们发现一些HILIC色谱柱可以在70% ACN左右有效地捕获糖肽。我们进一步证明了该系统可以识别传统方法无法识别的高亲水性糖肽。70% ACN富集方法的良好互补性极大地提高了人血n -糖蛋白组鉴定的覆盖率（> 20%）。应用该方法进一步研究了阿尔茨海默病（AD）和轻度认知障碍（MCI）患者血浆中n -糖基化的变化。研究发现，随着疾病的进展，集中化的糖肽上调，唾液化的糖肽下调。在许多位点特异性糖型中检测到糖基化模式的改变，这可能是进一步基于糖基化的AD进展的潜在有趣靶点。我们的研究结果表明，使用互补策略提供了一种全面的方法来研究n-糖蛋白组学，为深入的糖蛋白组学分析铺平了道路。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.