Boost-DiLeu: Enhanced Isobaric N,N-Dimethyl Leucine Tagging Strategy for a Comprehensive Quantitative Glycoproteomic Analysis

化学 糖基化 糖肽 聚糖 糖蛋白组学 糖组 计算生物学 糖蛋白 生物化学 生物 抗生素
作者
Danqing Wang,M Ma,Junfeng Huang,Ting‐Jia Gu,Yusi Cui,Miyang Li,Zicong Wang,Henrik Zetterberg,Lingjun Li
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:94 (34): 11773-11782 被引量:20
标识
DOI:10.1021/acs.analchem.2c01773
摘要

Intact glycopeptide analysis has been of great interest because it can elucidate glycosylation site information and glycan structural composition at the same time. However, mass spectrometry (MS)-based glycoproteomic analysis is hindered by the low abundance and poor ionization efficiency of glycopeptides. Relatively large amounts of starting materials are needed for the enrichment, which makes the identification and quantification of intact glycopeptides from samples with limited quantity more challenging. To overcome these limitations, we developed an improved isobaric labeling strategy with an additional boosting channel to enhance N,N-dimethyl leucine (DiLeu) tagging-based quantitative glycoproteomic analysis, termed as Boost-DiLeu. With the integration of a one-tube sample processing workflow and high-pH fractionation, 3514 quantifiable N-glycopeptides were identified from 30 μg HeLa cell tryptic digests with reliable quantification performance. Furthermore, this strategy was applied to human cerebrospinal fluid (CSF) samples to differentiate N-glycosylation profiles between Alzheimer's disease (AD) patients and non-AD donors. The results revealed processes and pathways affected by dysregulated N-glycosylation in AD, including platelet degranulation, cell adhesion, and extracellular matrix, which highlighted the involvement of N-glycosylation aberrations in AD pathogenesis. Moreover, weighted gene coexpression network analysis (WGCNA) showed nine modules of glycopeptides, two of which were associated with the AD phenotype. Our results demonstrated the feasibility of using this strategy for in-depth glycoproteomic analysis of size-limited clinical samples. Taken together, we developed and optimized a strategy for the enhanced comprehensive quantitative intact glycopeptide analysis with DiLeu labeling, showing significant promise for identifying novel therapeutic targets or biomarkers in biological systems with a limited sample quantity.
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