Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Genome and Systems Biology Degree Program, Academia Sinica and National Taiwan University, Taipei 10617, Taiwan.
Anal Chem. 2021 Dec 7;93(48):15931-15940. doi: 10.1021/acs.analchem.1c03224. Epub 2021 Nov 15.
Alterations of protein glycosylation are closely related with pathophysiological regulation. Due to the structural macro- and microheterogeneity, low stoichiometry, and low ionization efficiency of glycopeptides, high-performance tools to enrich glycopeptides, especially the negatively charged and labile sialoglycopeptides, are essential to enhance the identification of the underexplored glycoproteome. Here, we present the first implementation of zwitterionic hydrophilic interaction chromatography with the exposed choline group (ZIC-cHILIC) in StageTip for simultaneous enrichment and fractionation of intact glycopeptides. In a model study using lung cancer cells, early elution by a high percentage of acetonitrile prominently prefilters nonglycopeptides, facilitating high enrichment specificity for glycopeptides (92-96%) and sialoglycopeptides (77-89%) in the subsequent hydrophilic fractions. The stepwise elution shows a high glycopeptide fractionation efficiency by a <10% overlap of glycopeptides between adjacent fractions. Most importantly, the ZIC-cHILIC stepwise strategy demonstrated good reproducibility (>80% in triplicate analysis) as well as superior coverage of 4.6- to 12.0-fold and 2.1- to 35.6-fold more glycopeptides and sialoglycopeptides compared to conventional TiO and ZIC-HILIC, respectively. To the best of our knowledge, the result with 2742 sialoglycopeptides among 7367 unique glycopeptides and 166 glycans from 2434 N-glycosites of 1118 glycoproteins (Byonic score > 100) provides one of the deepest glycoproteomic profiles in single-cell type. Without the immunoprecipitation step, the large-scale glycoproteomic atlas also reveals site-specific glycosylation of many druggable receptor proteins, such as EGFR, MET, ERBB2, ERBB3, AXL, and IGF1R. The demonstrated high enrichment specificity and identification depth show that stepwise ZIC-cHILIC is an efficient method to explore the under-represented sialoglycoproteome.
蛋白质糖基化的改变与病理生理调节密切相关。由于糖肽的结构宏观和微观异质性、低计量、低离化效率，因此需要高性能的工具来富集糖肽，特别是带负电荷和不稳定的唾液酸糖肽，这对于增强对未充分探索的糖蛋白质组的鉴定至关重要。在这里，我们首次在 StageTip 中实现了带有暴露的胆碱基团的两性离子亲水相互作用色谱（ZIC-cHILIC），用于完整糖肽的同时富集和分级。在使用肺癌细胞的模型研究中，高比例乙腈的早期洗脱显著预过滤非糖肽，从而提高了糖肽（92-96%）和唾液酸糖肽（77-89%）在随后亲水馏分中的高富集特异性。分步洗脱显示出高的糖肽分级效率，相邻馏分之间的糖肽重叠<10%。最重要的是，与传统的 TiO 和 ZIC-HILIC 相比，ZIC-cHILIC 分步策略表现出良好的重现性（>80%的三重分析），并且分别覆盖了 4.6 到 12.0 倍和 2.1 到 35.6 倍更多的糖肽和唾液酸糖肽。据我们所知，该结果在 7367 个独特的糖肽中鉴定到 2742 个唾液酸糖肽，在来自 1118 个糖蛋白的 2434 个 N-糖基化位点中有 166 个聚糖（Byonic 得分>100），提供了单细胞类型中最深入的糖蛋白质组学图谱之一。无需免疫沉淀步骤，大规模的糖蛋白质组图谱还揭示了许多可靶向受体蛋白（如 EGFR、MET、ERBB2、ERBB3、AXL 和 IGF1R）的特定位点的糖基化。所证明的高富集特异性和鉴定深度表明，分步 ZIC-cHILIC 是一种有效的方法来探索代表性不足的唾液酸糖蛋白质组。
