Department of Chemistry, Graduate School of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Faculty of Pharmaceutical Sciences, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan.
Anal Chim Acta. 2024 Sep 1;1320:342990. doi: 10.1016/j.aca.2024.342990. Epub 2024 Jul 19.
N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required.
In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates.
In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.
糖基化是蛋白质翻译后的最重要修饰之一。由于生物样本中的 N-糖谱因病理状况而异且多样化，因此已经开发了各种分析方法，如液相色谱（LC）、毛细管电泳（CE）和质谱。然而，传统的分析方法在灵敏度和/或分辨率方面存在局限性，阻碍了对基本糖生物学研究和作为生物标志物的医学应用都很重要的次要但特定的 N-聚糖的发现。因此，需要一种高灵敏度和高分辨率的 N-糖谱分析方法。
在这项研究中，我们开发了一种新型的二维（2D）分离系统，该系统通过等速电泳（ITP）和浓缩（LDIS）的大体积双预浓缩将亲水相互作用液相色谱（HILIC）与毛细管凝胶电泳（CGE）相结合。由于 LDIS 的高效浓缩效率，在麦芽七糖的 2D 分析中，检测限达到 12 pM（60 amol，S/N=3），校准曲线线性良好（R>0.999）。最后，展示了从标准糖蛋白和细胞裂解物获得的 N-糖谱的 2D 分析。通过使用三重内标进行数据对齐，成功获得了高分辨率的 2D 图谱。根据聚糖大小、唾液酸数量、连接类型等，N-聚糖在 HILIC/CGE 2D 平面上分布良好。结果，成功鉴定了 HepG2 和 HeLa 细胞裂解物中的特定少量糖。
总之，HILIC/CGE 2D 分析方法对于从复杂的细胞样品中鉴定少量但特定的 N-聚糖具有足够的灵敏度和分辨率，表明其具有作为下一代糖组学工具的潜力。我们用于将 LC 和 CE 耦合的新方法也可以极大地提高其他分离模式的灵敏度，这可以成为一种新的 2D 生物分析标准，不仅适用于聚糖，还适用于代谢物、蛋白质和核酸等其他多样化的生物分子。
