背景:N-糖基化是蛋白质中最重要的翻译后修饰之一。由于生物样品中的N-聚糖谱是多样的,并且根据病理状况而变化,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,常规分析方法在灵敏度和/或分辨率方面存在局限性,阻碍发现次要但特异性的N-聚糖,这在基础糖生物学研究和作为生物标志物的医学应用中都很重要。因此,需要高灵敏度和高分辨率的N-聚糖谱分析方法。
结果:在这项研究中,我们开发了一种新颖的二维(2D)分离系统,它通过等速电泳和堆叠(LDIS)通过大容量双重预浓缩将亲水相互作用液相色谱(HILIC)与毛细管凝胶电泳(CGE)耦合。由于LDIS的有效预浓缩效率,检测限达到12pM(60amol,S/N=3)在麦芽七糖的2D分析中具有良好的校准曲线线性(R2>0.999)。最后,证明了从标准糖蛋白和细胞裂解物获得的N-聚糖的2D谱分析。通过使用三重内标进行数据比对,成功获得了高分辨率的2D轮廓。基于聚糖大小,N-聚糖在HILIC/CGE2D平面上分布良好,唾液酸的数量,联动类型,等等。因此,在HepG2和HeLa细胞裂解物中成功鉴定了特定的次要聚糖。
■总而言之,HILIC/CGE2D分析方法显示出足够的灵敏度和分辨率,用于从复杂的细胞样品中识别次要但特定的N-聚糖,表明了作为下一代N-糖工具的潜力。我们耦合LC和CE的新颖方法还可以显着提高其他分离模式的灵敏度,这可能是一种新的2D生物分析标准,不仅适用于聚糖,还有其他不同的生物分子,如代谢物,蛋白质,和核酸。
BACKGROUND: 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.
RESULTS: 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 (R2 > 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.
UNASSIGNED: 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.