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通过水友相互作用液体染色学和双堆叠毛细体凝电泳法对N结合甘进行高度敏感的二维分析

Takaya Miki¹, Sachio Yamamoto², Chenchen Liu¹

¹Department of Chemistry, Graduate School of Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.

Analytica chimica acta

|August 14, 2024

概括

一个新的二维 (2D) 分离系统,结合了水友互动液体染色学 (HILIC) 和毛细管凝电泳 (CGE),为N-甘氨酸分析提供了高灵敏度和分辨率. 这一进步使小N-甘氨酸的发现成为可能,这对于糖生物学研究和生物标志物开发至关重要.

科学领域:

生物化学生物化学
分析化学分析化学
分子生物学分子生物学

背景情况:

N-糖基化是一种关键的翻译后修改,影响蛋白质功能.
现有的N-甘氨酸分析方法缺乏发现小但重要的甘氨酸所需的灵敏度和分辨率.
识别特定的N-甘氨酸对于推进糖生物学和发现疾病生物标志物至关重要.

研究的目的:

开发一种高灵敏度和高分辨率的N-glycan分析方法.
将水友互动液体色谱 (HILIC) 与毛细管凝电泳 (CGE) 结合起来,以进行增强的N-甘氨酸分析.
为了能够在复杂的生物样本中识别小N-甘氨酸.

主要方法:

开发一种新的二维 (2D) 分离系统.
将HILIC与毛细管凝电泳 (CGE) 结合起来,使用大容量双重预度通过异位电泳和堆叠 (LDIS).
从标准葡萄糖蛋白和细胞溶解物中分析N-葡萄糖,并使用三重内部标准对数据进行对齐.

主要成果:

达到了对马尔托赫普塔糖的12 pM的检测极限,具有很好的线性 (R2 > 0.999).
生成了高分辨率的2DN-甘氨酸配置文件,甘氨酸根据大小,酸含量和结合类型分布.
在HepG2和HeLa细胞溶解物中成功识别了特定的小N-甘氨酸.

结论:

HILIC/CGE 2D 方法提供了足够的灵敏度和分辨率,用于在复杂样本中识别小N-甘氨酸.
这种方法代表了一种潜在的下一代N-glycomics工具.
LC-CE合技术可以提高对其他生物分子的敏感性,从而在二维生物分析中建立了一个新的标准.

关键词:

毛细管凝电泳发生在毛细管凝中.葡萄糖组分析分析水友互动液体色谱学液体色谱学大量的双重堆叠.二维分离的二维分离方式

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