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Updated: Jun 30, 2025

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

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Published on: October 24, 2018

固相微提取辅助毛细管区电泳-质谱:单个人类细胞的自下而上的蛋白质学

Jorge A Colón Rosado¹, Liangliang Sun¹

¹Department of Chemistry, Michigan State University, 578 S Shaw Lane, East Lansing, Michigan 48824, United States.

Journal of the American Society for Mass Spectrometry

|March 21, 2024

概括

我们开发了一种新方法,将固态微提取与毛细管区电泳质谱相结合起来,用于单细胞蛋白质组学. 这种技术显著增加了分析的样品的数量,使其适用于人类细胞分析.

科学领域:

分析化学分析化学
生物化学生物化学
蛋白质组学是指蛋白质组学.

背景情况:

毛细管区电泳质谱法 (CZE-MS) 对于分析像单细胞这样的质量有限样本至关重要.
由于CZE的样本负载能力较低,这阻碍了其在人类细胞单细胞蛋白质组学 (SCP) 中的广泛使用,分析限制在可用的<5%.

研究的目的:

开发和验证一个逆相固相微提取 (RP-SPME) -CZE-MS平台,以克服CZE对SCP的样本负载限制.
通过使用CZE-MS,使人类单细胞的有效和全面的蛋白质组分析成为可能.

主要方法:

在单个化毛细管内构建了一个集成的RP-SPME-CZE系统,其中包括一个现场合成的和一个C8珠包装的SPME部分.
开发了一种使用30%的乙二和50毫米乙酸 (pH6.5) 随后是基于动态pH连接的CZE-MS的化方案.
使用Q-Exactive高频质谱仪进行高分辨率和蛋白质识别.

主要成果:

RP-SPME-CZE-MS平台将样注射量增加到可用材料的近40%.
从相当于1个人类细胞 (0.1 ng注射) 的样本中平均鉴定出257 ± 24 种蛋白质和523 ± 69 种.
从最小的样本量 (0.25 ng总消化量,0.1 ng注射量) 证明了成功的蛋白质基因分析.

结论:

开发的RP-SPME-CZE-MS平台有效地解决了对SCP的传统CZE-MS的样本加载限制.
这项技术已准备好在人类细胞单细胞蛋白质组学中进行常规应用,从而使更深入的蛋白质组学见解成为可能.
代表了质量有限蛋白质组分析的重大进步,特别是在需要单细胞分辨率的领域.

关键词:

这就是CZE-MS.从底部向上的蛋白质组学.人类细胞是人类细胞.单细胞蛋白组学单细胞蛋白组学固体阶段微提取的微提取方法

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