Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones,Analytical Chemistry

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Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-03-02 , DOI: 10.1021/acs.analchem.0c04237
Daoyang Chen ₁ , Zhichang Yang ₁ , Xiaojing Shen ₁ , Liangliang Sun ₁

Affiliation

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μ_ef) of unmodified histone proteoforms can be predicted accurately (R² = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

中文翻译：

毛细管区带电泳-串联质谱法作为液相色谱-串联质谱法的替代方法，用于自上而下的组蛋白蛋白质组学分析

自上而下的蛋白质组学 (TDP) 是破译组蛋白密码的理想方法，通常采用反相液相色谱 (RPLC)-串联质谱 (MS/MS)。由于组蛋白在蛋白质型数量方面极其复杂，因此组蛋白的 TDP 需要具有高容量分离和高灵敏度蛋白质型检测的新分析工具。在这里，我们通过电动泵鞘流 CE-MS 接口展示毛细管区带电泳 (CZE)-MS/MS，用于大规模自上而下描绘组蛋白蛋白质形式。CZE-MS/MS 从小牛组蛋白样品中鉴定出与 RPLC-MS/MS 数量相当的蛋白质，样品消耗量减少了 30 倍以上（75 ng 与 3 μg），表明其灵敏度显着提高。我们使用二维尺寸排阻色谱 (SEC)-CZE-MS/MS 从小牛组蛋白样品中鉴定出约 400 种组蛋白蛋白质，消耗的蛋白质少于 300 ng。我们鉴定了携带各种尝试性翻译后修饰 (PTM) 的组蛋白蛋白质变体，例如乙酰化、甲基化（单、二和三）、磷酸化和琥珀酰化。根据我们最近的工作，使用优化的半经验模型可以准确预测未修饰组蛋白蛋白质形式的电泳迁移率 (μ _{ef ) (}R ^{2 = 0.98)。}结果使 CZE-MS/MS 成为在全球范围内以蛋白质型特异性方式破译组蛋白代码的有用工具。

更新日期：2021-03-16

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