Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples
Large-Scale Qualitative and Quantitative Top-Down Proteomics Using Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry with Nanograms of Proteome Samples
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DOI：
10.1007/s13361-019-02167-w
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发表时间：
2019-08-01
影响因子：
3.2
通讯作者：
Sun, Liangliang
中科院分区：
文献类型：
--
作者：
Lubeckyj, Rachele A.;Basharat, Abdul Rehman;Sun, Liangliang
毛细管区带电泳-电喷雾电离-串联质谱（CZE-ESI-MS/MS）最近在自上而下的蛋白质组学领域引起了人们的关注，因为它可以实现蛋白质的高效分离和非常灵敏的检测。然而，为了使用 CZE-MS/MS 获得更好的蛋白质组覆盖率，需要提高 CZE 的分离窗口和上样量。在这里，我们提出了一种改进的 CZE-MS/MS 系统，该系统实现了 180 分钟的分离窗口和 2 μL 的样品装载体积，用于自上而下的蛋白质混合物表征。该系统通过近百万个肌红蛋白理论板实现了蛋白质的高效分离，并实现了肌红蛋白三种不同蛋白形式的基线分离。 CZE-MS/MS 系统在单次运行中仅注射 250 ng 蛋白质，从大肠杆菌 (E. coli) 蛋白质组样本中鉴定出 797 21 蛋白质型和 258 7 蛋白质 (n = 2)。当每次运行仅注射 25 ng 蛋白质时，系统仍从大肠杆菌样品中鉴定出 449 +/- 40 种蛋白质形式和 173 +/- 6 种蛋白质 (n = 2)。对斑马鱼脑小脑 (Cb) 和视顶盖 (Teo) 区域的单次 CZE-MS/MS 分析分别识别出 1730 +/- 196 种蛋白质形式 (n = 3) 和 2024 +/- 255 种蛋白质形式 (n = 3)，每次运行仅上样 500 ng 蛋白质。斑马鱼大脑 Cb 和 Teo 区域的无标记定量自上而下蛋白质组学揭示了 Cb 和 Teo 之间在蛋白质丰度方面存在显着差异。与 Teo 相比，来自 131 种蛋白质的 700 多种蛋白质形式的 Cb 丰度明显更高，并且这些蛋白质在多种生物过程中高度富集，包括肌肉收缩、糖酵解过程和间质迁移。
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted attention recently for top-down proteomics because it can achieve highly efficient separation and very sensitive detection of proteins. However, separation window and sample loading volume of CZE need to be boosted for a better proteome coverage using CZE-MS/MS. Here, we present an improved CZE-MS/MS system that achieved a 180-min separation window and a 2-mu L sample loading volume for top-down characterization of protein mixtures. The system obtained highly efficient separation of proteins with nearly one million theoretical plates for myoglobin and enabled baseline separation of three different proteoforms of myoglobin. The CZE-MS/MS system identified 797 21 proteoforms and 258 7 proteins (n = 2) from an Escherichia coli (E. coli) proteome sample in a single run with only 250ng of proteins injected. The system still identified 449 +/- 40 proteoforms and 173 +/- 6 proteins (n = 2) from the E. coli sample when only 25ng of proteins were injected per run. Single-shot CZE-MS/MS analyses of zebrafish brain cerebellum (Cb) and optic tectum (Teo) regions identified 1730 +/- 196 proteoforms (n = 3) and 2024 +/- 255 proteoforms (n = 3), respectively, with only 500-ng proteins loaded per run. Label-free quantitative top-down proteomics of zebrafish brain Cb and Teo regions revealed significant differences between Cb and Teo regarding the proteoform abundance. Over 700 proteoforms from 131 proteins had significantly higher abundance in Cb compared to Teo, and these proteins were highly enriched in several biological processes, including muscle contraction, glycolytic process, and mesenchyme migration.