Tapered-Tip Capillary Electrophoresis Nano-Electrospray Ionization Mass Spectrometry for Ultrasensitive Proteomics: the Mouse Cortex
Tapered-Tip Capillary Electrophoresis Nano-Electrospray Ionization Mass Spectrometry for Ultrasensitive Proteomics: the Mouse Cortex
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DOI：
10.1007/s13361-016-1532-8
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发表时间：
2017-04-01
影响因子：
3.2
通讯作者：
Nemes, Peter
中科院分区：
文献类型：
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作者：
Choi, Sam B.;Zamarbide, Marta;Nemes, Peter
蛋白质组的超灵敏表征提高了理解差异基因表达如何协调大脑中细胞异质性的潜力。在这里，我们报告了一个微量分析毛细管电泳纳米流电喷雾电离（CE-nanoESI）接口的质谱，使有限数量的蛋白质在小鼠皮层的测量。我们的设计将定制的CE系统集成到同轴鞘流配置中的锥形尖端金属发射器。该接口可以使用现成的组件在15分钟内构建，便于广泛适应。锥形尖端CE-nanoESI通过可重复地锚定泰勒锥来产生稳定的电喷雾，最大限度地减少离子源中的样品稀释，并通过维持锥形喷射喷射方案来确保有效的离子产生。平行反应监测为血管紧张素II提供了260 zmol的检测下限（156，000拷贝）。CE能够在类似于330，000个理论塔板中解析复杂的肽混合物，并鉴定类似于15阿莫尔（类似于1 pg）的BSA或细胞色素c。超过30分钟的分离，1纳克蛋白质消化从小鼠皮质产生217个非冗余蛋白质，包括一个类似的3对数级的浓度范围内使用四极杆飞行时间质谱仪。所鉴定的蛋白质包括来自传统上用于标记少突胶质细胞、星形胶质细胞和小胶质细胞的基因的许多产物。最后，检测到参与神经退行性疾病的关键蛋白质（例如，帕金森综合征和痉挛性截瘫）。CE-nanoESI-HRMS具有足够的灵敏度，可检测有限数量的组织和细胞群中的蛋白质，以帮助了解基因表达差异如何维持大脑中的细胞异质性。
Ultrasensitive characterization of the proteome raises the potential to understand how differential gene expression orchestrates cell heterogeneity in the brain. Here, we report a microanalytical capillary electrophoresis nano-flow electrospray ionization (CE-nanoESI) interface for mass spectrometry to enable the measurement of limited amounts of proteins in the mouse cortex. Our design integrates a custom-built CE system to a tapered-tip metal emitter in a co-axial sheath-flow configuration. This interface can be constructed in < 15 min using readily available components, facilitating broad adaptation. Tapered-tip CE-nanoESI generates stable electrospray by reproducibly anchoring the Taylor cone, minimizes sample dilution in the ion source, and ensures efficient ion generation by sustaining the cone-jet spraying regime. Parallel reaction monitoring provided a 260-zmol lower limit of detection for angiotensin II (156,000 copies). CE was able to resolve a complex mixture of peptides in similar to 330,000 theoretical plates and identify similar to 15 amol (similar to 1 pg) of BSA or cytochrome c. Over 30 min of separation, 1 ng protein digest from the mouse cortex yielded 217 nonredundant proteins encompassing a similar to 3-log-order concentration range using a quadrupole time-of-flight mass spectrometer. Identified proteins included many products from genes that are traditionally used to mark oligodendrocytes, astrocytes, and microglia. Finally, key proteins involved in neurodegenerative disorders were detected (e.g., parkinsonism and spastic paraplegia). CE-nanoESI-HRMS delivers sufficient sensitivity to detect proteins in limited amounts of tissues and cell populations to help understand how gene expression differences maintain cell heterogeneity in the brain.