Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.
Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry.
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DOI：
10.1007/s13361-017-1781-1
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发表时间：
2017-12
影响因子：
3.2
通讯作者：
Ivanov AR
中科院分区：
文献类型：
--
作者：
Belov AM;Viner R;Santos MR;Horn DM;Bern M;Karger BL;Ivanov AR
天然质谱（MS）是分析蛋白质、蛋白质复合物和各种类型的大分子物质的快速发展的领域。迄今为止报告的大多数天然MS实验都是使用将纯化的分析物直接注入质谱仪进行的。在这项研究中，毛细管区带电泳（CZE）在线耦合到Orbitrap质谱仪使用商业无鞘接口，以实现高性能的分离，鉴定和结构表征有限数量的纯化蛋白质和蛋白质复合物，后者与保存的非共价缔合在天然条件下。使用已知形成非共价蛋白质-蛋白质和蛋白质-配体复合物的蛋白质标准品的混合物评估裸熔融二氧化硅和聚丙烯酰胺涂覆的毛细管的性能。本机复合物的高效分离证明使用这两种类型的毛细管，而中性涂层毛细管表现出更好的再现性和更高的效率，更复杂的样品。然后评价该平台用于测定单克隆抗体聚集和用于使用来自大肠杆菌的核糖体分离物分析有限复杂性的蛋白质组。杆菌天然CZE-MS，使用准确的单级和串联MS测量，能够在飞摩尔水平上鉴定蛋白质型和非共价复合物。这项研究表明，本地CZE-MS可以作为一个正交和互补的技术，传统的本地MS方法的优点，低样品消耗，最小的样品处理和损失，高通量和灵敏度。这项研究为核糖体和其他大分子复合物和细胞器的分析提供了一个新的平台，有可能发现定义细胞表型的新结构特征（例如专门的核糖体）。
Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g. specialized ribosomes).
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影响因子：
13.8
作者：
Gilbert, Wendy V.
通讯作者：
Gilbert, Wendy V.
影响因子：
7.4
作者：
Blackwell, Anne E.;Dodds, Eric D.;Wysocki, Vicki H.
通讯作者：
Wysocki, Vicki H.
DOI：
10.1016/j.jasms.2009.10.014
发表时间：
2010-02-01
影响因子：
3.2
作者：
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通讯作者：
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影响因子：
7.4
作者：
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影响因子：
7.4
作者：
Busnel, Jean-Marc;Schoenmaker, Bart;Mayboroda, Oleg A.
通讯作者：
Mayboroda, Oleg A.