Native protein MS and ion mobility large flying proteins with ESI.
Native protein MS and ion mobility large flying proteins with ESI.
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DOI：
10.1021/ac071878c
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发表时间：
2007-03-01
影响因子：
7.4
通讯作者：
Loo, Joseph A
中科院分区：
文献类型：
--
作者：
Kaddis, Catherine S;Loo, Joseph A
大分子生物分子的测量得益于与气相分析器(如质谱仪和离子迁移率光谱仪)相结合的电喷雾离子注入技术的发展。多亚单位的组装和聚集在正常的细胞过程和疾病中的作用为研究大分子复合体提供了一种实用的方法。X射线结晶学和核磁共振光谱提供了无与伦比的高分辨率结构信息。然而，蛋白质结晶传统上是耗时的；核磁共振受到蛋白质靶标大小的限制；与MS相比，这两种方法都需要大量的纯化分析物。在缺乏高分辨结构的情况下，ESI MS和ESI离子迁移率谱(IMS)可以提供有关蛋白质和蛋白质复合体的重要功能信息，如结合亲和力常数、组装状态、化学计量和构象变化[1]。这些细节可与现代分析超速离心技术、量热法(差示扫描和等温)、色谱方法(光散射和荧光)和表面等离子体共振(SPR)提供的细节相媲美。然而，ESI-MS/IMS分析不需要固定化，可视化也不需要蛋白质修饰。此外，ESI MS和ESI IMS可以单独使用或与其他分离方法结合使用，以鉴定和表征多相混合物中的蛋白质组分。MS更适合于这些测量，因为它测量蛋白质和蛋白质复合体的固有基本性质，即分子质量，其精度大大超过其他分析技术。在这篇文章中，我们描述了ESI-MS/IMS的发展，用于表征通过非共价键结合在一起的大的蛋白质复合体；我们还将重点介绍独特的应用。
The measurement of large biomolecules has benefited tremendously from the development of ESI coupled to gas-phase analyzers such as mass spectrometers and ion mobility spectrometers. The role of multisubunit assemblies and aggregation in normal cellular processes and diseases warrants a practical method for the study of large macromolecular complexes. X-ray crystallography and NMR spectroscopy provide unrivaled high-resolution structural information. However, protein crystallization is traditionally time-consuming; NMR is limited by the size of the protein target; and compared with MS, both methods require large quantities of purified analyte. In the absence of high-resolution structures, ESI MS and ESI ion mobility spectrometry (IMS) can provide crucial functional information about proteins and protein complexes, such as binding affinity constants, assembly states, stoichiometry, and conformational changes (1). These details are comparable to those offered by modern analytical ultracentrifugation techniques, calorimetry (differential scanning and isothermal), chromatographic methods (light scattering and fluorescence), and surface plasmon resonance (SPR). However, immobilization is not required for ESI-MS/IMS analysis, nor is protein modification required for visualization. Furthermore, ESI MS and ESI IMS may be used in isolation or coupled with other separation methods to identify and characterize protein components in heterogeneous mixtures. MS is more generally suited for these measurements because it measures an inherent fundamental property, the molecular mass, of proteins and protein complexes at an accuracy that substantially surpasses other analytical techniques. In this article, we describe the development of ESI-MS/IMS for characterizing large protein complexes held together by noncovalent bonds; we will also highlight unique applications.