Multiple Separations Facilitate Identification of Protein Variants by Mass Spectrometry
Multiple Separations Facilitate Identification of Protein Variants by Mass Spectrometry
复制标题
多重分离有助于通过质谱法鉴定蛋白质变体
作者：
Zhongli Zhang;David L. Smith;Jean B. Smith
从复杂的生物样品中鉴定变异蛋白将有助于我们对病理状态的病因学的理解。由于蛋白质序列的基因突变或翻译后修饰，变异的表征比目前大多数蛋白质组学研究中典型的简单蛋白质鉴定要困难得多。当目标是对蛋白质的修饰有一个完整的了解时，通过数据库检索来鉴定一些肽是不够的。虽然质谱法的一个优点是它能够获得对几种成分的特定反应，但生物样品的复杂性往往是压倒性的，导致光谱缺乏有用的信息。对于复杂的混合物，质谱分析之前的分离程序可能需要包括各种色谱和电泳分离技术。在本报告中，我们说明了如何几个准备步骤是必不可少的，以获得有关改性人晶状体β-晶体蛋白的信息。质谱分析前的制备技术包括粒径排除色谱、反相色谱、二维凝胶电泳、蛋白质和肽的原位消化、捕获和洗涤，然后进行最后的反相高效液相色谱在线分离到质谱仪。这种分离和分析的方法，当为其他蛋白质定制时，应该在许多需要识别复杂混合物的蛋白质变体的研究中找到应用。
Identification of variant proteins from complex biological samples promises to contribute much to our understanding of the etiology of pathological states. Characterization of variants, due either to genetic mutations in protein sequences or to post-translational modifications, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. Identification of a few peptides by database retrieval is not adequate when the goal is to have a complete understanding of the modifications of the protein. Although one advantage of mass spectrometry is its ability to obtain specific responses to several components, the complexity of biological samples is often overwhelming, resulting in spectra lacking useful information. For complex mixtures, isolation procedures before mass spectrometric analysis may need to include a variety of chromatographic and electrophoretic separation techniques. In this report, we illustrate how several preparative steps were essential for obtaining information about modified human lens β-crystallins. The preparative techniques prior to mass spectrometry included size-exclusion chromatography, reversed-phase chromatography, two-dimensional gel electrophoresis, in situ digestion of the proteins and peptide trapping and washing before a final reversed-phase high-performance liquid chromatographic separation on-line to the mass spectrometer. This approach for isolation and analysis, when customized for other proteins, should find application in many studies where protein variants of complex mixtures are to be identified.
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影响因子：
3.4
作者：
Lampi, KJ;Ma, ZX;David, LL
通讯作者：
David, LL
鉴定古代人类晶状体中高分子量晶状体蛋白的主要成分。
影响因子：
2
作者：
Yang,Z;Chamorro,M;Smith,DL;Smith,JB
通讯作者：
Smith,JB
影响因子：
7.4
作者：
Opiteck, GJ;Jorgenson, JW;Anderegg, RJ
通讯作者：
Anderegg, RJ
影响因子：
7.4
作者：
McCormack, AL;Schieltz, DM;Yates, JR
通讯作者：
Yates, JR
影响因子：
7.4
作者：
HOLLAND, HA;JORGENSON, JW
通讯作者：
JORGENSON, JW