复杂蛋白质混合物的凝胶内酶解-串联质谱分析
GeLC-MS/MS analysis of complex protein mixtures.
作者信息
Dzieciatkowska Monika, Hill Ryan, Hansen Kirk C
机构信息
Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, 80045, USA.
出版信息
Methods Mol Biol. 2014;1156:53-66. doi: 10.1007/978-1-4939-0685-7_4.
Abstract
Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we describe a general GeLC-MS/MS protocol and include technical advice and outline caveats to increase the probability of a successful analysis.
摘要
基于发现的蛋白质组学已在生物学研究的几乎每个方面占据一席之地。这种方法的一个关键目标是在很宽的浓度范围内最大化蛋白质的序列覆盖率。在蛋白质水平上对样品进行分级分离是应对样品复杂性挑战并提高蛋白质组覆盖率的最常用方法之一。在现有的方法中，一维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳后进行液相色谱 - 串联质谱分析（GeLC-MS/MS）是一种用于定性和定量蛋白质组分析的可靠且可重复的方法。在此，我们描述了一种通用的GeLC-MS/MS方案，并提供技术建议和列出注意事项，以提高成功分析的概率。
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