GeLCMS for In-Depth Protein Characterization and Advanced Analysis of Proteomes
GeLCMS for In-Depth Protein Characterization and Advanced Analysis of Proteomes
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DOI：
10.1007/978-1-61779-148-2_10
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Olsen, Jesper V.
中科院分区：
文献类型：
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作者：
Lundby, Alicia;Olsen, Jesper V.
近年来，用于解决分子和细胞生物学问题的质谱 (MS) 应用范围已大大扩展并持续增长。现代质谱仪可以对复杂生物样品中的蛋白质组成及其修饰进行识别、表征和定量。在 MS 分析之前，需要为实验正确准备任何生物样品。在这里，我们提出了一个方案，该方案结合了通过 ID 凝胶电泳预分离蛋白质，然后通过串联质谱 (MS/MS) 分析原位消化的蛋白质产物。详细解释了样品制备的所有步骤，并且该程序与任何可用质谱仪上的下游分析兼容。通过细微调整，该方案也可用于 2D 凝胶。提供的协议可用于分析特别感兴趣的特定蛋白质以及整个蛋白质组。如果在凝胶分离之前混合 SILAC 标记的蛋白质样品，则可以进一步准确地定量样品的蛋白质含量。
In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by ID gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments the protocol can be used with 2D gels as well. The protocol provided can be applied to analyze specific proteins of particular interest as well as entire proteomes. If SILAC-labeled protein samples are mixed prior to gel separation, the protein content of the sample can furthermore be accurately quantified.