Cramer Rainer, Saxton Malcolm, Barnouin Karin
Department of Biochemistry and Molecular Biology, University College London and Ludwig Institute for Cancer Research, London, England, UK.
Methods Mol Biol. 2004;261:499-510. doi: 10.1385/1-59259-762-9:499.
The identification and characterization of binding partners from protein complexes is increasingly undertaken by mass spectrometry because of its high sensitivity and expedient elucidation of protein structure by accurate mass measurement. A variety of affinity purification methods including immunoprecipitation and glutathione-S-transferase (GST) pull-downs are commonly employed for the isolation of protein complexes and coupled to gel electrophoresis for further separation and basic information with regard to their constituents. For the successful analysis of gel-separated proteins by mass spectrometry, additional sample preparation steps involving sample clean-up, proteolysis, and peptide recovery are essential. This chapter describes the important procedure of in-gel digestion with particular emphasis on maximum peptide recovery and compatibility for subsequent mass spectrometric analysis.
由于质谱具有高灵敏度以及能通过精确质量测量快速阐明蛋白质结构，因此越来越多地用于鉴定和表征蛋白质复合物中的结合伴侣。包括免疫沉淀和谷胱甘肽-S-转移酶（GST）下拉在内的多种亲和纯化方法通常用于分离蛋白质复合物，并与凝胶电泳相结合，以进一步分离其成分并获取有关成分的基本信息。为了通过质谱成功分析凝胶分离的蛋白质，涉及样品净化、蛋白酶解和肽回收的额外样品制备步骤至关重要。本章介绍凝胶内消化的重要程序，特别强调最大程度地回收肽以及与后续质谱分析的兼容性。
