Pourhaghighi Reza, Emili Andrew
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Department of Biology, Boston University, Boston, MA, USA.
Methods Mol Biol. 2019;1871:445-454. doi: 10.1007/978-1-4939-8814-3_26.
A high-resolution two-dimensional (2-D) proteomic fractionation technique for the systematic purification and subsequent mass spectrometry-based identification of endogenous protein macromolecular complexes is described. The method hyphenates preparative isoelectric focusing (IEF) with mixed-bed ion exchange chromatography (IEX) to efficiently separate cell- or tissue- derived soluble protein mixtures, allowing for more effective and less biased physiochemical characterization of stable multiprotein assemblies. After comprehensive 2D fractionation of cell-free lysates, each fraction is subjected to quantitative tandem mass spectrometry (MS/MS) and subsequent computational analysis to map high-confidence protein-protein interactions (PPIs). Herein, the experimental component (workflow protocols) for this global "interactome" network mapping platform is described.
本文描述了一种高分辨率二维（2-D）蛋白质组分级分离技术，用于系统纯化内源性蛋白质大分子复合物，并随后基于质谱进行鉴定。该方法将制备性等电聚焦（IEF）与混合床离子交换色谱（IEX）联用，以有效分离细胞或组织来源的可溶性蛋白质混合物，从而对稳定的多蛋白组装体进行更有效且偏差更小的物理化学表征。对无细胞裂解物进行全面的二维分级分离后，对每个级分进行定量串联质谱（MS/MS）分析及后续计算分析，以绘制高可信度的蛋白质-蛋白质相互作用（PPI）图谱。本文描述了这个全局“相互作用组”网络映射平台的实验组成部分（工作流程方案）。
