用于大分子蛋白质复合物全局蛋白质组学分析的二维生化纯化

Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

作者信息

Pourhaghighi Reza, Emili Andrew

机构信息

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.

Department of Biology, Boston University, Boston, MA, USA.

出版信息

Methods Mol Biol. 2019;1871:445-454. doi: 10.1007/978-1-4939-8814-3_26.

DOI:10.1007/978-1-4939-8814-3_26

PMID:30276754

Abstract

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.

摘要

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）。