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.
CITATION STYLE
Pourhaghighi, R., & Emili, A. (2019). Two-dimensional biochemical purification for global proteomic analysis of macromolecular protein complexes. In Methods in Molecular Biology (Vol. 1871, pp. 445–454). Humana Press Inc. https://doi.org/10.1007/978-1-4939-8814-3_26
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