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.
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Pourhaghighi, R., Emili, A. (2019). Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8814-3_26
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DOI: https://doi.org/10.1007/978-1-4939-8814-3_26
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8813-6
Online ISBN: 978-1-4939-8814-3
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