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Free keywords:
Cell Line, Tumor, HEK293 Cells, HeLa Cells, Humans, Mass Spectrometry, Multiprotein Complexes, Nuclear Envelope, Nuclear envelope isolation, Nuclear Pore, Nuclear Pore Complex Proteins, Nuclei isolation, Protein complex stoichiometry, Proteomics, SRM assays, Targeted proteomics
Abstract:
Accurate knowledge of the stoichiometry of protein complexes is a crucial prerequisite for understanding their structure and function. To purify or enrich large and intricate protein complexes such that their structure is preserved and to absolutely quantify all of their protein components is an enormous technical challenge. In this chapter, we describe how to purify nuclear envelopes from human tissue culture cells that are highly enriched for nuclear pore complexes. We use the nuclear pore as an example to discuss how the structural preservation of such preparations can be controlled. Furthermore, we give a practical guide how to develop and employ targeted proteomic assays for both, the absolute quantification of stoichiometries and the relative quantification of protein complex composition across multiple biological conditions. The concept discussed here is universally applicable to any protein complex.
