title: The use of targeted proteomics to determine the stoichiometry of large macromolecular assemblies.
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.
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117, Heidelberg, Germany.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ori
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117, Heidelberg, Germany.
authors.forename: Amparo
authors.initials: A
authors.lastname: Andrés-Pons
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117, Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beck
mesh_terms: D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D046912:Multiprotein Complexes; D028861:Nuclear Pore Complex Proteins
keywords: Nuclear envelope isolation; Nuclei isolation; Protein complex stoichiometry; SRM assays; Targeted proteomics