title: Extracting, enriching, and identifying nuclear body sub-complexes using label-based quantitative mass spectrometry.
abstract: Determining the proteome of a nuclear body is a crucial step toward understanding its function; however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-based affinity purification followed by quantitative mass spectrometry.
authors.affiliation: Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, the University of Western Australia, Crawley, Western Australia, 6009, Australia, archa.fox@perkins.uwa.edu.au.
authors.forename: Archa
authors.initials: A
authors.lastname: Fox
authors.forename: Virja
authors.initials: V
authors.lastname: Mehta
authors.forename: Severine
authors.initials: S
authors.lastname: Boulon
authors.forename: Laura
authors.initials: L
authors.lastname: Trinkle-Mulcahy
mesh_terms: D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D045586:Intranuclear Inclusion Bodies / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D009687:Nuclear Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
chemical_list: D009687:Nuclear Proteins