title: A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis.
abstract: Ubiquitination can have profound effects on the stability and function of cellular proteins. Mass spectrometry (MS) can be used to map the specific amino acid residues that are conjugated to ubiquitin in a target protein. However, the purification required for proteomic analysis can be challenging. In this paper, we describe a bimolecular affinity purification scheme for the isolation of a specific ubiquitinated protein in which affinity moieties are fused to ubiquitin and to a target protein of interest. After ubiquitin conjugation in vivo, the protein target acquires two affinity tags, allowing the specific purification of its ubiquitin-modified forms. To prevent deubiquitination after lysis or the copurification of interacting cofactors, this procedure is performed after protein denaturation using polyhistidine and biotinylation tags. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified in large amounts of sufficient purity for MS analysis and for mapping of ubiquitin acceptor sites.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: Gabriel N
authors.initials: GN
authors.lastname: Maine
authors.forename: Haiying
authors.initials: H
authors.lastname: Li
authors.forename: Iram W
authors.initials: IW
authors.lastname: Zaidi
authors.forename: Venkatesha
authors.initials: V
authors.lastname: Basrur
authors.forename: Kojo S J
authors.initials: KS
authors.lastname: Elenitoba-Johnson
authors.forename: Ezra
authors.initials: E
authors.lastname: Burstein
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000737:chemistry*; D057149:Ubiquitinated Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D057149:Ubiquitinated Proteins