A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis

Gabriel N. Maine, Haiying Li, Iram W. Zaidi, Venkatesha Basrur, Kojo S J Elenitoba-Johnson, Ezra Burstein

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1447-1459
Number of pages13
JournalNature Protocols
Volume5
Issue number8
DOIs
StatePublished - Aug 2010

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology

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