Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps)

• Published in: Nature protocols Vol. 11; no. 2; pp. 291 - 301
• Main Authors: Mark, Kevin G, Loveless, Theresa B, Toczyski, David P
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.02.2016
• Subjects: Affinity; Binding; Chromatography, Affinity - methods; Degradation; DNA repair; Endocytosis; Enrichment; Genetic engineering; Genetic research; Immunoprecipitation; Mass spectrometry; Mass spectroscopy; Observations; Polyubiquitin - genetics; Polyubiquitin - metabolism; Properties; Protein Binding; Protein purification; Proteins; Proteins - isolation & purification; Proteins - metabolism; Purification; Scientific imaging; Spectroscopy; Substrates; Ubiquitin; Ubiquitin-proteasome system; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.008

Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.