title: The use of ubiquitin lysine mutants to characterize E2-E3 linkage specificity: Mass spectrometry offers a cautionary "tail".
abstract: Oligomeric ubiquitin structures (i.e. ubiquitin "chains") may be formed through any of seven different lysine residues in the polypeptide, or via the amine group of Met 1. Different types of ubiquitin chains can confer very different biological outcomes to a protein substrate, yet the structural characteristics of E2s and E3s that determine ubiquitin linkage specificity remain poorly understood. In vitro autoubiquitylation assays combined with ubiquitin protein variants bearing individually mutated lysine residues ("K-to-R" mutants) have thus been widely used to characterize E2-E3 linkage specificity. However, how this type of assay compares to direct identification of ubiquitin linkage types using mass spectrometry (MS) has not been rigorously tested. Here, we characterize the linkage specificity of 12 different E2-E3 combinations using both approaches. The simple MS-based method described here is more robust, requires less material and is less prone to bias introduced by, e.g. the use of mutant proteins with unknown effects on E1, E2 or E3 recognition, antibodies with uncharacterized epitopes, the low dynamic range of X-ray film, and additional sources of experimental error. Indeed, our results suggest that the K-to-R assay be approached with some caution.
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, and Department of Medical Biophysics, University of Toronto, Toronto, ON, CANADA.
authors.forename: Jenny H
authors.initials: JH
authors.lastname: Hong
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, and Department of Medical Biophysics, University of Toronto, Toronto, ON, CANADA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Ng
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, and Department of Medical Biophysics, University of Toronto, Toronto, ON, CANADA.
authors.forename: Tharan
authors.initials: T
authors.lastname: Srikumar
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, and Department of Medical Biophysics, University of Toronto, Toronto, ON, CANADA.
authors.forename: Brian
authors.initials: B
authors.lastname: Raught
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000235:genetics*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D053719:Tandem Mass Spectrometry / Q000379:methods; D025801:Ubiquitin / Q000235:genetics*; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; C577447:CDC34 protein, human; C097309:UBE2D2 protein, human; C106347:UBE2E2 protein, human; C544962:Ube2S protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
keywords: Mass spectrometry; Spectral matching; Technology; Ubiquitin; Ubiquitin chains; Ubiquitin linkages