title: Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination.
abstract: Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N(epsilon) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular z+1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins.
authors.affiliation: Oxford Centre for Gene Function/OXION, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. frank.sobott@dpag.ox.ac.uk
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Watt
authors.forename: Julia
authors.initials: J
authors.lastname: Smith
authors.forename: Mariola J
authors.initials: MJ
authors.lastname: Edelmann
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D014357:Trypsin