title: Top-down 193-nm ultraviolet photodissociation mass spectrometry for simultaneous determination of polyubiquitin chain length and topology.
abstract: Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an in vitro-formulated ubiquitin chain attached to a substrate protein.
authors.affiliation: Department of Chemistry, University of Texas , 105 E. 24th St., Austin, Texas 78712, United States.
authors.forename: Joe R
authors.initials: JR
authors.lastname: Cannon
authors.forename: Kirby
authors.initials: K
authors.lastname: Martinez-Fonts
authors.forename: Scott A
authors.initials: SA
authors.lastname: Robotham
authors.forename: Andreas
authors.initials: A
authors.lastname: Matouschek
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
mesh_terms: D000818:Animals; D006801:Humans; D008239:Lysine / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D010782:Photolysis; D025821:Polyubiquitin / Q000737:chemistry*; D014466:Ultraviolet Rays
chemical_list: D025821:Polyubiquitin; D008239:Lysine