title: Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry.
abstract: One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393, USA.
authors.forename: G E
authors.initials: GE
authors.lastname: Reid
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: P A
authors.initials: PA
authors.lastname: Chrisman
authors.forename: J M
authors.initials: JM
authors.lastname: Wells
authors.forename: S A
authors.initials: SA
authors.lastname: McLuckey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008969:Molecular Sequence Data; D011522:Protons; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014452:Ubiquitins / Q000032:analysis*
chemical_list: D011522:Protons; D014452:Ubiquitins