Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the; major factors governing the "top-down" sequence analysis of:int act multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions., To mor e fully understand this effect electrospray ionization coupled to a quadrup ole ion trap mass spectrometer, collision-induced dissociation, and gas-pha se ion/ion reactions have been employed to examine the fragmentation of the [M + 12H](12+), [M + H](+) ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed c leavage at:aspartic acid residues was observed. At intermediate charge stat es, (+7, +8, and +9), extensive nonspecific fragmentation of the protein ba ckbone 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 pr oline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton m odel", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquit in in the gas phase, examined by ion mobility and hydrogen/deuterium exchan ge measurements.