Protein Ion Charge State Research Articles

Collisionally activated dissociation of supercharged proteins formed by electrospray ionization.

The dissociation of protein ions formed by ESI ranging in size from 12 to 29 kDa using sustained off-resonance irradiation collisional activation was investigated as a function of charge state in a 9.4-T Fourier transform mass spectrometer. Addition of m-nitrobenzyl alcohol to denaturing solutions of proteins was used to form very high charge states of protein ions for these experiments. For all proteins in this study, activation of the highest charge state results in a single dominant backbone cleavage, often with less abundant cleavages at the neighboring 3-5 residues. This surprising phenomenon may be useful for the "top-down" identification of proteins by producing sequence tags with optimum sensitivity. There is a slight preference for cleavage adjacent to acidic residues and proline. Solution-phase secondary structure does not appear to play a significant role. The very limited dissociation channels observed for the supercharged ions may be due, in part, to the locations of the charges on the protein.

Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure.

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.