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Collisional activation (CA) and collision-activated dissociation (CAD) of multiply protonated molecular ions produced by electrospray ionization using an atmospheric pressure source are described. A TAGA 6000E triple-quadrupole mass spectrometer, in both unmodified and differentially pumped inlet arrangements, was used to investigate CA and CAD during transfer through the atmosphere-vacuum interface and subsequent CAD in the tandem instrument. Melittin, which has a molecular weight (M r ) of 2846, is efficiently dissociated in the interface at higher nozzle-skimmer voltages, yielding fragmentation that can be assigned to the various charge states. Selection of such product ions formed in the interface for subsequent tandem mass spectrometry allows confirmation of earlier sequence assignments and extends the utility of these methods. Various charge states of larger polypeptides, such as human parathyroid hormone (1-44) (M r 5064), can be efficiently collisionally dissociated in the second (rf-only) quadrupole. However, for molecular ions of this size, the low-energy collisions used for CAD yield only partial sequence information. For large molecules such as horse heart myoglobin (M r 16,951), the effects of nozzle-skimmer bias are explored, and it is shown that higher charge states (at ≤ m/z 1400) can be effectively dissociated in the interface. Initial results for both metastable (unimolecular) and CAD for myoglobin are reported. The potential and limitations of CAD for large biomolecular ions are discussed. The feasibility of fingerprinting for proteins is illustrated by the CAD spectra of cytochrome c from nine species.

Collisional activation and collision-activated dissociation of large multiply charged polypeptides and proteins produced by electrospray ionization