The gas-phase fragmentation reactions of a series of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase have been examined by multistage tandem mass spectrometry (MS/MS and MS³) in a linear quadrupole ion trap in order to explore the utility of this instrumentation for routine â top-downâ recombinant protein characterization. Following a rapid low resolution survey of the fragmentation behavior of the precursor ions from the wild type (WT) protein, selected charge states were subjected to detailed structural characterization by using high resolution â zoomâ and â ultrazoomâ resonance ejection MS/MS product ion scans. Dissociation of the [M + 18H]¹⁸⁺ charge state yielded a range of product ions from which extensive sequence information could be derived. In contrast, dissociation of the [M + 20H]²⁰⁺ charge state resulted in a single dominant y96 product ion formed by fragmentation between adjacent Ile/Gly residues, with only limited sequence coverage. Further extensive sequence information was readily obtained however, by MS³ dissociation of this initial product. From the combined MS/MS and MS³ spectra an overall sequence coverage of 66.9%, with fragmentation of 85 of the 127 amide bonds within the WT protein, was obtained. MS/MS and MS³ of three of the four site-directed mutagenesis products (E29A), (Y61F) and (E81A) were found to yield essentially identical product ion spectra to the WT protein, indicating that these modifications had no significant influence on the fragmentation behavior. The specific site of modification could be unambiguously determined in each case by characterization of product ions resulting from fragmentation of amide bonds on either side of the mutation site. In contrast, MS/MS and MS³ of the K107A mutant led to significantly different product ion spectra dominated by cleavages occurring N-terminal to proline, which restricted the ability to localize the modification site to within only an 8 amino acid region of the sequence. This work highlights the need for further studies to characterize the charge state, sequence and structural dependence to the low energy collision induced dissociation reactions of multiply protonated intact protein ions.