Disulfide bonds formed between cysteine residues stabilize the native structure of many proteins.[1] These covalent crosslinkages are part of the tertiary structure and introduce conformational constraints to the polypeptide architecture, thus improving the thermodynamic stability. Mass spectrometry has become increasingly important in the characterization and identification of proteins as a result of its capability for highly specific measurements on relatively small amounts of analyte. Protein characterization and identification relies on database searches using primary sequence information obtained by tandem mass spectrometry (MS n or MS/MS). The top-down approach [2] applied to intact proteins ions is an efficient MS/MS method which is potentially suited for the high-throughput characterization of proteins. The problems addressed by the top-down approach include verification of the protein sequence, localization of errors in DNA-predicted sequences, de novo sequencing, as well as the analysis of pre-or posttranslational modifications (PTMs). However, such PTMs as disulfide bonds still remain a challenge for traditional MS/MS techniques that utilize vibrational excitation (VE), as they break peptide bonds preferentially in protonated polypeptides and leave the disulfide bonds intact.[3] As a result, information on the primary sequence is limited to parts of the protein that is not constrained by disulfide bonds. This restriction complicates primary sequence elucidation and limits the utility of automated database searches. The problem of disulfide bonds is solved conventionally by their reduction and alkylation [4] prior to MS/MS measurements. However, the additional step involving solution-phase chemistry, including subsequent purification, greatly increases the analysis time and the risk of sample loss. Another approach is to use an alternative iondissociation strategy. Chrisman and McLuckey have shown that VE of deprotonated polypeptides induced by collisionally activated dissociation (CAD) [5] breaks disulfide bonds with high efficiency.[6] However, CAD of peptide anions often produces complicated mass spectra that, together with backbone cleavages, contain significant amounts of neutral mass losses and rearrangement products which make such MS/MS spectra of limited value for sequencing. Electron-capture dissociation (ECD) [7] stands alone in its ability to cleave SĂS bonds preferentially in peptide polycations (Scheme 1).[3] A drawback of this technique is that the capture of a low-energy electron by multiply charged polypeptides results in charge reduction prior to bond cleavage, which in turn leads to a charge decrease in one of the two fragments. As species need to contain at least one charge to be detectable in mass spectrometry, the charge reduction in ECD can lead to a loss of information. Previously, when magnetic sector instruments were widely used, high-energy CAD (HE CAD) was utilized to produce cleavage of numerous disulfide bonds in peptides.[8] The abundance of this fragmentation channel comp...