Fragmentation of peptide polyanions by electron detachment dissociation (EDD) has been induced by electron irradiation of deprotonated polypeptides [M-nH](n-) with >10 eV electrons. EDD has been found to lead preferentially to a* and x fragment ions (C(alpha)-C backbone cleavage) arising from the dissociation of oxidized radical anions [M-nH]((n-1)-*. We demonstrate that C(alpha)-C cleavages, which are otherwise rarely observed in tandem mass spectrometry, can account for most of the backbone fragmentation, with even-electron x fragments dominating over radical a* ions. Ab initio calculations at the B3 LYP level of theory with the 6-311+G(2 p,2 d)//6-31+G(d,p) basis set suggested a unidirectional mechanism for EDD (cleavage always N-terminal to the radical site), with a*, x formation being favored over a, x* fragmentation by 74.2 kJ mol(-1). Thus, backbone C(alpha)-C bonds N-terminal to proline residues should be immune to EDD, in agreement with the observations. EDD may find application in mass spectrometry for such tasks as peptide sequencing and localization of labile post-translational modifications, for example, those introduced by sulfation and phosphorylation. EDD can now be performed not only in Fourier transform mass spectrometry, but also in far more widely used quadrupole (Paul) ion traps.

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 SÀS 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...

The adverse influence of the radio frequency (RF) voltage on electrons has been the main obstacle for the implementation of electron capture dissociation (ECD) in three-dimensional quadrupole ion traps (3D QITs). Here we demonstrate that the use of axial magnetic field, together with the injection of low-energy (<5 eV) electrons, in the beginning of the positive RF semi-period achieves trapping of electrons for a period of time comparable with the semi-period duration. Importantly, the energy of the electrons remains low during most of the trapping period. With this technique, which we call "magnetized electrons, in-phase injection" (MEPhI), ECD and other ion-electron reactions have become possible in a 3D QIT. Initial ECD results, including single-scan data, were obtained with dications of Substance P. The observed secondary fragmentation of ECD fragments indicates that the trapped electrons are still somewhat hotter than desired.

A new method of detecting the presence of deprotonation and determining its position in gas-phase polypeptide cations is described. The method involves 157-nm ultra-violet photodissociation (UVPD) and is based on monitoring the losses of CO2 (44 Da) from electronically excited deprotonated carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+)...(-)...(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding within polypeptide clusters. Collision-activated dissociation (CAD) of decarboxylated cations localizes the position of deprotonation. Fragment abundances can be used for the semiquantitative assessment of the branching ratio of deprotonation among different acidic sites, however, the mechanism of the fragment formation should be taken into account. Cations of Trp-cage proteins exist preferentially as zwitterions, with the deprotonation position divided between the Asp9 residue and the C terminus in the ratio 3:2. The majority of dications of the same molecule are not zwitterions. Furthermore, 157-nm UVPD produces abundant radical cations M*+ from protonated molecules through the loss of a hydrogen atom. This method of producing M*+ ions is general and can be applied to any gas-phase peptide cation. The abundance of the molecular radical cations M*+ produced is sufficient for further tandem mass spectrometry (MS/MS), which, in the cases studied, yielded side-chain loss of a basic amino acid as the most abundant fragmentation channel together with some backbone cleavages.

A new technique for fragmentation of cations and anions of peptides stored in ion traps including radiofrequency devices is described. The technique involves irradiation of peptide ions by a beam of particles generated by a fast atom bombardment (FAB) gun. This irradiation leads to fragmentation of N--C(alpha) backbone bonds (c- and z-fragments) and S--S bonds for cations and C(alpha)-C backbone bonds (a- and x-fragments) for anions of peptides. The fragmentation patterns observed are hypothesized to be due to the interaction of peptide ions with metastable, electronically excited species generated by the FAB gun. Interaction of a metastable atom A* with a peptide n-cation M(n+) leads to the electron transfer from the metastable atom to the polycation through the formation of an ion-pair collision complex A(+.) . . . M((n-1)+.) and subsequent fragmentation of the peptide cation. Thus, for polycations, this metastable-induced dissociation of ions (MIDI) is similar to the phenomenon of electron capture dissociation (ECD). Interaction of A* with an anion leads to the deexcitation of the metastable species and detachment of an electron from the anion. This in turn leads to backbone fragmentation similar to that in electron detachment dissociation (EDD). The MIDI technique is robust and efficient, and it is applicable to peptides in as low charge states as 2+ or 2-.