Electron Capture in Spin-Trap Capped Peptides. An Experimental Example of Ergodic Dissociation in Peptide Cation-Radicals

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4′-carboxypyrid-2′-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH2, pepy-AQEFGEQGQKALKQL-NH2, and pepy-AQEGSEQAQKFFKQL-NH2. Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b11-14 + 1)+· fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1′ positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol−1) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol−1, which exceeded the hydrogen atom affinity of the backbone amide groups (21–41 kJ mol−1). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD

Hydrogen Bonding Constrains Free Radical Reaction Dynamics at Serine and Threonine Residues in Peptides

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal C_α–C or N–C_α bond rather than the typical Cα–C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the β-carbon and the N–C_α bond, leading to low-barrier β-cleavage of the N–C_α bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z• ions, whereas C-terminal interaction leads to effective cleavage of the C_α–C bond through rapid loss of isocyanic acid. Dissociation of the C_α–C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes