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

Abstract

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-alpha-C or N-C-alpha bond rather than the typical C-alpha-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 pi orbital on the beta-carbon and the N-C-alpha bond, leading to low-barrier beta-cleavage of the N-C-alpha bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z(center dot) ions, whereas C-terminal interaction leads to effective cleavage of the C-alpha-C bond through rapid loss of isocyanic acid. Dissociation of the C-alpha-C bond may also occur via water loss followed by beta-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.