posted on 2014-09-18, 00:00authored byDaniel
A. Thomas, Chang Ho Sohn, Jinshan Gao, J. L. Beauchamp
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
Cα–C or N–Cα 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–Cα bond, leading
to low-barrier β-cleavage of the N–Cα 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
Cα–C bond through rapid loss of isocyanic
acid. Dissociation of the Cα–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.