Novel Cβ–Cγ Bond Cleavages of Tryptophan-Containing Peptide Radical Cations

In this study, we observed unprecedented cleavages of the Cβ–Cγ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M – 116]+ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent Cβ–Cγ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH – 43]+ and [WGGGH – 116]+, obtained from the CID of [LGGGH]•+ and [WGGGH]•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]•-2), in agreement with the CID data for [WGGGH]•+ and [W1-CH3GGGH]•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from Cβ–Cγ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the Cβ–Cγ bond and, therefore, decreases the dissociation energy barrier dramatically

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