Temperature of Radicals Produced by Hydrogen Atom Attachment to Protonated Peptides in a Quadrupole Ion-Trap Mass Spectrometer

Tandem mass spectrometry equipped with hydrogen attachment dissociation (HAD) has been developed for peptide and protein analysis. This study investigates gas-phase fragmentation induced by hydrogen-atom attachment to peptides containing S-carbamidomethylated cysteine residues. The attachment of hydrogen atoms to peptides induces cleavage of either the N-Cα or Cα-C bond. The resulting α-carbon-centered radical intermediates undergo either side-chain loss or a further reaction with hydrogen atoms. Different precursor ions generate α-carbon-centered radical intermediates with varying stabilities, even when their chemical compositions are identical. This difference is due to the effective temperature of the resultant peptide cation radicals depending on the precursor ion. During hydrogen attachment to protonated peptides in the gas phase, the kinetic energy of the hydrogen atom and reaction enthalpy are converted to the internal energy of the resulting peptide cation radical. The effective temperature of the radicals is influenced by their number of degrees of freedom, as the excess energy from the reaction is distributed across these number of degrees of freedom. The fragmentation efficiency of HAD decreases with increasing peptide mass due to the reduced effective temperature. Supplemental activation through ion trap heating promoted fragmentation by HAD, which is especially useful for the analysis of large peptides.