Isomerization and fragmentation of isomeric peptide radical cations

Abstract

Tandem mass spectrometry is an important tool for examining the fragmentation processes of peptide ions, laying the scientific foundation for gas phase peptide sequencing—one of the key analytical techniques in proteomics applications.

The first part of the thesis (Chapter 3) describes the systematic examination of laser-induced dissociation (LID) spectra using a modified hybrid linear ion trap (LIT) mass spectrometer. The LID spectra of even-electron protonated peptides (〖[M + H]〗^+) featured fragment ions that were both similar to and different from those found in collision-induced dissociation (CID) spectra of analogous odd-electron radical cations (M^(●+)), generated through dissociative electron transfer in ternary metal–ligand–peptide complexes or through laser photolysis (at 266 nm) of iodopeptides, within the same instrument.

The second part of the thesis (Chapters 4and 5) describes how the modified hybrid LIT mass spectrometer was used to examine the isomerization and fragmentation reactions of α-, β-, and ε-carbon–and π–centered radical cations of aromatic-containing peptides in combination with low-energy CID and ion–molecule reactions. The fragmentations of the ε-carbon–centered radical cations were in some cases substantially different from those of their π-centered isomeric analogues, but both were similar to, yet different from, those of their α-carbon–centered isomeric analogues; this behavior is affected by the proximity of the ε-and α-radical centers. Density functional theory calculations and Rice–Ramsperger–Kassel–Marcus modeling were employed for systematic mechanistic investigations of selected isomerization and fragmentation reactions about model tyrosine-containing tripeptides(ε-and α-carbon–and π–centered radical cations). Direct interconversion from the ε-carbon–centered radical of the tyrosine residue to the α-carbon–centered radical of the terminal glycine residue is favorable both energetically and kinetically; the activation barriers for isomerizations along the peptide backbones of those isomers are higher than those of competitive direct dissociation pathways. The close proximity of the α-CH hydrogen atom of the terminal glycine residue to the ε-carbon–centered radical in the tyrosine residue leads to ready direct interconversion and facile radical migrations, as confirmed through isotopic labeling experiments. Subsequent investigations provided the first experimental evidence for the existence of stable α-and β-carbon–and π–centered radical cationic peptide isomers in the gas phase.

Ion–molecule reactions have been used (Chapter 5)to monitor the radical site mobility in the selected radical cationic peptides; the mass spectra of stable open-shell precursors were recorded to map their structures after reacting with 〖NO〗_2● in the gas phase. Radical recombination was evident for the three isomers having radical sites at the α-and β-carbon atoms and the π-center, producing corresponding even-electron protonated nitrated precursors. The transitory nature of both the α-and β-carbon–centered radical cationic peptides led to facile losses of neutral NO2H to form an N=Cα double bond in the peptide backbone and a Cα=Cβ double bond in the tyrosyl side chain, respectively; in contrast, the π-centered radical generated a stable ortho-nitro derivative of the tyrosyl residue.