Covalent Modification of Amino Acids and Peptides Induced by Ionizing Radiation from an Electron Beam Linear Accelerator Used in Radiotherapy

Abstract

To identify modifications to amino acids that are directly induced by ionizing radiation, free amino acids and 3-residue peptides were irradiated using a linear accelerator (Linac) radiotherapy device. Mass spectrometry was performed to detail the relative sensitivity to radiation as well as identify covalent, radiation-dependent adducts. The order of reactivity of the 20 common amino acids was generally in agreement with published literature except for His (most reactive of the 20) and Cys (less reactive). Novel and previously identified modifications on the free amino acids were detected. Amino acids were far less reactive when flanked by glycine residues in a tripeptide. Order of reactivity, with GVG most and GEG least, was substantially altered, as were patterns of modification. Radiation reactivity of amino acids is clearly and strongly affected by conversion of the α-amino and α-carboxyl groups to peptide bonds, and the presence of neighboring amino acid residues.

Figure 1.

A, Raw signal loss for unmodified amino acids at 50 μM in H₂O. B, Linearization from 0Gy to 200Gy dosage, the linear range of reactant loss. C, Relative reactivities of amino acids charted altogether and compared to reactivities determined using previous techniques^17,20,31. D, The cluster of aromatic ring-containing residues. E, Modification events identified and their abundance on Tyr, from ESI-TOF MS.

Figure 2.

Generalized reaction scheme for the most common oxidative modifications to amino acids and proteins. Following hydrogen abstraction at the α-carbon, carbon or oxygen radical species can further react to produce hydroxylation (+16), peroxidation (+32), or carbonylation (+14).

Figure 3.

Raw signal loss for tripeptides at 50 μM in H₂O. B, Linearization from 0Gy to 1000Gy dosage, the linear range of reactant loss. C, Comparison of relative reactivity rates of free amino acids and tripeptides. D, Reaction scheme for C-terminal decarboxylation. R represents the first and second residues, and the sidechain and C-terminus of the third Gly residues are shown in full.

Figure 4.

Example MS/MS spectra from unmodified tripeptide GCG and triply oxidized version demonstrating fragment ion identification and localization of oxidation to middle C residue. A, B, and Y refer to classic fragment ion products generated by collisionally-induced dissociation within the mass spectrometer.