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 directly induced by ionizing radiation (IR), free amino acids and 3-residue peptides were exposed to radiation generated by a linear accelerator (Linac) used in radiotherapy. Mass spectrometry was performed to detail the relative sensitivity to radiation as well as identify covalent, IR-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. IR 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.

Ionizing Radiation-induced Proteomic Oxidation in Escherichia coli

“…These were combined and subjected to high-pH fractionation, and fractions were analyzed using MS (MS). Database searches on a whole proteome level focused on previously identified IR-induced amino acid modifications, including carbonylation (+13.98 Da), hydroxylation (+15.99 Da), dioxidation (+31.99 Da), and trioxidation (+47.99 Da) ( 25 , 29 ). We note here that although we classify +31.99 Da as dioxidation, such a mass shift could also be peroxidation.…”

“…+47.99 Da on Cys results in a sulfonic acid chemical group, +13.98 Da on Pro can represent conversion to pyroglutamic acid, etc.). We additionally attempted to identify several previously reported, less-common modifications ( 25 , 29 , 30 ); however, none of these appeared to any significant degree in our data sets.…”

“…Our current understanding of IR-induced damage to cellular macromolecules has been primarily based on in vitro studies, in which nucleotides, amino acids, peptides, or proteins have been exposed to IR in solution and the products characterized via MS (MS) ( 1 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ). The few in vivo studies that detect and quantify IR-induced protein damage within intact cells have primarily relied on antibody-mediated detection of chemically derived adducts from initial oxidative modifications, primarily carbonylation ( 31 , 32 ).…”

“…Thus, with this methodology alone, one can miss many types of oxidation found on proteins ( 1 , 33 , 34 ) and be misdirected to others. Previous studies suggest that IR-induced oxidative damage to protein results in covalent modifications such as hydroxylation and carbonylation to amino acid side chains, as well as peptide bond hydrolysis ( 1 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 35 ). However, there have been no reports identifying the proteins that are the immediate in vivo targets of IR-mediated oxidation.…”

Proteome Damage Inflicted by Ionizing Radiation: Advancing a Theme in the Research of Miroslav Radman

“…To survey and quantify as much IR-induced oxidation as possible, peptides from ten replicates (five treated and five mock-treated) were analyzed using mass spectrometry (MS). The proteome databases were searched systematically for previously identified IR-induced amino acid modifications, including carbonylation (+13.98 Da), hydroxylation (+15.99 Da), dioxidation (+31.99 Da), and trioxidation (+47.99 Da) [ 26 , 36 ]. We note here that although we classify +31.99 Da as dioxidation, such a mass shift could also be peroxidation.…”