Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals

Analytical and Bioanalytical Chemistry, Jan 2017

Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32–55% for BSA, ∼10–21% for OVA) were substantially higher than those for the other identified amino acids (∼5–12% for BSA, ∼4–6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

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Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals

Anal Bioanal Chem Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals Fobang Liu 0 1 2 3 4 5 Senchao Lai 0 1 2 3 4 5 Haijie Tong 0 1 2 3 4 5 Pascale S. J. Lakey 0 1 2 3 4 5 Manabu Shiraiwa 0 1 2 3 4 5 Michael G. Weller 0 1 2 3 4 5 Ulrich Pöschl 0 1 2 3 4 5 Christopher J. Kampf 0 1 2 3 4 5 0 Department of Chemistry, University of California , Irvine, 1102 Natural Sciences II, Irvine, CA 92697-2025 , USA 1 School of Environment and Energy, South China University of Technology, Higher Education Mega Center , Guangzhou 510006 , China 2 Multiphase Chemistry Department, Max Planck Institute for Chemistry , Hahn-Meitner-Weg 1, 55128 Mainz , Germany 3 Institute for Organic Chemistry, Johannes Gutenberg University Mainz , Duesbergweg 10-14, 55128 Mainz , Germany 4 Institute for Inorganic and Analytical Chemistry, Johannes Gutenberg University Mainz , Duesbergweg 10-14, 55128 Mainz , Germany 5 Division 1.5 Protein Analysis, Federal Institute for Materials Research and Testing (BAM) , Richard-Willstätter-Str. 11, 12489 Berlin , Germany Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals. Peptides; Proteins; Oxidation; Hydroxyl radicals; HPLC-MS; Amino acid analysis Introduction Reactive oxygen species (ROS) have been associated with various diseases (e.g., diabetes and cancer), as they can cause oxidative stress, biological aging, and cell death [ 1–7 ]. The hydroxyl radical (OH), the most reactive form of ROS, can oxidize most organic compounds such as proteins and DNA [ 8 ]. Hydroxyl radicals can be generated in biological systems endogenously and exogenously [ 9 ], and the sources include a variety of different processes such as cellular metabolic processes, radiolysis, photolysis, and Fenton chemistry [ 10–12 ]. Elucidation of the OH-induced oxidation mechanism of amino acids, peptides, and proteins is of exceptional importance for physiological chemistry (e.g., for understanding the relationship between protein oxidation and aging) [ 13–16 ] and also of considerable interest for the Earth’s atmosphere [ 17, 18 ]. Hydroxyl radicals undergo several types of reactions with amino acids, peptides, and proteins. Typical reactions include addition, electron transfer, and hydrogen abstraction [ 14, 15 ]. The OH radicals can attack both amino acid side chains and the peptide backbone, generating a large number of different radical derivatives of proteins [ 19, 20 ]. With respect to the peptide backbone cleavage, the main reaction pathway is initiated by an H abstraction at the α-carbon position. This is followed by a reaction with O2 to give a peroxyl radical, which ultimately results in fragmentation and cleavage of the backbone of the protein, thereby mainly forming amide and carbonyl fragments [ 11, 21 ]. Several studies have demonstrated that the H abstraction from the α-carbon position is the dominant pathway for the OH-mediated fragmentation of proteins and occurs at specific sites or amino acid residues as shown by computational and experimental investigations [ 9, 22, 23 ]. Also, the metal-catalyzed oxidation (MCO) of proteins was found to be an important pathway for protein degradation, as metal ions preferentially bind particular sites of proteins, resulting in selective damage [ 14, 24–26 ]. Among the multiple oxidation products, carbonyl compounds, peptide-bound hydroperoxides, and larger protein fragments were predominantly identified [ 27–30 ]. For example, Morgan et al. [28] investigated the site selectivity of peptide-bound hy (...truncated)


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Fobang Liu, Senchao Lai, Haijie Tong, Pascale S. J. Lakey, Manabu Shiraiwa, Michael G. Weller, Ulrich Pöschl, Christopher J. Kampf. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals, Analytical and Bioanalytical Chemistry, 2017, pp. 2411-2420, Volume 409, Issue 9, DOI: 10.1007/s00216-017-0188-y