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

• Published in: Analytical and bioanalytical chemistry Vol. 409; no. 9; pp. 2411 - 2420
• Main Authors: Liu, Fobang, Lai, Senchao, Tong, Haijie, Lakey, Pascale S. J., Shiraiwa, Manabu, Weller, Michael G., Pöschl, Ulrich, Kampf, Christopher J.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.03.2017; Springer; Springer Nature B.V
• Subjects: Alanine; Amino acids; Analytical Chemistry; Arrays; Asparagine; Aspartic acid; Biochemistry; Bovine serum albumin; Characterization and Evaluation of Materials; Chemical properties; Chemical research; Chemistry; Chemistry and Materials Science; Fluorescence; Food Science; Free radicals; Glycine; High performance liquid chromatography; Hydrogen peroxide; Hydroxyl radicals; Iron; Laboratory Medicine; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Monitoring/Environmental Analysis; Nitrogen; Ovalbumin; Oxidation; Oxidation-reduction reactions; Peptides; Proteins; Radicals (Chemistry); Research Paper; Serum albumin; Proteins; Hydroxyl radicals; Oxidation; Peptides; Amino acid analysis; HPLC-MS
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-017-0188-y

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.