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

Fobang Liu,Pascale S J Lakey,Michael G Weller,Manabu Shiraiwa,Christopher J Kampf,Senchao Lai,Ulrich Pöschl,Haijie Tong

doi:10.1007/s00216-017-0188-y

Abstract

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.

Highlights

Reactive oxygen species (ROS) have been associated with various diseases, as they can cause oxidative stress, biological aging, and cell death [1,2,3,4,5,6,7]
LC-mass spectrometry (MS)/ MS analysis of underivatized samples further confirmed the free amino acid glycine to be an oxidation product of proteins and peptides reacting with hydroxyl radicals
Free amino acids were identified as products in the OHinduced oxidation of proteins and peptides by LC-MS/MS analysis

Summary

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 [1,2,3,4,5,6,7]. Hydroxyl radicals undergo several types of reactions with amino acids, peptides, and proteins. We focus on the identification and quantification of amino acids as oxidation products of proteins and peptides generated by hydroxyl radicals from the Fenton reaction. For this purpose, we introduced two robust analytical methods based on mass spectrometry and liquid chromatography, which have been widely used for the determination of amino acids in various environments (e.g., plasma and plant extracts) [33, 34].

Results and discussion

Conclusions

Compliance with ethical standards