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)