Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals
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 School of Environment and Energy, South China University of Technology, Higher Education Mega Center , Guangzhou 510006 , China
1 Multiphase Chemistry Department, Max Planck Institute for Chemistry , Hahn-Meitner-Weg 1, 55128 Mainz , Germany
2 Christopher J. Kampf
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
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 . Hydroxyl radicals can be generated in
biological systems endogenously and exogenously , 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.  investigated the site selectivity of peptide-bound
hydroperoxide and alcohol group formation, as well as
fragment species formed through protein oxidation by OH/O2
using a mass spectrometry (MS) approach.
The high reactivity of proteins with OH radicals, however,
may result in various products due to different reaction
mechanisms [31, 32]. In this study, 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]. These methods provide
analytical evidence for the release of amino acids due to the
OH-mediated oxidation of peptides and enable their yields to
Bovine serum albumin (BSA) and ovalbumin (OVA) were
used as model proteins, and tripeptides with varying amino
acid sequences were used to study yields and site selectivity
for reactions with OH radicals. The amino acids consisted of
the tripeptides (glycine (Gly), alanine (Ala), serine (Ser), and
methionine (Met)) were chosen due to their reactivity towards
OH radicals; i.e., Gly, Ala, and Ser show a low reactivity
towards OH, while the rate constant of Met with OH is about
2 orders of magnitude higher . Oxidation products were
analyzed by high-performance liquid chromatography tandem
mass spectrometry (HPLC-MS/MS) using a Q-ToF mass
spectrometer 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. We report the release of
free amino acids in the OH radical-induced oxidation of
peptides and proteins. Furthermore, effects of amino acid side
chains on the release are discussed with regard to product
identification and site selectivity.
BSA (A5611), OVA (grade V, A5503), Gly-Gly-Gly ((Gly)3,
G1377), Met-Ala-Ser (M1004), NaH2PO4·H2O (71504), OPA
(P0657), 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl,
23186), 3-mercatopropionic acid (63768), acetonitrile (ACN,
34998), methanol (MeOH, 494291), amino acid standards
(AAS18), asparagine (A0884), glutamine (49419), tryptophan
(93659), sodium tetraborate decahydrate (Na2B4O7·10H2O,
S9640), FeSO4·7H2O (F7002), H2O2 solution (30%, w/v,
16911), and HCl solution (0.1 M, 318965) were purchased
from Sigma-Aldrich (Germany). Sodium hydroxide (NaOH,
0583) was from VWR (Germany). Met-Gly-Ala,
Gly-AlaMet, and Ala-Met-Gly were obtained from GeneCust
(Luxembourg) and were delivered in the desalted form with
a purity >95%. High purity water (18.2 MΩ cm) was taken
from an ELGA LabWater system (PURELAB Ultra, ELGA,
UK) and autoclaved before use if not specified otherwise.
Protein/peptide oxidation reactions
Reaction mixtures of proteins/peptides (structures shown in
Fig. 1) with Fenton oxidants (FeSO4-H2O2) were stirred
(Multistirrer 15, Fischer Scientific, Germany) in closed
screw-cap vials at room temperature. Hydroxyl radicals were
generated under two oxidation conditions, and the estimated
effective OH concentrations are listed in Table 1. The pH of
the reaction solutions was adjusted to 3 by adding 1 M NaOH
and measured by a pH meter (Multi 350i; WTW, Weilheim,
Germany). Although ethylenediaminetetraacetic acid (EDTA)
is a common chelator to stimulate the generation of radicals
under physiological pH conditions (pH 6–8) , no EDTA
was added in this study as glycine was found to be one of the
degradation products of EDTA in the presence of OH .
For protein oxidation reactions, the proteins BSA and OVA
were pretreated with a size-exclusion column (PD-10, GE
Healthcare, Germany) using ultrapure H2O to remove low
molecular components (<5 kDa). From the purified
25 mg mL−1 protein solutions, 100-μL aliquots were added
to the Fenton oxidant solutions to a final volume of 2.5 mL.
After the respective reaction times, the oxidized samples were
immediately eluted on a PD-10 column pre-equilibrated with
ultrapure H2O to separate the protein and the low molecular
Fig. 1 Structures of the
investigated peptides (a) and
proteins (b) in this study. The
molecular structures of proteins
(BSA, PDB accession number
3V03; OVA, PDB accession
number 1OVA) were created
using the RCSB PDB protein
workshop (4.2.0) software
weight fraction (<5 kDa). For peptide oxidation reactions,
100 μL of 100 mM solutions of the investigated peptides were
added as described before. Control reactions were performed
using either H2O2 or FeSO4 alone at the same concentrations
and pH conditions, adjusted by 0.1 M HCl and 1 M NaOH,
In addition, oxidation experiments were performed for
peptides with UV-induced OH generation via the homolysis of
H2O2 in aqueous solution. Briefly, 4 mM (Gly)3 were mixed
with 50 mM H2O2 or 200 mM H2O2 in a 10 × 10 × 40 mm UV
quartz cuvette (Hellma Analytics, Müllheim, Germany) and
subsequently irradiated by four UV lamps (wavelength of
254 nm, LightTech, Hungary) for 1 h. The pH of these
samples was also adjusted to 3 by adding 0.1 M HCl. Control
samples were either treated the same way as described above,
but without UV irradiation, or prepared without H2O2 and
irradiated for 1 h.
All experiments described above were performed in
duplicate, and the samples were lyophilized (−40 °C, ∼12 h)
immediately after reaction to stop the reaction by removing the
hydrogen peroxide. The dry residues were stored at −20 °C
and redissolved in 100 μL H2O for analysis.
The oxidized peptides and low molecular weight fraction of
proteins were analyzed with the HPLC-DAD-FLD system
(Agilent Technologies 1200 Series) consisting of a binary
pump (G1312B), a four-channel microvacuum degasser
(G1379B), a column thermostat (G1316B), an autosampler
with a thermostat (G1330B), a photo-diode array detector
(DAD, G1315C), and a fluorescence detector (FLD,
G1321A). ChemStation software (version B.03.01, Agilent)
was used to control the system and for the data analysis.
Table 1 Oxidation conditions for
the generation of OH radical in
pH (adjusted by 1 M NaOH)
[OH] (molecule cm−3)a
a The decay of (Gly)3 was monitored and allowed for an estimation of the effective OH concentration based on a
pseudo-first-order kinetic rate function: [(Gly)3] = [(Gly)3]0e(−k[OH]t) , where [(Gly)3] is the recovery of (Gly)3,
[(Gly)3]0 is the initial recovery (i.e., 100%), k (1.2 × 10−12 cm3 s−1 ) is the second-order rate constant for the
reaction of OH with (Gly)3 , [OH] is the effective concentration of hydroxyl radical (assuming it remains
constant during the reaction), and t is the reaction time. The fitting curves are shown in Fig. S6 in ESM
Chromatographic conditions were in accordance with the
instructions by Agilent Technologies . Briefly, automatic
pre-column derivatization with OPA and FMOC was
performed at room temperature, according to the injector
programs (for details, see Table S1 in Electronic Supplemental
Material (ESM)) listed in Henderson et al. . After
derivatization, an amount equivalent to 0.5 μL of each sample was
injected on a Zorbax Eclipse amino acid analysis (AAA)
column (150 mm × 4.6 mm i.d., 3.5 μm, Agilent) at a
temperature of 40 °C. Mobile phase A was 40 mM NaH2PO4 (aq),
adjusted to pH 7.8 with 10 N NaOH (aq), while mobile phase
B was acetonitrile/methanol/water (45:45:10, v/v/v). The flow
rate was 2 mL min−1 with a gradient program that started with
0% B for 1.9 min followed by a 16.2-min step that raised
eluent B to 57%. Then, eluent B was increased to 100% within
0.5 min and kept for another 3.7 min. The mobile phase
composition was reset to initial conditions within 0.9 min, and the
column was equilibrated for 2.8 min before the next run.
Primary amino acids were detected by monitoring the UV
absorbance at 338 nm, with a reference at λ = 390 nm,
bandwidth = 10 nm, slit of 4 nm, and peak width of >0.1 min,
simultaneously detected by FLD with excitation 340 nm,
emission 450 nm, and photomultiplier tube (PMT) gain of
10. Secondary amino acids were detected by FLD with
excitation 266 nm, emission 305 nm, and PMT gain of 9. A
mixture of 20-amino acid standards (see ESM Table S2) was used
to obtain calibration curves for quantification as illustrated in
Fig. S1 in ESM. The limits of detection (LODs, defined as a
signal-to-noise ratio of 3) for 20 individual amino acids are in
the range of 0.1 to 5 pmol. Linearity is demonstrated for the
concentration range of 20 to 500 μM for all amino acids by
detection using a DAD or FLD.
Identification of OH-mediated reaction products of peptides
and the low molecular weight fraction of proteins was also
carried out using an HPLC-MS/MS system (Agilent). The
LC-MS/MS system consists of a quaternary pump
(G5611A), an autosampler (G5667A) with a thermostat
(G1330B), a column thermostat (G1316C), and an
electrospray ionization (ESI) source interfaced to a Q-ToF
mass spectrometer (6540 UHD Accurate-Mass Q-ToF,
Agilent Technologies). All modules were controlled by
MassHunter software (Rev. B. 06.01, Agilent). The LC
column was a Zorbax Extend-C18 Rapid Resolution HT
(2.1 × 50 mm, 1.8 μm) and was operated at a temperature of
30 °C. Eluents used were 3% (v/v) acetonitrile (Chromasolv,
Sigma, Seelze, Germany) in water/formic acid (0.1% v/v,
Chromasolv, Sigma, Seelze, Germany) (eluent A) and 3%
water in acetonitrile (eluent B). The flow rate was
0.2 mL min−1 with a gradient program starting with 3% B
for 1.5 min followed by an 18-min step that raised eluent B
to 60%. Further, eluent B was increased to 80% at 20 min and
returned to initial conditions within 0.1 min, followed by
column re-equilibration for 9.9 min before the next run. The
sample injection volume was 1–5 μL.
The ESI-Q-TOF instrument was operated in the positive
ionization mode (ESI+) with a drying gas temperature of
325 °C, 20 psig nebulizer pressure, 4000 V capillary voltage,
and 75 V fragmentor voltage. Fragmentation of protonated
ions was conducted using the targeted MS/MS mode with a
collision energy of 10 V (16 V for m/z 76). Spectra were
recorded over the mass range of m/z 50–1000 for MS mode
and m/z 20–1000 for MS/MS mode. Data analysis was
performed using the qualitative data analysis software (Rev. B.
Results and discussion
Identification of amino acid products in the hydroxyl
radical-induced oxidation of peptides and proteins
Figure 1 shows the tripeptides and proteins investigated in this
study. The oxidation products generated by OH radicals from
the Fenton reaction were analyzed by AAA and LC-MS/MS
in order to identify and quantify amino compounds and, in
particular, amino acid products.
Figure 2 shows the exemplary AAA chromatograms of an
amino acid standard, as well as protein and peptide samples
oxidized by OH radicals. The signal corresponding to
glycineOPA derivative at a retention time (RT) of 7.8 min was
detected in all oxidized samples of glycine-containing peptides and
proteins. Moreover, the peak was absent when the oxidized
peptide did not contain glycine (i.e., Met-Ala-Ser). LC-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. Figure 3 shows
the MS/MS spectra of a glycine standard (m/z 76) and those of
precursor ions with m/z 76 found in oxidized BSA, (Gly)3, and
Ala-Met-Gly samples. In all cases, identical fragmentation
patterns were observed and the loss of 16 Da from the
precursor ions corresponds to the loss of NH2 . In addition, the
signal intensity of extracted ion chromatograms (EICs) for m/z
76 in the oxidized samples increased significantly compared
to the control samples (see ESM Fig. S2), indicating the
formation of an OH-mediated reaction product with m/z 76 in
these samples. Thus, glycine, which does not contain an
oxidation sensitive side chain, could be identified as a product of
all studied reaction systems of peptides and proteins
comprising glycine in their amino acid sequences.
In addition to glycine, three other peaks exhibiting the RT
of OPA derivatives of aspartic acid (Asp), asparagine (Asn),
and Ala were detected in the AAA of oxidized protein (BSA
and OVA) samples, i.e., at 2.1 min for Asp, 6.4 min for Asn,
Fig. 2 Amino acid analysis (AAA) with fluorescence detection of OPA-derivatized amino acids: (A) 200 μM of a 20-amino acid standard; (B) 15 μM
BSA, Ox2, 24 h; (C) 4 mM tri-Gly, Ox1, 0.25 h; and (D) 4 mM Ala-Met-Gly, Ox1, 19 h. The dotted box indicates the signal of glycine in all samples
and 9.2 min for Ala, as illustrated in Fig. 2B. The LC-MS/MS
analysis of reference compounds and samples confirmed the
identity of the amino acids as shown in Fig. S3 in ESM [34,
38]. It should be noted that the four free amino acids (Asp,
Asn, Gly, and Ala) identified in oxidized protein samples, all
exhibit a low rate constant for reactions with OH [19, 39],
resulting in a higher stability towards further reactions with
OH radicals and enabling their identification in the analysis.
Fig. 3 The MS2 spectra of m/z 76
in (A) 1 mM Gly, (B) oxidized
BSA sample in Ox2 condition,
(C) (Gly)3 in Ox1 condition, and
(D) Ala-Met-Gly in Ox1
condition (Ox1, 5 mM FeSO4–
50 mM H2O2; Ox2, 5 mM
FeSO4–150 mM H2O2). The
oxidized samples show an
accurate mass of precursor ion
m/z 76 with the glycine standard,
and they exhibit the same
fragments of m/z 60. The
extracted ion chromatograms
(EICs) of m/z 76 for the above
samples are shown in Fig. S2 in
Furthermore, Ala and Asp were unambiguously identified by
LC-MS/MS in the oxidized Met-Gly-Ala and Gly-Ala-Met
samples. Exemplary MS2 spectra of reference standards and
samples are shown in Fig. S4 in ESM. The presence of Asp in
the tripeptide samples can be explained by the OH-induced
oxidative modification of methionine (Met), as suggested by
Xu and Chance  and illustrated in Fig. S5 in ESM. Note
that Asp was not identified in the oxidized Ala-Met-Gly
sample. This discrepancy may be explained by the formation of
other oxidation products of Met, which can be formed when
Met is located in the middle of the peptide, as Met is highly
reactive towards OH and the reaction could result in different
oxidized species . In the oxidized Met-Ala-Ser sample, the
amino acids Asp, Ala, and Ser were identified. Here, Ser could
be released directly from the C-terminal position or it could be
formed by the oxidation of the methyl side chain of Ala
released from the peptide . Therefore, from the combined
AAA and LC-MS/MS results, we can confirm that free amino
acids are products in the OH-induced oxidation of proteins
Quantification and site selectivity of amino acid formation
Figure 4 shows the molar yields of free amino acids for the
OH oxidation of two model proteins (BSA and OVA)
quantified by AAA, whereby yields increased with increasing
oxidant concentrations. The yields of Gly were found to be the
highest among the quantified amino acids and ranged from
∼32 to 55% for BSA and from ∼10 to 21% for OVA.
Notably, the Gly yield of BSA was approximately two to three
times higher than that of OVA under the same conditions,
despite the higher number of Gly residues in OVA (19)
compared to BSA (17). The factors influencing the yields of
individual free amino acids in the studied reactions might be
multiple, including different tertiary and primary structures and
thus different numbers of accessible sites available for the
OH attack, as well as differences in adjacent amino acids in
BSA and OVA, influencing OH site selectivity .
Fig. 4 Molar yields of amino acids obtained in the oxidation of BSA and
OVA samples with different concentrations of oxidants (50 and 150 mM
H2O2 with 5 mM FeSO4, respectively)
Figure 5 shows the temporal evolution of the Gly yield
during the oxidation of (Gly)3 by OH radicals. The
corresponding recovery of (Gly)3 (see ESM Fig. S6) was
obtained through AAA analysis using a calibration curve
made by a set of (Gly)3 solutions (see ESM Fig. S7). We
found that the recovery of (Gly)3 has declined to 50%
after 1 h of reaction (see ESM Fig. S6), while the molar
yield of glycine only reached 6% of (Gly)3. Additionally,
t h e m o l a r r a t i o o f f r e e G l y t o r e a c t e d ( G l y ) 3
(Δ(Gly)3 = (Gly)3, t = 0 − (Gly)3, t = x) was relatively stable
over the reaction time with a value of ∼12%. These
results indicate that other reaction products than Gly are
accounting for ∼88% of the reacted peptide. These
products may include, e.g., carbonyl species known to be
products of the α-carbon H abstraction pathway .
To exclude an influence of acidic or basic hydrolysis on
the observed formation of glycine , control
experiments were conducted, in which (Gly)3 was incubated
under acidic (pH 2) and basic (pH 12) conditions for
24 h, respectively. No glycine formation was observed
in these experiments. Furthermore, we found that amino
acids were also released in the absence of iron ions. This
was confirmed through control experiment, in which OH
radicals were generated by the photolysis of H2O2, and a
positive relationship between glycine yield and H2O2
concentrations was observed (see ESM Fig. S8).
Furthermore, we found the amino acid yields of three
small peptides (Ala-Met-Gly, Met-Gly-Ala, and
Gly-AlaMet) are dependent on the sequence of Gly, Ala, and Met,
as shown in Fig. 6. The highest yields of Gly and Ala
were obtained when they were located at the C-terminus,
followed by the mid-chain position and the N-terminal
site. While the Gly concentration was increasing with
reaction time, the Ala concentration already showed a
reduction after 2 h of reaction time when located at the
C-terminal site (Met-Gly-Ala), which may be due to
further oxidation of free Ala by OH radicals. Besides,
comparing the results in the case of Gly and Ala both located
in the same position of the respective tripeptide, the yield
of Gly was about 50% higher than that of Ala when they
are located C-terminally. For mid-chain and N-terminal
sites, their yields were more comparable. These results
suggest that the OH attack for the release of free amino
acids preferably occurs at Gly, particularly for Gly
located at the C-terminal site and, to a less extent, at Ala.
Previous studies have suggested that OH-mediated
fragmentation of proteins likely occur at specific sites rather
than giving rise to random fragments [23, 28, 42].
Glycine residues could be favorable sites for OH
attacking the polypeptide backbone due to its low steric
hindrance . It should be noted that the highest molar
yield of Gly was found to be ∼2% of the corresponding
tripeptide (Ala-Met-Gly), confirming free amino acids to
Fig. 5 The temporal evolution of molar yield Gly/(Gly)3 (blue dots) and
the product ratio of Gly to Δ(Gly)3 (red dots) in the oxidation of 4 mM
(Gly)3 with 5 mM FeSO4–50 mM H2O2 condition (Ox1). Δ(Gly)3 was
quantified by a calibration curve made by a set of (Gly)3 solutions (see
ESM Fig. S7) monitored at a UV absorbance of 338 nm. The solid line
(blue) is fitted with a pseudo-first-order kinetic rate function:
[AA] = a[TriPep]0(1 − e− k[OH]t), as discussed in the BQuantification and
site selectivity of amino acid formation^ section
be low yield products and explaining the lack of reports
in the literature.
Aspartic acid, the OH oxidation product of Met, was found
in Met-Gly-Ala and Gly-Ala-Met. In contrast to the observed
increasing yield of Gly and Ala for the C-terminal site, the Asp
yields were found to be higher for the N-terminal site than for
the C-terminal site, i.e., 0.7% in Met-Gly-Ala and only 0.1%
in Gly-Ala-Met. The site selectivity for the OH attack at Gly
may also explain why the yield of Asp was higher for Met at
the N-terminal site than at the C-terminal site, since in
MetGly-Ala, the attack on Gly may lead to the formation of Met or
its oxidized product as a Bbyproduct^. Additionally, the
temporal evolution of release for amino acids in Figs. 5 and
6 can be fitted with a pseudo-first-order rate function:
[AA] = a[TriPep]0(1 − e− k[OH]t), where the coefficient a
stands for the maximum molar yield for the release of the
specific amino acid, k is the second-order rate coefficient, t
is the reaction time, and [AA], [TriPep]0, and [OH] are the
concentrations of amino acids, tripeptide (4 mM), and OH
(1.5 × 108 mol cm−3, assuming [OH] is constant), respectively.
The second-order rate coefficient for the release of amino
acids from the four investigated tripeptides is in the order of
magnitude of 10−12 cm3 s−1. The maximum molar yield for all
the amino acids was from 0.0014 ± 0.0018 to 0.0709 ± 0.0011,
with the highest found for Gly in (Gly)3 (0.0709 ± 0.0011); the
detailed coefficients from fittings can be found in Table S3 in
ESM. The kinetics and mechanism will be further investigated
in follow-up studies.
Fig. 6 Temporal evolution of the
concentration (left axis) and molar
yield (right axis) of glycine (A),
alanine (B), and aspartic acid (C)
from Gly-Ala-Met, Met-Gly-Ala,
and Ala-Met-Gly subjected to the
oxidation with 5 mM FeSO4–
50 mM H2O2 (Ox1). The solid
lines are fitted with a
pseudo-firstorder kinetic rate function:
[AA] = a[TriPep]0[1 − e− k[OH]t],
as discussed in the
BQuantification and site
selectivity of amino acid
formation^ section. For the fitting
for Ala in Ala-Met-Gly, it is only
fitted for the first two data points
Free amino acids were identified as products in the
OHinduced oxidation of proteins and peptides by LC-MS/MS
analysis. In addition, the molar yields of the formation of
amino acids were quantified by AAA analysis. Glycine was
released at higher yields than the other identified amino acids,
which is likely to be due to the absence of a side chain
resulting in low rate constants for further reactions with OH
and low steric hindrance of the initial radical generation on the
peptide backbone, especially when Gly was in the C-terminal
position. Note that the molar yields and production rates of
amino acids for different peptides and proteins cannot be
interchangeably used, as release of amino acids is not equal to
their presence in the solution due to possible side chain
oxidations of amino acids.
The formation of free amino acids, however, has not been
reported for the main backbone cleavage process through
αcarbon H abstraction, which results in the formation of amide
and carbonyl products, as outlined in the BIntroduction^
section. Thus, another reaction pathway may be responsible for
the formation of free amino acids. The peptide which was only
composed of glycine ((Gly)3) appears to be a good candidate
for the investigation of such pathways, because H abstraction
by OH radicals can only occur at the α-carbon and the amide
nitrogen. For other amino acids, however, hydroxyl radicals
can attack at the side chain and polypeptide backbone sites,
complicating investigations of the reaction mechanism. In
previous studies, Štefanić et al.  determined that the amide
nitrogen is the preferred site for OH attack through pulse
radiolysis on free glycine and a glycine anion, whereas Doan
et al.  concluded that H abstraction from the peptide
nitrogen atom is the least preferred site for OH attack at the peptide
backbone by ab initio calculations. The key difference for the
contradiction in the above two studies is that the former
investigated isolated amino acids while the latter used peptide
systems for their calculation methods. Also, the electron transfer
between sites resulting in secondary fragmentation or
rearrangement [14, 44], should be considered for the formation
of nitrogen-centered radicals. Further verification of the
generation of nitrogen-centered radicals and the investigation of
their role for the release of amino acids via protein/peptide
oxidation by hydroxyl radicals could be obtained by
techniques such as electron paramagnetic resonance (EPR)
spectroscopy in follow-up studies [45, 46].
Acknowledgements Open access funding provided by Max Planck
Society. F.L. and S.L. acknowledge the financial support from the
China Scholarship Council (CSC), and C.J.K. acknowledges the support
by the Max Planck Graduate Center (MPGC) with the Johannes
Gutenberg University Mainz and the financial support by the German
Research Foundation (DFG, grant no. KA4008/1-2).
Compliance with ethical standards
Ethical approval This article does not contain any research with
human participants or animals.
All authors of this manuscript were informed and agreed for
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