Boron-doped diamond electrodes for the electrochemical oxidation and cleavage of peptides

Article abstract of DOI:10.1021/ac303795c

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

Full text of DOI:10.1021/ac303795c

Article
pubs.acs.org/ac
Boron-Doped Diamond Electrodes for the Electrochemical Oxidation
and Cleavage of Peptides
Julien Roeser, Niels F. A. Alting, Hjalmar P. Permentier, Andries P. Bruins, and Rainer Bischoff*
Analytical Biochemistry and Mass Spectrometry Core Facility, Department of Pharmacy, University of Groningen, A. Deusinglaan 1,
9713 AV Groningen, The Netherlands
S
* Supporting Information
ABSTRACT: Electrochemical oxidation of peptides and proteins is traditionally
performed on carbon-based electrodes. Adsorption caused by the affinity of
hydrophobic and aromatic amino acids toward these surfaces leads to electrode
fouling. We compared the performance of boron-doped diamond (BDD) and glassy
carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An
optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on
BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals
took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to
be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of
small peptides, some of which had been previously shown to undergo electrochemical
cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon
electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower
conversion yields due to a change in surface termination. Cathodic pretreatment of
BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable
reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced
adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The
fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as
they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in
protein and peptide chemistry and analytics.
arbon-based electrodes are widely used in electro-
chemistry and are often preferred to noble metal
BDD electrodes have become attractive materials for
electrochemical applications in place of glassy carbon or
graphite due to their wider potential window, low background
currents, higher chemical inertness, high thermal conductivity,
and high mechanical stability. The high overpotential for both
oxygen and hydrogen evolution are responsible for the wide
potential window (the widest so far measured in aqueous
electrolytes) and allows conversion of molecules with high
oxidation and reduction potentials. Chemical inertness and low
background currents stem from the C-sp3 hybridization of
diamond which has a low capacitance and prevents surface
oxide formation.1 Owing to these properties, BDD is a material
well-known for its low adsorption, resistance to (bio)fouling,
excellent response stability, and high current signal-to-noise
ratio which made it popular for a wide range of electroanalytical
applications for both inorganic and organic compounds.4,8
Electrochemical properties and performance of BDD electro-
des depend on several factors such as doping levels,
nondiamond impurities (C-sp2 carbon) and surface termination
(hydrogen or oxygen). The latter has a great influence on
charge transfer rates at the electrode surface which decrease
C
electrodes for oxidation of organic and biological molecules.1
The main benefits of such materials are their electrocatalytic
activity for a variety of redox reactions, their wide potential
window, low cost, and the possibility of preparing porous
materials with large surface areas (porous graphite carbon).
Graphite and glassy carbon electrodes are, however, also known
to suffer from surface fouling and from oxide formation by
reaction with oxygen and water. These surface reactions can
have a significant effect on adsorption, electron-transfer kinetics
and electrocatalysis.2
The introduction of diamond-based materials as electrodes in
the late 1980s by Pleskov et al.3 was a major advance in
electrochemistry. Natural diamond is an electrically insulating
material that cannot be used as an electrode but the
introduction of impurities within the sp3-hybridized tetrahedral
lattice of diamond makes it conductive. Boron is by far the most
widely used dopant and yields p-type semiconductors or
materials with metal-like electronic properties depending on the
doping levels.4 Boron-doped diamond (BDD) thin-films are
mostly prepared by chemical vapor deposition (CVD) on
silicon or metallic substrates under a hydrogen atmosphere
yielding H-terminated surfaces. Preparation techniques, char-
acterization, surface modifications, and electrochemical proper-
ties of BDD electrodes have been reviewed elsewhere.1,2,4−8
Received: December 30, 2012
Accepted: June 12, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
upon the gradual replacement of superficial hydrogen by
oxygen-terminated sites during repeated anodic oxidation or
prolonged exposure to ambient air.9,10 BDD electrode
pretreatment methods at high anodic or cathodic potentials
in an acidic environment were shown to allow tuning of surface
termination states in order to study reaction kinetics.10,11 Both
anodic and cathodic pretreatment may improve the voltam-
metric response of analytes (ref 11 and references therein).
Cathodic pretreatment can for instance be used for the
regeneration of an electrode surface that was passivated due to
analyte adsorption on an O-terminated surface, as has been
shown in the context of the electrochemical oxidation of
proteins.12
Anodic hydroxyl radical production by oxidation of water can
occur very effectively on BDD electrodes due to the high
overpotential for oxygen evolution. Hydroxyl radical generation
at BDD electrodes is very specific for this material and is widely
used for water treatment and destruction of organic or
inorganic pollutants, which is by far the most investigated
application area for BDD electrodes. Formation of hydroxyl
radicals and even methoxy radicals has also been applied to
other fields such as protein footprinting13 and organic
synthesis.14−16
Electrochemical oxidation of peptides and proteins has been
shown to yield specific cleavage of the peptide bonds C-
terminal to Tyr and Trp residues and holds promise to become
an instrumental alternative to chemical and enzymatic protein
cleavage.17−19 Electrochemical cleavage has so far mainly been
carried out on purely carbon-based materials. Adsorption and
fouling of carbon electrodes are aggravating issues when
working with large (bio)molecules and impair repeatability and
reproducibility, or even prevent oxidation to occur at all.18
Moreover, Tyr dimer formation by cross-linking reactions20,21
occurs on carbon-based materials22 due to strong affinity and
adsorption of the phenolic ring to the electrode surface.23 Such
drawbacks contribute to the limited cleavage yields. BDD is
thus a promising electrode material for electrochemical
oxidation of peptides and proteins, owing to its limited
adsorption. Studies involving a single amino acid or amino
acid mixtures have shown improved performance for electro-
chemical detection in terms of lower adsorption and improved
electrode stability, although in some cases fouling and electrode
passivation have been observed when working with concen-
trated solutions in the millimolar range.24−30 BDD electrodes
have also been investigated for the electrochemical detection of
peptides and proteins (both metallo- and nonmetal-containing
proteins)12,31−38 and for studies on resistance to protein
fouling.39−42
We report here the first comparison and evaluation of the
performance of BDD versus GC electrodes for the electro-
chemical oxidation and cleavage of peptides. Products were
monitored by LC−MS which provided detailed information
about the reactions occurring at the electrode surfaces. The
potential regions of the two different oxidation mechanisms
occurring on BDD electrodes, i.e. direct electron transfer
processes and hydroxyl radical formation, were investigated as
well as methods to regenerate the electrode surface after
performance loss upon prolonged oxidation experiments.
(ACTH) 1−10 (SYSMEHFRWG) and formic acid (HCOOH)
were purchased form Sigma-Aldrich (Steinheim, Germany).
Water was purified by an Arium Ultrapure water system
(conductivity 18.2 MΩ.cm, Sartorius Stedim Biotech, Gottin-
̈
gen, Germany). HPLC supra gradient acetonitrile was
purchased from Merck (Darmstadt, Germany).
Electrochemical Oxidation of Peptides. Stock solutions
of LYL, LWL, LFL, angiotensin I, and ACTH 1-10 were
prepared at a concentration of 1 mM in 90/10/1 (v/v/v)
ultrapure water/acetonitrile/formic acid and diluted to a final
concentration of 5 μM prior to oxidation.
The tripeptide solutions were oxidized with a Flexcell thin-
layer cell (Antec Leyden, Leiden, The Netherlands) with Magic
Diamond (BDD) and glassy carbon (GC) working electrodes
(8 mm diameter, surface area of 50.3 mm2), and a palladium
(Pd/H2) reference electrode (Hy-REF). Cathodic pretreatment
of BDD electrodes was performed prior to all experiments at a
constant potential of −3000 mV for 1 h in the presence of 0.5
M sulfuric acid in water at a flow rate of 5 μL/min. Prior to use,
BDD electrodes were washed with methanol and ultrapure
water. GC electrodes were polished using diamond spray (1 μm
particles) and subsequently rinsed with ultrapure water.
The potentials were controlled with a homemade potentio-
stat controlled by a MacLab system (ADInstruments, Castle
Hill, NSW, Australia) and EChem software (eDAQ, Denistone
East, NSW, Australia). The anodic currents obtained in the
course of the experiments are reported in Table S3 in the
Supporting Information (SI).
Cyclic Voltammetry. Cyclic voltammograms (CVs) were
recorded with the thin-layer cell (Flexcell, Antec Leyden), with
either a BDD or GC working electrode. The GC electrode was
cleaned by polishing with diamond slurry according to the
manufacturer’s protocol. The BDD electrode was cleaned by
cathodic pretreatment for 1 h at −3000 mV with 0.5 M
sulphuric acid in water at a flow rate of 5 μL/min.
Samples with or without peptide in H2O/CH3CN/FA
89:10:1 were infused at 5 μL/min. Peptide concentration was
1 mM. The potentials were controlled with a homemade
potentiostat controlled by a MacLab system (ADInstruments,
Castle Hill, NSW, Australia) and EChem software (eDAQ,
Denistone East, NSW, Australia). Ten consecutive CVs were
recorded at a scan rate of 100 mV/s. For GC the potential
range was from 0 to 2000 mV, for BDD from 0 to 3500 mV.
Liquid Chromatography−Mass Spectrometry (LC−
MS). Liquid chromatography was performed on an Ultimate
plus system (Dionex-LC Packings, Amsterdam, The Nether-
lands) equipped with an Ultimate gradient pump and Famos
well plate Microautosampler. A Vydac RP-C18 column (150
mm × 1 mm i.d., 5 μm particles, 300 Å pore size, Grace Vydac)
was used for chromatographic separation at a flow rate of 50
μL/min. Mobile phase A consisted of ultrapure water with 0.1%
formic acid. Mobile phase B was acetonitrile with 0.1% formic
acid.
For analysis of the peptide-derived reaction products, 50 μL
injections of the collected oxidized solutions were performed,
and separation was achieved with a gradient of B (5−50% at
1%/min). The column was directly coupled to an API365 triple
quadrupole mass spectrometer (AB-Sciex, Concord, Ontario,
Canada) upgraded to EP10+ (Ionics, Bolton, Ontario, Canada)
and equipped with a TurboIonSpray source for product
detection in the positive ion mode.
EXPERIMENTAL SECTION
■
Chemicals. The tripeptides LYL, LWL and LFL were
obtained from Research Plus Inc. (Barnegat, NJ, USA).
Angiotensin I (DRVYIHPFHL), Adrenocorticotropic hormone
B
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
a
Scheme 1. Hydroxyl-Radical-Induced Oxidation of the Tripeptide LFL on BDD electrodes
a
Para-hydroxylation of LFL yields the tripeptide LYL which can undergo further direct electron transfer reactions including cleavage of the peptide
bond C-terminal to Tyr.
RESULTS AND DISCUSSION
■
Direct Oxidation vs Electrochemical Hydroxyl Radical
Formation. The high overpotential for oxygen evolution on
BDD electrodes allows efficient anodic oxidation of water to
hydroxyl radicals. Generation of hydroxyl radicals on BDD
electrodes takes place at elevated potentials which can vary
from one electrode to another, depending on the physical,
chemical, and electronic properties of the material as was
observed when comparing BDD electrodes from different
manufacturers. Experiments performed with a similar thin-layer
cell (5040 cell, ESA Inc., Bedford, MA, U.S.A.) showed that less
positive potentials were required to generate hydroxyl radicals
on a BDD electrode compared to the Antec thin-layer cells
used in the present study. We presume that the difference of
potential between the two systems stems from the different
doping levels of the BDD electrodes (the latter information is
not known to us).
The tripeptide LFL (1) (Scheme 1) was used to determine
Figure 1. LC−MS chromatograms of reaction products obtained after
oxidation of 5 μM LFL in H2O/CH3CN/HCOOH (89:10:1) at a flow
rate of 5 μL/min on a BDD electrode. Base-peak chromatograms
(BPC) obtained after oxidation at (A) 2000 mV and (B) 2500 mV.
(C) Extracted ion chromatograms (XICs) of the oxidation products
obtained at 2500 mV.
the potential limit at which hydroxyl radicals are generated in
our system, since the aromatic ring of phenylalanine (Phe)
cannot undergo direct oxidation by electron transfer reactions
but is also a target for hydroxylation by reactive oxygen
species.5 Parts A and B of Figure 1 compare the base peak
chromatograms obtained after oxidation of LFL (1) at a BDD
electrode at 2000 mV and 2500 mV vs Pd/H2. The formation
of hydroxyl radicals by oxidation of water occurred at 2500 mV
as indicated by the presence of LFL oxidation products eluting
prior to the unoxidized tripeptide in LC−MS (Figure 1B). The
combined extracted ion chromatograms in Figure 1C illustrate
in more detail the range of products obtained for LFL (1) at
2500 mV. The presence of products with a mass increase of 16
Da (m/z 408) compared to LFL (m/z 392) indicates the
formation of hydroxylated LFL that occurs via hydroxyl radical
attack. Despite their broad range of reactivity, hydroxyl radicals
preferably target oxidation-sensitive amino acid side chains (i.e.,
Cys, Met, Trp, Tyr, Phe, and His) within peptides and
proteins.5,43 The presence of three peaks with a mass increment
of 16 Da suggests that oxidation in the ortho-, meta-, and para-
positions of the aromatic ring occurred, yielding hydroxylated
LFL (2), (3), and (4), respectively (Scheme 1). The high peak
intensities of LFL+32 Da products, also eluting as three
chromatographic peaks, show that a second hydroxylation step
is favored under these conditions. This secondary oxidation
step can occur via two different mechanisms: a second hydroxyl
radical attack or direct electrochemical oxidation of hydroxy-
lated LFL. Indeed, hydroxylation of Phe in the para-position
leads to the formation of Tyr and in this case the formation of
the tripeptide LYL (4), which can react further by direct
electron transfer reactions. As described in previous work19 and
shown in Scheme 1, LYL (4) can undergo dehydrogenation,
hydroxylation, or a combination of those reactions yielding
products with a mass decrease of 2 Da or a mass increase of 16
and 14 Da, respectively. These products are all observed in
Figure 1C and are labeled LFL+14, LFL+32, and LFL+30,
respectively. Finally, cleavage of the peptide bond next to Tyr
by direct electrochemical oxidation followed by a secondary
hydrolysis step19 yielding the dipeptide LY-2 (5) occurs as well
as shown by the presence of the early eluting m/z 293 product
(Figure 1C).
Oxidation products of LFL were absent at 2000 mV (Figure
1A). Therefore, anodic oxidation of water leading to the
C
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
formation of hydroxyl radicals did not occur at 2000 mV,
making this potential suitable when only direct electrochemical
oxidation reactions are desired, such as the electrochemical
peptide cleavage C-terminal to Tyr or Trp.
low μL/min range. Oxidation of LYL started at 1000 mV vs
Pd/H2 but with a low oxidation yield. Maximal conversion of
30−40% was reached at a potential of 1500 mV (Figure 3A)
Cathodic Pretreatment of BDD Electrodes. The surface
termination of BDD affects the electrochemical behavior of the
electrode. Charge transfer is enhanced when working with
hydrogen-terminated surfaces. Oxygen-terminated sites are
formed during oxidation at positive potential in the presence
of oxygen. Reversion to an H-terminated surface is possible by
cathodic pretreatment at high negative potentials in an acidic
environment.10
Deterioration of BDD-electrode performance was observed
after repeated peptide oxidation over a period of several days by
a progressive decrease of oxidation yields to 20−30%. Figure 2
Figure 2. Influence of cathodic pretreatment on the performance of a
BDD electrode for the oxidation of 5 μM LYL in H2O/CH3CN/
HCOOH (89:10:1) at a flow rate of 5 μL/min at 2000 mV. Base peak
chromatograms of the reaction products (A) prior to and (B) after
cathodic pretreatment at a constant potential of −3000 mV for 1 h in
the presence of 0.5 M sulfuric acid.
Figure 3. LC−MS chromatograms of reaction products obtained after
electrochemical oxidation of 5 μM LYL in H2O/CH3CN/HCOOH
(89:10:1) at a flow rate of 5 μL/min. (A) Base peak chromatogram
and (B) combined extracted ion chromatograms (XICs) of the main
oxidation and cleavage products obtained on a GC electrode at 1500
mV. Base peak chromatogram obtained on a BDD electrode at (C)
2000 mV and (D) 3500 mV. Note that there is no cleavage at 3500
mV.
illustrates the conversion of the tripeptide LYL prior to and
after 1 h of cathodic pretreatment. Electrode pretreatment was
performed by applying a constant potential of −3000 mV in the
presence of 0.5 M sulfuric acid and resulted in oxidation yields
of 60−70%, which are comparable to the values observed
originally. This is attributed to the replacement of O-terminated
by H-terminated sites. Cathodic pretreatment has to be carried
out regularly in order to ensure reliable and reproducible results
for the present set of peptides. No significant drop in
performance was observed after one day of oxidation of either
LYL or blank solution (Figure S2 in SI) indicating that peptide
adsorption is very low and that daily regeneration the electrode
is sufficient for reproducible results.
Peptide Cleavage on a Thin-Layer GC Electrode. For
comparison with the BDD electrode, electrochemical oxidation
was performed with a GC electrode of identical size (8 mm
diameter). The tripeptide LYL was used to determine the
optimal potential for maximum conversion by ramping the
oxidation potential in 100 mV increments and following
product distribution by LC−MS analysis. All experiments were
performed at 5 μM peptide concentration with flow rates in the
which preceded a gradual decrease of peptide signal when
increasing the potential above 1500 mV. Cyclic voltammetry
experiments (Figure S1 of SI) confirmed the onset of LYL
oxidation at 1000 mV (Figure S1, panel C in SI) and revealed
that water oxidation was initiated at 1500 mV (Figure S1, panel
A in SI) which is in agreement with the optimal potential
deduced from LC−MS experiments. The range of oxidation
and cleavage products detected on the GC electrode in a thin-
layer cell is detailed in Figure 3B and is in accordance with the
range of products obtained in previous work performed with
porous graphite electrodes (ESA 5021 cell).19 However, the
amount of products was much higher in the coulometric ESA
cell due to the much larger surface area of the porous graphite
electrode (more than 50 times the area of the flat GC
D
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
electrode). Dimers formed by Tyr cross-linking reactions are
the most abundant oxidation products of the tripeptide LYL on
GC, suggesting strong adsorption of the peptide to the working
electrode surface. Dimerization of and cross-linking within Tyr-
containing peptides and proteins44,45 readily occurs at the
surface of carbon electrodes due to strong adsorption of the
phenolic ring.23 This constitutes a major drawback of GC
electrodes, since dimers are formed at the expense of other
oxidation products, notably cleavage products.
peptide at the electrode surface. Angiotensin I was successfully
oxidized at the BDD electrode in the thin layer cell with a
conversion yield of 20−30% (Figure 4A), which was lower than
Peptide Cleavage on a Thin-Layer BDD Electrode.
Since formation of hydroxyl radicals on the BDD electrode was
observed at a potential of 2500 mV, we choose a potential of
2000 mV as the upper limit for evaluating BDD for the
electrochemical oxidation and cleavage of peptides by direct
electron transfer.
Hydrodynamic voltammetry performed by ramping the
potential in 100 mV increments and following the product
distribution by LC−MS showed that oxidation of LYL on BDD
was initiated at 1300 mV with a conversion of around 5% and
no detectable cleavage products. Both oxidation and cleavage
yields increased with higher potentials, and maximum
conversion of 50% was obtained at 2000 mV. Cyclic
voltammetry experiments performed on BDD (Figure S1 in
SI) confirmed the results obtained by LC−MS, i.e. onset of
peptide oxidation at 1300 mV (Figure S1, panel D in SI) and
water oxidation at potentials greater than 2000 mV (Figure S1,
panel B in SI). The cleavage and noncleavage oxidation
products are presented in Figure 3C and correspond closely to
those obtained on a porous graphite electrode as previously
described.17,19 Since complete conversion can be reached with
peptide concentrations of up to 50 μM on the porous graphite
electrode, BDD electrodes with larger surface areas are needed
to reach similar conversion levels.
Two main differences were observed between BDD and the
pure carbon electrodes. First, there is no dimer formation on
BDD while this is a significant reaction pathway when working
with carbon electrodes as reported above for the oxidation of
LYL with a GC disk electrode and in previous work with a
porous graphite electrode in a coulometric cell.19 Dimer
formation likely occurs on the electrode surface due to the high
local concentration of adsorbed LYL. The observation that LYL
does not dimerize even at high positive potentials indicates that
adsorption to BDD electrodes is much lower. We therefore
used the extent of Tyr-mediated dimer formation as an indirect
measure of peptide adsorption. Second, when potentials greater
than 2000 mV were applied, a progressive decrease of the
cleavage yield with a concomitant increase of the M+16
hydroxylated products was observed (see Figure 3D for the
reaction products observed at 3500 mV). This observation
confirms the oxidation pathway by reaction with hydroxyl
radicals at very positive potentials on the BDD electrode and
shows that this goes at the expense of cleavage product
formation.
Figure 4. LC−MS chromatograms of reaction products obtained after
oxidation of 5 μM angiotensin I (DRVYIHPFHL) in H2O/CH3CN/
HCOOH (89:10:1) at a flow rate of 5 μL/min at (A) 2000 mV on
BDD and (C) 1500 mV on GC electrodes. (B) Extracted ion
chromatograms (XICs) of the main oxidation and cleavage products
obtained on the BDD electrode at 2000 mV.
for LYL. Lower conversion yields for larger peptides may be
explained by their lower diffusion rates. While oxidation yielded
mainly noncleavage products, listed as A1−2, A1+14, A1+16 in
panels A and B of Figure 4, the cleavage products DRVY-2 and
IHPFHL were clearly detectable (Figure 4B). When the
performance of the BDD and the GC electrodes in the thin-
layer cell was compared with respect to the conversion of
angiotensin I (Figure 4C), a significantly lower yield was
observed with the GC electrode. This lower efficiency may
again be explained by adsorption on the surface of the GC
electrode leading to a low recovery of oxidation products.
Dimer formation was, however, not detected, possibly due to
the low oxidation yields or to adsorption of the dimer on the
electrode surface.
The second peptide we evaluated was ACTH 1-10
(SYSMEHFRWG). This peptide has been tested earlier with
porous graphite electrode-containing coulometric cells, but no
products were detected, presumably due to very strong
adsorption on the porous graphite electrode.18 It was thus of
interest to determine whether lower adsorption on the BDD
electrode would allow identification of oxidation or cleavage
products. Another interesting feature of this peptide is that it
contains three residues, i.e. Tyr, Met, and Trp, that can be
readily oxidized electrochemically by direct electron transfer
reactions. This peptide is therefore useful to determine which
oxidation pathways occur preferentially, and whether cleavage
will occur next to both Tyr and Trp under the same conditions.
Oxidation of ACTH 1-10 at 2000 mV at the BDD working
electrode showed noncleavage oxidation products (Figure 5A)
containing the expected ACTH-2, ACTH+14, ACTH+16,
ACTH+32, ACTH+48 in analogy to those obtained with LYL
and LWL.19 In addition, two out of the three possible cleavage
products were detected as shown in Figure 5B, namely the C-
terminal peptide formed by cleavage next to Tyr
Electrochemical Oxidation of Larger Peptides. Two
decapeptides, angiotensin I and adrenocorticotropic hormone
(ACTH) 1-10, were analyzed to compare BDD and GC
electrodes with larger peptides in view of the adsorption issues
encountered previously on porous graphite electrodes.17,18
Angiotensin I (DRVYIHPFHL) contains a single Tyr residue
that is amenable to direct electrochemical oxidation. Earlier
experiments with porous graphite electrodes17 showed that it is
possible to cleave this peptide C-terminal to the Tyr residue but
with poor reproducibility, presumably due to adsorption of the
E
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
maximal conversion rates and to obtain reliable and
reproducible results even after repeated use of the electrode.
Investigation of the larger decapeptides angiotensin I and
ACTH 1-10 demonstrated the benefit of BDD in comparison
to GC in achieving cleavage of the peptide bond C-terminal to
Tyr and Trp.
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: +31 50 363 3338. Fax: +31 50 363 7582. E-mail: r.p.h.
bischoff@rug.nl.
Notes
Figure 5. LC−MS chromatograms of reaction products obtained after
oxidation of 5 μM ACTH 1-10 (SYSMEHFRWG) in H2O/CH3CN/
HCOOH (89:10:1) at a flow rate of 5 μL/min at (A) 2000 mV on
BDD and (C) 1500 mV on GC electrodes. (B) Extracted ion
chromatograms (XICs) of the cleavage products obtained on the BDD
electrode at 2000 mV.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Dutch Technology Foundation (STW) is gratefully
acknowledged for financial support (Grant 07047). Further
financial support was obtained from Astra Zeneca (Molndal,
̈
Sweden) and Organon, (Oss, The Netherlands; a former
subsidiary of Merck & Co. Inc., Whitehouse Station, NJ,
U.S.A.). Electrochemical cells were donated by ESA Inc.
(Bedford, MA, U.S.A.) and LC equipment by LC-Packings
(Amsterdam, The Netherlands; now both part of Thermo
Scientific, Sunnyvale, CA, U.S.A.).
(SMEHFRWG) and the N-terminal peptide formed by
cleavage next to Trp (SYSMEHFRW+14). The corresponding
small cleavage products (SY-2 and G) were not detected,
probably due to poor retention on the reversed-phase column
during LC−MS analysis. The higher intensity of the Trp
cleavage product suggests that this reaction is favored under
these conditions. Interestingly, the Trp cleavage product eluted
in two peaks, suggesting the formation of isomers (Figure 5B).
This has not been reported in our earlier study of Trp-
containing tripeptides (performed on porous carbon electro-
des).19 This might indicate the formation of conformational
isomers that can be detected due to the higher oxidation and
cleavage yields obtained on BDD. Conformational isomers have
also been observed after chemical labeling of Trp-containing
peptides after electrochemical cleavage.46 Modification of
oxidizable amino acids present in the cleavage products of
ACTH 1-10 did not always take place. S-oxidation of Met
within the N-terminal peptide SMEHFRW+14 was not
detected. On the other hand, oxidation within the C-terminal
peptide obtained after cleavage next to Tyr (SMEHFRWG+16)
was observed, most likely due to hydroxylation of Trp. The
thin-layer cell containing a GC electrode of identical
dimensions resulted in lower conversion and cleavage yields
possibly due to peptide adsorption as indicated by dark deposits
on the GC electrode (Figure 5C).
REFERENCES
■
(1) McCreery, R. L. Chem. Rev. 2008, 108, 2646−2687.
(2) Luong, J. H. T.; Male, K. B.; Glennon, J. B. Analyst 2009, 134,
1965−1979.
(3) Pleskov, Y. V.; Sakharova, A. Y.; Krotova, M. D. J. Electroanal.
Chem. 1987, 228, 19−27.
(4) Kraft, A. Int. J. Electrochem. Sci. 2007, 2, 355−385.
(5) Xu, G.; Chance, M. R. Chem. Rev. 2007, 107, 3514−3543.
(6) Pleskov, Y. V. Russ. J. Electrochem. 2002, 38, 1275−1291.
(7) Swain, G. M. In Electroanalytical Chemistry; Bard, A. J.,
Rubinstein, I., Eds.; Marcel Dekker: New York, 2004; Vol. 22.
(8) Peckova,
39, 148−172.
́ ́
K.; Musilova, J.; Barek, J. Crit. Rev. Anal. Chem. 2009,
(9) Granger, M. C.; Witek, M.; Xu, J.; Wang, J.; Hupert, M.; Hanks,
A.; Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek,
J. W.; Swain, G. M. Anal. Chem. 2000, 72, 3793−3804.
(10) Salazar-Banda, G. R.; Andrade, L. S.; Nascente, P. A. P.; Pizani,
P. S.; Rocha-Filho, R. C.; Avaca, L. A. Electrochim. Acta 2006, 51,
4612−4619.
(11) Suffredini, H. B.; Pedrosa, V. A.; Codognoto, L.; Machado, S. A.
S.; Rocha-Filho, R. C.; Avaca, L. A. Electrochim. Acta 2004, 49, 4021−
4026.
CONCLUSIONS
■
Boron-doped diamond (BDD) was investigated as electrode
material in comparison to the more commonly used glassy
carbon (GC) electrodes with respect to the electrochemical
oxidation and cleavage of peptides. Lower adsorption of
peptides on BDD was indicated by the absence of Tyr dimer
formation at the electrode surface, even at elevated potentials as
opposed to high amounts of Tyr dimers on GC. The maximal
potential on BDD was limited to 2000 mV vs Pd/H2 to prevent
hydroxyl radical formation occurring at 2500 mV vs Pd/H2.
Cathodic pretreatment of the BDD electrode was shown to be
effective in regenerating the electrode surface to ensure
(12) Chiku, M.; Ivandini, T. A.; Kamiya, A.; Fujishima, A.; Einaga, Y.
J. Electroanal. Chem. 2008, 612, 201−207.
(13) McClintock, C.; Kertesz, V.; Hettich, R. L. Anal. Chem. 2008,
80, 3304−3317.
(14) Sumi, T.; Saitoh, T.; Natsui, K.; Yamamoto, T.; Atobe, M.;
Einaga, Y.; Nishiyama, S. Angew. Chem., Int. Ed. 2012, 51, 5443−5446.
(15) Iniesta, J.; Michaud, P. A.; Panizza, M.; Comninellis, C.
Electrochem. Commun. 2001, 3, 346−351.
(16) Fardel, R.; Griesbach, U.; Putter, H.; Comninellis, C. J. Appl.
̈
Electrochem. 2006, 36, 249−253.
(17) Permentier, H. P.; Jurva, U.; Barroso, B.; Bruins, A. P. Rapid
Commun. Mass Spectrom. 2003, 17, 1585−1592.
F
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
Article
(18) Permentier, H. P.; Bruins, A. P. J. Am. Soc. Mass Spectrom 2004,
15, 1707−1716.
(19) Roeser, J.; Permentier, H. P.; Bruins, A. P.; Bischoff, R. Anal.
Chem. 2010, 82, 7556−7565.
(20) Eickhoff, H.; Jung, G.; Rieker, A. Tetrahedron 2001, 57, 353−
364.
(21) Giulivi, C.; Traaseth, N. J.; Davies, K. J. A. Amino Acids 2003,
25, 227−232.
(22) Zhang, M.; Mullens, C.; Gorski, W. Anal. Chem. 2005, 77,
6396−6401.
(23) Lund, H.; Hammerich, O. Organic Electrochemistry; Marcel
Dekker: New York, 2001.
(24) Foord, J. S.; Chatterjee, A. Phys. Status Solidi A 2005, 202,
2110−2115.
(25) Zhao, G.; Qi, Y.; Tian, Y. Electroanalysis 2006, 18, 830−834.
(26) Szunerits, S.; Coffinier, Y.; Galopin, E.; Brenner, J.;
Boukherroub, R. Electrochem. Commun. 2010, 12, 438−441.
(27) Spataru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A.
Anal. Chem. 2001, 73, 514−519.
(28) Terashima, C.; Rao, T. N.; Sarada, B. V.; Fujishima, A. Chem.
Lett. 2003, 32, 136−137.
(29) Terashima, C.; Rao, T. N.; Sarada, B. V.; Kubota, Y.; Fujishima,
A. Anal. Chem. 2003, 75, 1564−1572.
(30) Chailapakul, O.; Aksharanandana, P.; Frelink, T.; Einaga, Y.;
Fujishima, A. Sens. Actuators B 2001, 80, 193−201.
(31) Ivandini, T. A.; Sarada, B. V.; Terashima, C.; Rao, T. N.; Tryk,
D. A.; Ishiguro, H.; Kubota, Y.; Fujishima, A. J. Chromatogr., B 2003,
791, 63−72.
(32) Chiku, M.; Nakamura, J.; Fujishima, A.; Einaga, Y. Anal. Chem.
2008, 80, 5783−5787.
(33) Chiku, M.; Horisawa, K.; Doi, N.; Yanagawa, H.; Einaga, Y.
Biosens. Bioelectron. 2010, 26, 235−240.
(34) Iniesta, J.; Esclapez-Vicente, M. D.; Heptinstall, J.; Walton, D. J.;
Peterson, I. R.; Mikhailov, V. A.; Cooper, H. J. Enzyme Microb. Technol.
2010, 46, 472−478.
(35) Zhang, J. D.; Oyama, M. Microchem. J. 2004, 78, 217−222.
(36) McEvoy, J. P.; Foord, F. S. Electrochim. Acta 2005, 50, 2933−
2941.
(37) Marken, F.; Paddon, C. A.; Asogan, D. Electrochem. Commun.
2002, 4, 62−66.
(38) Haymond, S.; Babcock, G. T.; Swain, G. M. J. Am. Chem. Soc.
2002, 124, 10634−10635.
(39) Shin, D.; Tryk, D. A.; Fujishima, A.; Merkoci̧ , A.; Wang, J.
Electroanalysis 2005, 17, 305−311.
(40) Witek, M.; Swain, G. M. Anal. Chim. Acta 2001, 440, 119−129.
(41) Rao, T. N.; Yagi, I.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal.
Chem. 1999, 71, 2506−2511.
(42) Trouillon, R.; O’Hare, D. Electrochim. Acta 2010, 55, 6586−
6595.
(43) Stadtman, E. R.; Levine, R. L. Amino Acids 2003, 25, 207−218.
(44) Burzio, L. A.; Waite, J. H. Protein Sci. 2001, 10, 735−740.
(45) Michon, T.; Chenu, M.; Kellershon, N.; Desmadril, M.;
Gueguen, J. Biochemistry 1997, 36, 8504−8513.
(46) Roeser, J.; Alting, N. F. A.; Permentier, H. P.; Bruins, A. P.;
Bischoff, R. Rapid Commun. Mass Spectrom. 2013, 27, 546−552.
G
dx.doi.org/10.1021/ac303795c | Anal. Chem. XXXX, XXX, XXX−XXX