Analysis of protein-phenolic compound modifications using electrochemistry coupled to mass spectrometry

Article abstract of DOI:10.3390/molecules23020264

In the last decade, electrochemical oxidation coupled with mass spectrometry has been successfully used for the analysis of metabolic studies. The application focused in this study was to investigate the redox potential of different phenolic compounds such as the very prominent chlorogenic acid. Further, EC/ESI-MS was used as preparation technique for analyzing adduct formation between electrochemically oxidized phenolic compounds and food proteins, e.g., alpha-lactalbumin or peptides derived from a tryptic digestion. In the first step of this approach, two reactant solutions are combined and mixed: one contains the solution of the digested protein, and the other contains the phenolic compound of interest, which was, prior to the mixing process, electrochemically transformed to several oxidation products using a boron-doped diamond working electrode. As a result, a Michael-type addition led to covalent binding of the activated phenolic compounds to reactive protein/peptide side chains. In a follow-up approach, the reaction mix was further separated chromatographically and finally detected using ESI-HRMS. Compound-specific, electrochemical oxidation of phenolic acids was performed successfully, and various oxidation and reaction products with proteins/peptides were observed. Further optimization of the reaction (conditions) is required, as well as structural elucidation concerning the final adducts, which can be phenolic compound oligomers, but even more interestingly, quite complex mixtures of proteins and oxidation products.

Full text of DOI:10.3390/molecules23020264

molecules
Communication
Analysis of Protein-Phenolic Compound
Modifications Using Electrochemistry Coupled to
Mass Spectrometry
Constanze Kallinich, Simone Schefer and Sascha Rohn *
Institute of Food Chemistry, Hamburg School of Food Science, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany; constanze.kallinich@chemie.uni-hamburg.de (C.K.);
simone.schefer@studium.uni-hamburg.de (S.S.)
*
Correspondence: rohn@chemie.uni-hamburg.de; Tel.: +49-40-428-387-979
Received: 13 December 2017; Accepted: 24 January 2018; Published: 29 January 2018
Abstract: In the last decade, electrochemical oxidation coupled with mass spectrometry has
been successfully used for the analysis of metabolic studies. The application focused in this
study was to investigate the redox potential of different phenolic compounds such as the very
prominent chlorogenic acid. Further, EC/ESI-MS was used as preparation technique for analyzing
adduct formation between electrochemically oxidized phenolic compounds and food proteins,
e.g., alpha-lactalbumin or peptides derived from a tryptic digestion. In the first step of this approach,
two reactant solutions are combined and mixed: one contains the solution of the digested protein,
and the other contains the phenolic compound of interest, which was, prior to the mixing process,
electrochemically transformed to several oxidation products using a boron-doped diamond working
electrode. As a result, a Michael-type addition led to covalent binding of the activated phenolic
compounds to reactive protein/peptide side chains. In a follow-up approach, the reaction mix was
further separated chromatographically and finally detected using ESI-HRMS. Compound-specific,
electrochemical oxidation of phenolic acids was performed successfully, and various oxidation
and reaction products with proteins/peptides were observed. Further optimization of the reaction
(
conditions) is required, as well as structural elucidation concerning the final adducts, which can be
phenolic compound oligomers, but even more interestingly, quite complex mixtures of proteins and
oxidation products.
Keywords: adduct formation; protein interaction; electrochemical oxidation; phenolic compounds
1
. Introduction
Despite many years of research on protein-phenolic compound interactions, predictions of protein
adduct formation between various food ingredients are still challenging and not yet satisfyingly
solved [ ]. Proteins provide reactive side chains that might act as nucleophiles for a Michael-type
1
reaction. Here, especially amino, indole, and thiol groups seem to be pre-requisite reaction sites.
Especially covalently bound phenolic compounds might significantly affect physicochemical protein
properties such as the hydrophobicity/hydophilicity balance, correspondingly affecting the protein
structure, as well the technofunctional properties (e.g., solubility). In addition, influences on the
biological properties of proteins and enzymes, such as enzyme activity [
quality [ ], and changes in the allergenicity of the considered components, seem to be more or
less obvious. For example, studying the allergenicity of milk proteins is still of certain interest, as it
might be influenced during processing and dairy product development [ ]. In this regard, one can
think of products in which milk or whey fractions are combined with ingredients derived from fruits
2], nutritional protein
3
4
Molecules 2018, 23, 264; doi:10.3390/molecules23020264
www.mdpi.com/journal/molecules

Molecules 2018, 23, 264
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and vegetables that provide high concentrations of plant phenolic compounds (e.g., whey beverages
on a fruit juice basis).
Due to their chemical structure, plant phenolic compounds are highly reactive and can be
easily oxidized enzymatically and non-enzymatically [5]. Autoxidation can lead to dimers and even
higher oligomers. The very complex brown polymers, so called melanins, are formed preferably
during enzymatic oxidation (‘apples turn brown when they are cut’). At the beginning of such
reactions, quinones are formed, which are considered to be key-elements of follow-up reactions [
6
].
].
Intermediates are usually part of polymerization, hydration, and disproportionation reactions [
7
The oxidation products can react with each other, interact with other compounds, and remain even reactive.
Nevertheless, the complex reaction system of the phenolic compound leads to complex structures [5,8].
The reaction between phenolic compounds and proteins is even more complex. Reaction sites are
differentially distributed all over the molecule (depending on the genetically-coded amino acid sequence).
The state of oxidation of the phenolic compound might be different due to endogenous factors such as the
pH value, temperature, etc. So far, protein derivatives have been synthesized (for characterization)
very traditionally by incubating proteins and phenolic compounds under alkaline conditions [9].
However, this leads to a very crude mix of reaction products. Not surprisingly, the characterization of
specific adducts and reaction sites is not yet satisfying. In many studies, only the extent of modification
was studied. In a kind of indirect approach, free amino or thiol groups remaining after the reaction
were determined photometrically [9]. In the meantime, several further analytical techniques, such as
capillary electrophoresis, electrospray mass spectrometry, high-performance affinity chromatography,
NMR spectroscopy, fluorescence quenching, and multi-spectroscopic methodologies have been developed
to characterize protein-phenol interactions [1,10]. However, identification of specific binding sites of
small molecules within a protein sequence (and the corresponding conformational position) is still
challenging. Moreover, reaction kinetics have to be considered as well. In the past, it was often believed
that a single oxidized phenolic compound reacts directly with a protein side chain, but it has to be also
considered that the (oxidized) phenolic compounds might react initially with themselves, before reacting
with the nucleophilic, sometimes sterically hindered protein side chains [11]. An important aim of
this research topic is the development of methods for explaining the structural conditions of reaction
products, assignments of binding sites, and reaction mechanisms. The development of new synthesis
strategies in combination with a direct characterization using high resolution mass spectrometry offers
new possibilities and technological advances in the analytical tasks mentioned.
The applicability of electrochemistry with regard to the investigation of the reactivity and coupling
possibilities of different substance classes has already been successfully tested [12,13]. With regard
to phenolic compounds, previous research has so far been able to demonstrate an electrochemical
oxidation of caffeic acid by means of a flow-through system [14]. In contrast to the prevalent oxidability
of some phenolic representatives like caffeic acid, there are also those phenolic compounds that are
not suitable for a complete oxidation due to structural conditions and the redoxpotential necessary
for a transformation. Those phenolic compounds comprise, for example, representatives that have
structural elements such as methoxy or keto groups and only one hydroxyl group such as ferulic
acid [15,16], in which the formation of semiquinones with different possibilities for follow-up reactions
has to be considered, as well [5].
This present study focused on the comparison of the redox potential of various phenolic
compounds and the characterization of oxidation products with regard to their ability to act as reaction
partners for food proteins or peptides thereof using electrochemistry. It aimed to evaluate the different
redox potentials for forming either monomeric, dimeric, or even oligomeric oxidation products and
their reactivity towards peptides resulting from a tryptic digest of the whey protein alpha-lactalbumin.

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2
. Results
2
.1. Electrochemical Oxidation of Phenolic Acids by Means of EC/ESI-MS
In the first step of this study, the applicability of an electrochemical oxidation for transforming
phenolic compounds was evaluated. Exemplarily, the electrochemical oxidation of chlorogenic acid
was optimized by varying the voltage from 0.3 V to 3 V in steps of 0.3 V using a boron-doped diamond
®
electrode and a Roxy potentiostat (Antec Scientific, Zoeterwoude, The Netherlands). In the following
mass spectrometry, the scan mode was used to identify the optimal potential concerning maximum
recovery of the oxidation products. For chlorogenic acid, voltages from 0.6 V up to 1.8 V led to a bundle
of oxidation products. Consequently, 1.8 V were chosen for the further experiments due to reproducible
reaction product formation, diversity, and intensity. The resulting mass spectra are shown in Figure 1.
Figure 1. Full scan mass spectra of (
A
) intact chlorogenic acid and (
B
) oxidized chlorogenic acid using
a potential of 1.8 V.
−
While the intensity of monomeric chlorogenic acid (m/z 353.7 [M
−
H] ) and dimeric
−
−
derivatives (m/z 707.0 [M
−
H] ), as well as caffeic acid (m/z 179.0 [M
−
H] ) and quinic acid
−
(
m/z 191.1 [M − H] ), decreased during the oxidation, the ion intensity of three, so far unknown,
−
−
−
H] ), as well as the intensity of
compounds (m/z 381.5 [M − H] ; m/z 735.2 [M
−
H] ; m/z 765.2 [M
−
−
the trimeric derivative (m/z 1054.3 [M − H] ), increased significantly.
2
.2. Comparison of the Oxidation of Various Phenolic Acids
To achieve comparability of the oxidability of the several phenolic compounds of interest,
the electrochemical oxidation of four phenolic acids was evaluated. Besides chlorogenic acid,
ferulic acid, caffeic acid, and sinapinic acid were oxidized electrochemically with a potential of 1.8 V
analogously to chlorogenic acid. In all four reactions, a formation of dimeric and oligomeric products
was observed. Considering caffeic acid and chlorogenic acid, the formation of oligomeric products was
−
−
already detected at 0 V (caffeic acid: monomer (m/z 180.8 [M
− H] ), dimer (m/z 363.0 [M − H] ),
−
and trimer adduct (m/z 583.0 [M + 2Na
−
H] ). In addition, adduct and cleavage products are already
−
recognizable: (caffeic acid: 3,4-dihydroxystyren (m/z 137.0 [M
−
H] ); chlorogenic acid: caffeic acid
−
−
(m/z 179.0 [M
−
H] ), and quinic acid (m/z 191.1 [M
−
H] ). Upon application of 1.8 V, an increase of

Molecules 2018, 23, 264
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the oligomeric products (e.g., trimer of chlorogenic acid with m/z 1054.3 [M − H]−), and a decrease of
the initial signals was observed. With regard to ferulic acid and sinapinic acid, only the monomeric
molecules and various adducts or cleavage products were detected at 0 V (monomeric ferulic acid
−
−
H] )). There were no dimeric or oligomeric
(m/z 194.2 [M
−
H] ) or sinapinic acid (m/z 224.2 [M
−
products detectable above the limit of detection. Upon application of 1.8 V, an increase of the
corresponding dimers (ferulic acid: m/z 389.1 [M
oligomeric products was recognized, analogously to chlorogenic acid and caffeic acid.
−
−
− H] ; sinapinic acid: m/z 445.0 [M − H] ) and
2
.3. Investigation of Adduct Formation of Chlorogenic Acid and Alpha-Lactalbumin Using EC/LC/ESI-MS
For evaluating the reactivity of the electrochemically generated oxidation products of chlorogenic
acid towards tryptic peptides of alpha-lactalbumin, the reaction of those two reactants was initiated via
a reaction coil. Alpha-lactalbumin is a major component in mammalian milk. It is a whey protein with
allergenic properties and, therefore, a very well-studied protein, and many epitopes have already been
elucidated [17]. It is an acidic globular protein with a moderate size of 123 amino acids and a molecular
mass of 14.2 kDa. The two-domain protein is stabilized by 4 disulfide bonds: The alpha-domain has
disulfide bonds at positions Cys6-Cys120 and Cys28-Cys111, and the beta-domain has Cys61-Cys77
and Cys73-Cys91 disulfide bonds [18]. With its amino acids cysteine (in total 8) and lysine (in total 12),
it provides several potentially reactive amino acid side chains, which could serve as binding sites
for activated, electrophilic quinones or semiquinones. Defined volumes of both reactant solutions,
phenolic acid and tryptic peptides, were injected to the electrochemical system via a syringe
pump and a flow of 20
µL/min. Afterwards, the reaction products were directly transferred
into an LC/ESI-MS system for chromatographic separation and detection. Negative ion mode in
a scan range from 100 Da to 2000 Da was used to identify protein-phenol-adducts. Quinic acid,
monomeric chlorogenic acid, and polymerization products were observed at a retention time of
−
−
1
3.5 min (m/z 192 [M − H] (quinic acid; 192 g/mol), m/z 354 [M
−
H] (monomeric chlorogenic
−
−
acid; 354 g/mol), m/z 711 [M − H] (dimeric chlorogenic acid 711 g/mol), and m/z 1063 [M
−
H]
(
trimeric chlorogenic acid; 1063 g/mol) (Figure 2). It was even possible to detect the tetrameric
−
derivative of chlorogenic acid with an m/z 1417 [M − H] (1417 g/mol)) (Figure 2).
Figure 2. Full scan mass spectra at a retention time 13.5 min of oxidized chlorogenic acid.
−
At 18.5 min, a signal with m/z 967.5 [M
−
H] was detected (Figure 3B), being assumed
−
to be a reaction product of the monomeric form (m/z 354 [M
−
H] ) and one of the peptides
−
(
m/z 616 [M − H] ; Glu-Gln-Leu-Thr-Lys) of alpha-lactalbumin. This assignment can be supported by
the fact that the peptide mentioned contains lysine residues and thus represents a very potent reaction
−
−
H] )
partner. Furthermore, there were two signals at 14.6 min (m/z 1260.3 [M
Figure 3A). The second signal correlates with an adduct formation between the dimer of chlorogenic
−
H] ; m/z 1321.6 [M
−
(
−
−
−
H]
H] and
acid (m/z 711 [M
−
H] ) and the peptide m/z 616 [M
−
H] . In contrast, signal m/z 1260.3 [M
is different. It was hypothesized to be a condensation product of the peptides m/z 749 [M
−
−
−
−
m/z 544 [M − H] due to their reactivity instead of a reaction product with a phenolic compound.

Molecules 2018, 23, 264
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Figure 3. Full scan mass spectra after chromatographic separation via UPLC and detection using
ESI-MS. ( ) Potential adduct formation at retention time 14.6 min; ( ) Potential adduct formation at
retention time 18.5 min.
A
B
3
. Discussion
Electrochemical systems provide a well-controlled application for generating activated metabolites
and for further inducing redox-based adduct-formation. The prediction of the reaction mechanisms of
highly reactive compounds is still challenging. Electrochemistry coupled to mass spectrometry is suitable
to characterize optimum reaction conditions of the oxidation of phenolic compounds. Consequently, it is
possible to generate starting material for follow-up reactions of the quinones that result. By controlling the
reaction conditions and the use of on-line monitoring with, e.g., mass spectrometry, the adduct formation
can be studied, and so far non-considered intermediates can be identified.
Phenolic compounds are known to be highly reactive. Several studies already shown
an electrochemical oxidation of phenolic compounds such as caffeic acid and the formation of reactive
intermediates [14,19]. In the present study, the structurally similar phenolic acids chlorogenic acid,
caffeic acid, ferulic acid, and sinapinic acid were activated electrochemically for further adduct formation
with proteins (or peptides thereof). The applicability of electrochemical oxidation by means of EC/ESI-MS
concerning the four mentioned phenolic acids was successful. Oxidation products were investigated
with regard to diversity and intensity in order to compare oxidative reactivities. Caffeic acid, as well as
chlorogenic acid, are assigned to the group of hydroxycinnamic acids. These are characterized by
an ortho-hydroxyl group and a conjugated double-bond in the side chain. They are able to form metastable
radicals and reactive quinones and are therefore very accessible to nucleophilic attacks. As a result,
an increased reactivity compared to further prominent phenolic acids is assumed. With regard to ferulic
acid and sinapinic acid, a complete oxidation and formation of o-quinones is not possible with those
chemical structures, as there is only one hydroxyl group. Instead of quinones, only semiquinones
are formed [20]. As quinones are a key element of follow-up reactions of phenolic compounds,

Molecules 2018, 23, 264
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especially flavonoids [6], ferulic and sinapinic acid provide a lower reactivity than caffeic acid or
chlorogenic acid. Proposed reactivities were confirmed by electrochemical application. The preference of
phenolic compounds for oligomerization reactions was confirmed, as well. Various cleavage products and
adducts were detected. The level of oligomerization depends on the structure of the phenolic compound.
Caffeic acid and chlorogenic acid showed a higher degree of oligomerization and higher intensities than
those of ferulic acid and sinapinic acid.
The reaction of the electrochemically oxidized products with peptides of a previously tryptically
digested protein was performed in this study. This reaction was carried out exemplarily with chlorogenic
acid and the peptides of the tryptically-digested protein alpha-lactalbumin. Previous research has already
been successful in adjusting coupling reactions and generating adduct formation as well as protein labeling
reactions [12,19,21]. Chlorogenic acid, an ester of caffeic and quinic acid, is a highly reactive secondary plant
metabolite and is discussed as a potentially prevention of cardiovascular disease and because of its high
antioxidant activity [22]. It is well known that chlorogenic acid tends to undergo oligomerization reactions
prior to binding to other substrates such as biomolecules [23]. The use of the terms “monomer”, “dimer”,
and “trimer” is not consistently synonymous with a regular and similar reaction of two or more of the
exact initial substrate molecules in terms of oligomerization. With regard to the studies mentioned above,
a reaction of two, three, or more initial phenolic molecules is assumed. Reactions can occur irregularly and
differently and lead to several forms such as C-C or C-O bonds with a diverse bunch of leaving groups
resulting in a variety of reaction products [24]. For instance, fragment of the monomeric chlorogenic
−
−
acid is m/z 355.7 [M
−
H] ; the ‘dimer-like’ product is m/z 712.1 [M
−
H] , providing a difference of
−
m/z 356.4 [M
−
H] . At this point, it is not clear to which extent the initial reactants undergo changes
such as dehydration, oxidation, or binding to breakdown products prior to follow up reactions. Also, with
regard to the further reaction partners (e.g., peptides), preliminary breakdown reactions or transformation
cannot be ruled out. For example, chemical stability of peptides depends on the amino acid sequence and
the side chains. Transformations such as hydrolysis, oxidation, or deamidation are possible. For this reason,
irregularities in the mass differences of the reaction products may occur. Furthermore, data from mass
spectrometric analysis may not lead to a structural elucidation, but rather give tentative indications.
According to the allergenic property of alpha-lactalbumin, it is of particular interest to study
post-translational modifications of the protein, for example, resulting from a reaction with phenolic
compounds. Therefore, it is necessary to investigate adduct formations with components of any
kind and to investigate the effect on the binding properties of the epitope. Previous research has
already investigated many reactions and their effect on the allergenicity of milk proteins such as
phosphorylation [25].
In order to evaluate adduct formation between phenolic compounds and further biomolecules,
reaction mechanisms must be clarified. On the one hand, a simple reaction of a phenolic acid monomer
respective of its quinoid structure with a reactive side chain of a peptide can take place (Figure 4A).
Furthermore, it is also possible that not only monomers, but also polymerization products such
as dimers, trimers, etc., can undergo reactions with the nucleophilic reaction partners (Figure 4B).
However, it cannot be distinguished by mass spectrometry whether a reaction took place with either
two monomers or a dimer when two reactive side chains are present in one protein/peptide. This issue
−
can be seen in the case of the signal m/z 1321.6 [M
−
H] in the present study. Here, a reaction of
a dimer of chlorogenic acid is assumed, but it could be obviously also a reaction of two monomers
with the corresponding peptide. Even in this case, it is not clear if a reaction of the monomers can take
place at different parts of the peptide at the same time or the monomer associated with the peptide
reacts again with another phenolic compound in a subsequent step (Figure 4C). Previous research
using size exclusion chromatography successfully demonstrated that phenolic acids seem to favor
oligomerization reactions prior to binding to the proteins [26].

Molecules 2018, 23, 264
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Figure 4. Varying reaction possibilities between peptides and phenolic compounds. (
phenolic compound bound to a single amino acid residue of a peptide; ( ) binding of different
polymerization products of phenolic compounds to a peptide; ( ) further reaction between bound
phenolic compound with a further activated phenolic compound.
A) monomeric
B
C
Besides the aspects mentioned so far, the elucidation of the interactions between phenolic
compounds and proteins is still challenging due to the size and structure of the proteins.
In previous studies, reactions between amino acids, peptides, or proteins, and reactive o-quinones or
o-semiquinones, have been described [27–29]. In order to enable a better detection, enzymatic digestion
of the target protein by using, e.g., trypsin, is recommended. Of note, the resulting peptides may also
−
show crosslinking behavior. With regard to the detected signal m/z 1260.3 [M
−
H] in this study,
it can be assumed that a reaction between two peptides occurred due to their reactivity.
The EC/LC/ESI-MS method introduced in this study indicated a significant contribution of
the components to addition reactions. Many of the polymerization products could be assigned;
three signals could be assigned to potential protein-phenol-adducts. However, many signals still
could not be finally clarified at this time. Structural elucidation of the detected compounds by
means of NMR is recommended. It is mandatory to isolate and separate the structures, for example,
by means of preparative HPLC in advance. Due to the reactivities of the analytes, many signals
were generated that could not yet be assigned. At this point, reactions between peptides next to
peptide-phenol-interactions can be assumed. In comparison to the signals of the tryptic peptides of the
protein digest, many previously detectable peptide signals of the tryptic digestion of alpha-lactalbumin
could no longer be perceived with comparable intensity after the chromatographic separation of
the electrochemically generated reaction products. This indicated that multiplicities of the tryptic
peptides are significantly involved in reactions that may contribute to polymerization or adduct
formation. Resulting from these investigations, it was shown that it is possible to produce and identify
reaction products under controlled and defined conditions using electrochemistry coupled with mass
spectrometry. Compared to traditional methods for the preparation of reaction products between
phenolic compounds and peptides (pH value, temperature), reaction parameters could be optimized
and the applicability of the electrochemical oxidation of phenolic compounds and subsequent coupling
with another flow system to verify interactions could be demonstrated.

Molecules 2018, 23, 264
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4
. Materials and Methods
4
.1. Chemicals
Sinapinic and ferulic acid were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany);
caffeic acid, chlorogenic acid, alpha-lactalbumin (bovine), and ammonium acetate were purchased from
Sigma Aldrich Chemie GmbH (Steinheim, Germany). Methanol and acetonitrile were purchased from
Carl Roth GmbH & Co. KG (Karlsruhe, Germany). All chemicals were used in highest quality available.
Water was purified before utilization via Direct-Q 3 UV-R system (Merck KGaA, Darmstadt, Germany).
4
.2. Electrochemical Oxidation of Phenolic Acids by Means of EC/MS
Electrochemical oxidation of phenolic acids was performed using a preparative electrochemical
thin-layer cell (µPrepCell, Antec, Leyden, Netherlands), consisting of a boron-doped diamond working
electrode, a titanium counter electrode, and a Pd/H reference electrode. Potential was controlled
2
®
using a Roxy potentiostat. A solution of 2 mM phenolic acid (in 90% MeOH (v/v) and 10% purified
and double-distilled water (v/v) with 20 mM ammonium acetate) was injected into the electrochemical
cell using a flow rate of 10
thickness (here: 150
µ
L/min. Total volume of
µ
-prep cell is 11
µ
L depending on effective spacer
◦
µ
m). Temperature of the electrochemical cell was set to 20 C. A constant potential
of 1.8 V was applied. Detection of oxidation products was performed with an ESI-MS ion trap mass
analyzer in negative ion mode (amazon speed ETD, Bruker Daltonik GmbH, Bremen, Germany),
◦
with following mass spectrometer settings: ion spray voltage: 4.5 kV; ion source heater: 350 C;
source gas: 55 psi. For instrumental setup see Figure 5.
Figure 5. Instrumental setup for oxidation of phenolic acids by EC/ESI-MS. Phenolic acids are oxidized
electrochemically via thin-layer cell including boron-doped diamond working electrode and are directly
infused to ESI-MS.
4
.3. Tryptic Digestion of Alpha-Lactalbumin
Tryptic digestion of alpha-lactalbumin was performed with an aqueous solution of alpha-lactalbumin
(Sigma Aldrich Chemie GmbH, Steinheim, Germany) in a concentration of 1 mg/mL protein (in purified
and double-distilled water). It was digested tryptically (trypsin from porcine pancreas; Sigma Aldrich
◦
Chemie GmbH, Steinheim, Germany) for 16 h at 37 C (protein-enzyme ratio 100:1). Digestion samples
were used after SPE cleaning procedure via RP18ec SPR cartridges (Macherey Nagel GmbH & Co. KG,
Düren, Germany) with 60% ACN for conditioning and washing steps and 0.2% aqueous formic acid for
equilibration and elution steps. Afterwards, samples were dried using gaseous nitrogen and re-dissolved
in a defined volume using 0.2% aqueous formic acid. Tryptic digest was analyzed using ESI-MS ion trap
mass analyzer in negative ion mode (amazon speed ETD, Bruker Daltonik GmbH, Bremen, Germany),
◦
with following mass spectrometer settings: ion spray voltage: 4.5 kV; ion source heater: 350 C; source gas:
55 psi. An assignment of signals and a resulting identification of the peptides were performed using the
UniProtKB database (http://www.uniprot.org/) and the SIB Bioinformatics Resource Portal ExPASy

Molecules 2018, 23, 264
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(https://www.expasy.org/). Mentioned databases provide a comparison with the theoretical tryptic
digestion of sequences. The peptides detected during this analysis are shown in Table 1.
Table 1. Fragments, position in protein, and sequence of detectable peptides after tryptic digestion.
Detection by use of high resolution mass spectrometry.
Fragments [m/z]
198.6
Position
Sequence
1
118-127
113-118
25-29
20-24
78-81
114-117
30-32
33-35
Val-Gly-Ile-Asn-Tyr-Trp-Leu-Ala-His-Lys
Glu-Leu-Lys-Asp-Leu-Lys
Cys-Glu-Val-Phe-Arg
Glu-Gln-Leu-Thr-Lys
Ile-Trp-Cys-Lys
7
49.4
52.3
16.3
44.3
86.3
87.2
73.2
6
6
5
4
3
3
Ile-Leu-Asp-Lys
Glu-Leu-Lys
Asp-Leu-Lys
4
.4. Investigation of Adduct Formation of Chlorogenic Acid and Alpha-Lactalbumin Using EC/LC/ESI-MS
For considering adduct formation of oxidized chlorogenic acid and alpha-lactalbumin-deriven
peptides, chlorogenic acid was oxidized as mentioned above (4.2). Subsequently, a second flow
system was used to combine both analyte solutions. The ratio of both analytes in the reaction cell
was 1:1 (v/v) regarding mentioned concentrations. A chromatographic separation was done on
®
®
a Phenomenex (Phenomenex Inc., Torrance, CA, USA) reversed-phase HPLC column (Kinetex
.6 m RP 18 100 Å, 150 2.1mm) equipped with a guard column of the same material. A Dionex
UltiMate 3000 UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used.
Detection was performed with a diode-array-detector as well as an ESI-MS ion trap mass analyzer
amazon speed ETD, Bruker Daltonik GmbH, Bremen, Germany), recording mass spectra in negative
2
µ
×
™
(
ion mode. The mobile phase A was water, and B was acetonitrile, both containing 0.1% formic acid.
The gradient elution started with 95% A for 10 min, linearly increased to 60% B in 20 min and further
up to 95% B in 3 min, and kept constant for 10 min. It was brought back to 95% A in 2 min followed
by 10 min of re-equilibration. Injection volume was 1 µL using a flow rate of 200 µL/min for all
samples. LC-MS system was controlled by HyStar 3.2 (Bruker Daltonik GmbH, Bremen, Germany).
For instrumental setup see Figure 6.
Figure 6. EC/LC/ESI-MS instrumental setup for oxidation, reaction, and separation of potentially
phenol-protein-adducts. Phenolic acids are oxidized electrochemically via a thin-layer cell including
a boron-doped diamond working electrode. A second flow system consists of a solution of tryptic
peptides. Both solutions were combined using a three-way valve and a reaction coil and afterwards
infused into the LC/ESI-MS system.

Molecules 2018, 23, 264
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5
. Conclusions
Electrochemistry coupled with mass spectrometry was successfully used to investigate the
redoxpotential of phenolic acids such as chlorogenic acid. It was possible to create optimized
conditions for the oxidation of chlorogenic acid to generate a wide variety of oxidation products.
Electrochemically oxidation of chlorogenic acid, caffeic acid, ferulic acid, and sinapinic acid generated
a variety of reactive intermediates. Feasibility of this technique for the research topic was shown.
Structural elucidation using NMR after isolation of generated oxidation products is planned in
further studies to determine reaction mechanisms. The applicability of electrochemistry coupled
with mass spectrometry for investigating reaction products of phenolic compounds and proteins could
be demonstrated by the reaction of oxidized chlorogenic acid with peptides of alpha-lactalbumin.
The method is suitable for generating adducts between proteins and phenolic components and is able to
supplement or replace traditional methods. A detailed clarification of the resulting chemical structures
of the products was not possible at this time. For this purpose, a synthesis of higher amounts of reaction
products for an analysis with NMR is mandatory. With regard to a potential change of the protein
allergenicity due to modification of the protein, methods such as ELISA, HPTLC-immunostaining,
or HPTLC-aptastaining can be used [30,31].
Acknowledgments: The authors gratefully thank ERC GmbH (München, Germany) for supplying the
electrochemically equipment.
Author Contributions: C.K. and S.R. planned and designed the experiments; C.K. and S.S. performed the
experiments and analyzed the data; C.K. and S.R. contributed reagents and analysis tools; C.K. and S.R. wrote
the paper.
Conflicts of Interest: The author declares no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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