Enzymatically modified peptide surfaces: towards general electrochemical sensor platform for protein kinase catalyzed phosphorylations

Abstract

We hereby present an electrochemical approach for monitoring the three protein kinases sarcoma-related kinase (Src), extracellular signal-regulated kinase 1 (Erk1), and cyclin A-dependent kinase 2 (CDK2/cyclin A). The electrochemical sensor is based on the ability of kinases to transfer a redox-labeled phosphoryl group to surface-bound peptides that are highly specific substrates for the particular protein kinase (EGIYDVP, EPLTPSG, and HHASPRK, respectively). The detection method relies on the use of 5′-γ-ferrocenoyl-ATP (Fc-ATP) as a co-substrate for peptide phosphorylation. The peptides themselves are attached to a Au substrate, which acts as the working electrode. In this process a Fc-phosphoryl group is transferred to the peptide and the presence of the redox active Fc group is detected electrochemically. All peptide films were fully characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Particular attention was given to the electron transfer rates, k_ET, in peptide films after Fc-phosphorylation which were found to be on the order of seconds. The slow ET kinetics is presumably a result of the negative charge on the phosphoryl group. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) experiments based on the peptide modified Au surfaces reveal significant ferrocene and phosphate group content introduced using the kinase-catalyzed phosphorylation reaction.

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1. Introduction

Phosphorylation is a major driving process for many biological functions and protein kinases are the primary agents responsible for this event.¹ Amongst hundreds of protein kinases recently discovered, enzymes of interest are those involved directly in the cell cycle, such as cyclin-dependent kinases,^2–5 and those proteins related to the mitogen-activated protein kinase (MAPK) signalling pathway, such as sarcoma-related (Src)⁶ and extracellular signal-regulated kinase 1 (Erk1) proteins.⁷ The MAPK cascade is based on sequential phosphorylation reactions and along with the cell cycle regulates every aspect of cell life. Hyperactivity of such protein kinases favours tumour development by inducing uncontrollable cell division. Naturally, an abnormal cellular signalling upon kinase-catalyzed phosphorylation can lead to diseases, including cancer.¹ Hence, monitoring and quantifying kinase activity is a promising approach to early-on-set diagnosis and drug target screening.

Protein kinase activity has been studied extensively by electrochemical,^8–12 optical^13–17 and radiolabeling methods.^18,19 Recently, alternative techniques such as electrochemical impedance spectroscopy (EIS),^20–22surface plasmon resonance (SPR),²³time-of-flight secondary ion mass spectrometry (TOF-SIMS)^24,25 and X-ray photoelectron spectroscopy (XPS)¹⁸ have been found useful for the study of kinase-based biological processes.

Earlier, we reported on the use of a new electrochemical approach for the detection of kinase-catalyzed phosphorylation reactions exploiting 5′-γ-ferrocenoyl-ATP conjugate (Fc-ATP), as the co-substrate.^26,27 Here, we expand on this research and report results employing the three protein kinases Src, Erk1 and CDK2/Cylin A. In addition, the electron transfer process was investigated in peptide films following Fc-phosphorylation by electrochemical means for the first time. In addition, detailed surface characterizations of these systems were performed by CV, EIS, TOF-SIMS and XPS techniques.

2. Experimental procedure

2.1 General experimental conditions

All reagents and solvents were used without further purification unless otherwise specified. The substrate peptides EGIYDVP and EPLTPSG were purchased from BioBasic Inc (Markham, Ontario) and vSrc kinase and Erk1 kinase were purchased from Cell Signaling (New England Biolabs Ltd., Pickering, Ontario). CDK2 peptide substrate, HHASPRK, and CDK2/Cyclin A complex were both purchased from Enzo Life Sciences. All organic solvents were freshly distilled and the experiments in aqueous solutions were prepared using ultra-pure water (18.3 MΩ cm) from Millipore Milli Q system. Adenosine 5′-[γ-ferrocene] triphosphate (Fc-ATP) was synthesized according to the procedure published elsewhere.²⁶

2.2 Immobilization of the peptide substrates

The sputtering gold on silicon chips (Ti 6 nm, Au 140 nm, 0.2 cm² surface area, fabricated at Western's Nanofabrication Facility) were cleaned by 3 min etching in 1 M H₂SO₄ (aq), 5 min sonication in distilled water and 5 min sonication in freshly distilled ethanol. Next, the gold electrodes were incubated with 2 mM N-hydroxysuccinimide lipoic acid active ester ethanolic solution for 3 days. Then, the electrodes were washed with ethanol and incubated with 0.1 mM (Millipore water) peptide solution (EGIYDVP, HHASPRK or EPLTPSG, in milliQ water) for 20 h at 273 K. Following the peptide incubation the electrodes were rinsed with milliQ water and blocked with ethanolic 100 mM ethanolamine solution (1 h) followed by ethanolic 10 mM dodecanethiol solution (20 min) for the back-filling of the remaining Au surfaces.

2.3 Phosphorylation reaction on the surface

The activities of protein kinases were measured using the specific kinase assay buffer to maximize the phosphorylation process. The Src kinase assay buffer consisted of 5 mM MOPS (pH 7.5), 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 2.5 mM MnCl₂ and 4 mM MgCl₂. CDK2/Cyclin A complex assays were performed in 60 mM HEPES (pH 7.5), 3 mM MnCl₂, 3 mM MgCl₂, 0.05 μg μL⁻¹ PEG 20000 and 3 μM sodium ortho-vanadate. The activity of Erk1 kinase was measured in 5 mM MOPS (pH 7.2), 2.5 mM β-glycerophosphate, 1 mM EGTA and 5 mM MgCl₂. Peptide modified gold electrodes were incubated in a 20 μL reaction volume in the presence of protein kinase (1μg mL⁻¹ or 10 μg mL⁻¹) and Fc-ATP (0.2 mM). After 6 h of incubation at 37 °C in a heating block (VWR Scientific, USA), the electrodes were washed using the kinase assay buffer prior to measurement.

2.4 Electrochemical studies

All electrochemical experiments including cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) were carried out using a CHInstrument potentiostat 660B (Austin, TX). In a typical electrochemical experimental set up, a peptide-modified gold electrode was used as a working electrode, Ag/AgCl in 3 M KCl as the reference electrode, which was connected with the electrolyte via a salt bridge, and platinum wire as the counter electrode. All CV electrochemical measurements were carried out in the presence of 0.1 M sodium phosphate buffer (pH 7.4) and at a scan rate of 0.1 V s⁻¹ in the potential range of 0.2 to 0.6 V. SWV were performed at a pulse amplitude of 25 mV in the same buffer. Values of electron-transfer rates in each peptide film were obtained from normalized CV spectra and the corresponding Tafel plots.²⁸ The surface density, Γ_Fc, was calculated from the charge, Q, determined by integrating the redox peaks in the CV. At least three measurements were performed for all experiments. Biosensor characterization studies were carried out by running the CV experiments in 5 mM solution of K₄[Fe(CN)₆]·3H₂O and K₃[Fe(CN)₆]·3H₂O in 0.1 M sodium phosphate buffer (pH 7.4). All AC impedance spectra were acquired at the formal potential of the [Fe(CN)₆]^3−/4−redox couple (0.25 V vs.Ag/AgCl) at 5 mV amplitude and in the 0.1 Hz to 100 kHz range. All impedance spectra are represented as Nyquist plots with real impedance (Z_re) vs. imaginary impedance (Z_img) values. The experimental EIS data were fitted to an appropriate equivalent circuit by using the ZSimpWin 2.0 (EChem software).

2.5 Time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments

TOF-SIMS experiments were performed with TOF-SIMS IV (ION-TOF GmbH, Munster, Germany) which was equipped with a Bi liquid metal ion source. For all measurements, a 25 keV Bi₃⁺ cluster primary ion beam with a pulse width of 12 ns was employed (target current of ∼1 pA). The cycle time for the processes of bombardment and detection was 100 μs (or 10 kHz). A pulsed, low energy electron flood was used to neutralize sample charging. For each sample, spectra were collected from 128 × 128 pixels over an area of 500 μm × 500 μm for 60 s. Positive and negative ion spectra were internally calibrated by using H⁺, H₂⁺ and CH₃⁺ and H⁻, C⁻ and CH⁻ signals, respectively. Two spots per sample were analyzed by using a random approach.

2.6 X-Ray photoelectron spectroscopy (XPS) experiments

The XPS analyses were carried out with a Kratos Axis Ultra spectrometer using a monochromatic Al K(alpha) source (15 mA, 14 kV). The instrument work function was calibrated to give a binding energy (BE) of 83.96 eV for the Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give a BE of 932.62 eV for the Cu 2p3/2 line of metallic copper. The Kratos charge neutralizer system was used on all specimens. Survey scan analyses were carried out with an analysis area of 300 × 700 microns and a pass energy of 160 eV. High resolution analyses were carried out with an analysis area of 300 × 700 microns and a pass energy of 20 eV. Spectra have been charge corrected to the main line of the carbon 1s spectrum (adventitious carbon) set to 284.8 eV. Spectra were analysed using CasaXPS software (version 2.3.14).

3. Results and discussion

3.1 Electrochemical protein kinase assays

Hereby, special attention was given to the evaluation of Fc-ATP approach to the detection of the following protein kinases Src, Erk1 and CDK2/Cyclin A complex.^26,27 The preparation of the electrochemical sensor surface is illustrated in Scheme 1. Briefly, after the immobilization of N-hydroxysuccinimide lipoic acid active ester (NHS-ester) on the Au surface (A), the target peptide was coupled to the sublayer via an amide linkage (B). Blocking of unreacted NHS-ester with ethanolamine and back-filling with dodecanethiol was important in order to avoid unwanted chemisorption and thus risk potential loss of the protein kinase. Following the attachment of EGIYDVP, HHASPRK or EPLTPSG peptides, the tyrosine, serine or threonine residues in these peptides served as the target sites for the kinase-catalyzed Fc-phosphorylation reactions (C), respectively.

Scheme 1 Schematic illustration of the stepwise assembly of the electrochemical biosensor surface for detection of protein kinase-catalyzed Fc-phosphorylation using Fc-ATP. (A) Initial formation of a thin film of NHS-ester on Au surfaces is followed by (B) peptide incubation resulting in the formation of a film containing target peptides chemically bound to the transducer surface. (C) After blocking with 100 mM ethanolamine, which reacts with the excess NHS ester on the surface, and back-filling with 10 mM dodecanethiol, the protein kinase-catalyzed Fc-phosphorylation reaction was carried out in the presence of kinase and Fc-ATP co-substrate.

As a first step in our investigation, we investigated the sensor surface prior to the enzymatic transformation.

For this purpose, CV and EIS studies were performed in 5 mM [Fe(CN)₆]^3−/4− in 0.1 M sodium phosphate buffer. Fig. 1 summarizes the results of CV and EIS studies. The redox bahaviour of the [Fe(CN)₆]^3−/4−redox couple is fully reversible at the bare gold electrode (Fig. 1A). Upon incubation of the Au surface in 2 mM NHS-ester solution, the current signal is reduced. The reversibility of the redox reaction is further suppressed after peptide immobilization. Subsequent treatment with 100 mM ethanolamine solution followed by 10 mM dodecanethiol solution led to a complete surface blocking as evidenced by the lack of redox probe signal.

Fig. 1 Surface characterization of the peptide biosensor surface in a 0.1 M sodium phosphate buffer (pH 7.4) containing 5 mM [Fe(CN)₆]^3−/4−redox probe. (A) Cyclic voltammograms and (B) Nyquist plots: (a) Bare Au electrode, (b) after modification with NHS-ester, (c) following immobilization of peptide substrate, and d) after blocking with 100 mM ethanolamine and back-filling with 10 mM dodecanethiol. Inset shows the equivalent circuit used to fit the impedance spectra. The electrochemical impedance spectra were acquired at the formal potential of the [Fe(CN)₆]^3−/4−redox couple (0.25 V vs.Ag/AgCl) at 5 mV amplitude and in the 0.1 Hz to 100 kHz range.

To further explore the electrochemical characteristics of the films, we carried out EIS studies. Fig. 1B shows the Nyquist plots of impedance spectra for different films on Au surface. All impedance curves were fitted to an equivalent circuit model that included, a solution resistance, constant phase element and Warburg constant. Incubation of electrodes with NHS-ester and peptide substrate solutions resulted in a significant increase in the charge transfer resistance (R_CT) from the redox couple to the electrode surface and led to the appearance of the ‘semi-circle’ portion at the high frequencies. The charge transfer process was further reduced, as expected, by blocking with ethanolamine and back-filling with dodecanethiol. Notably, all films are characterized by a significant contribution from a diffusion controlled electrochemical process at low frequencies.

Next, we investigated the kinase-catalyzed Fc-phosphorylation of the peptide-modified sensor surfaces for the three protein kinases and their corresponding peptide targets. The peptide-modified Au electrodes were incubated in 0.2 mM Fc-ATP solution in the presence of the corresponding protein kinase (Src, CDK2/CylinA or Erk1). The phosphorylation reactions were monitored electrochemically by CV and SWV. A typical CV for the Src target peptide EGIYDVP film on the Au surface following the kinase-catalyzed phosphorylation reaction is shown in Fig. 2A. A current density response for the Fc-phosphorylation is characterized by a reversible couple at ∼ 395 mV (vs.Ag/AgCl reference electrode) which is attributed to the formal potential E° of γ-Fc-phosphate group transferred to the tyrosine residue of the surface-bound peptide, as evidenced from the linear current vs. scan rate dependence, suggesting a surface-controlled process (Fig. S1, ESI†). Fig. 2B depicts a typical SWV response of a peptide-modified electrode in the presence and absence of Src kinase. Similar electrochemical responses were observed for peptide biosensors in the presence of Erk1 or CDK2/Cyclin A with their coresponding target peptides (Fig. S2–S5, ESI†). Importantly, when phosphorylation reaction was performed in the absence of protein kinase, no Faradaic response was observed in CV and SWV (Fig. 2). This demonstrates that the redox signal is attributed to the Fc group directly attached to the peptide surface. Table 1 summarizes the electrochemical properties of the peptide films following the enzymatic Fc-phosphorylation reactions. All peptide films have several characteristics in common, including the peak separation ΔE in the 40–60 mV range which suggests one electron redox process in all films. By integration of the reduction peaks, the surface coverage was determined for all three films and was found to be in 2 – 6 × 10⁻¹¹ mol cm⁻² range. The peak full width at half maximum, ΔE_fwhm, provides a measure of organizational behaviour of monolayers in the electrochemical biosensors.^29,30 The values of ΔE_fwhm ∼ 90 mV observed for all three films clearly suggest that the monolayers are characterized by minimal disorder around the redox active probe and within the film.

Fig. 2 Cyclic voltammograms (A) and square-wave voltammograms (B) of peptide-modified Au electrode for detection of Src protein kinase (1 μg ml⁻¹) (a) in the absence of Src and (b) in the presence of Src. Measurements were taken in 0.1 M sodium phosphate buffer (pH 7.4) solution vs.Ag/AgCl as reference electrode and Pt wire as counter electrode at a scan rate of 100 mV s⁻¹.

Table 1 Electrochemical properties of peptide films on Au surface following the kinase-catalyzed Fc-phosphorylation (E°, ΔE and ΔE_fwhm (oxidation peak) in mV, k_ET in s⁻¹, Γ_Fc in ×10⁻¹¹ mol cm⁻², supporting electrolyte 0.1 M sodium phosphate buffer (pH 7.4), Ag/AgCl reference and Pt counter electrode, values are taken from CV data)

Protein kinases	E°	ΔE	ΔEfwhm	i pa /ipc	k ET	Γ Fc
Src	392 ± 11	55 ± 4	93 ± 3	0.89 ± 0.1	13	2.68 ± 0.29
Erk1	401 ± 4	56 ± 3	90 ± 2	0.81 ± 0.1	8.8	5.42 ± 0.52
CDK2 /Cyclin A	394 ± 2	41 ± 6	94 ± 10	0.94 ± 0.2	3.4	4.36 ± 0.81

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Next, we investigated the rate of electron transfer (ET) from the ferrocene moiety in the peptide film to Au surface following the Fc-phosphorylation reactions in the presence of different protein kinases. Asymmetric Tafel plots can be attributed to the challenges associated with the limited diffusion of solvent and counter ions from solution into the films as well as increased molecular mobility of Fc-group.³¹ A dynamic model for the ET process can be assumed which occurs through the collision of the terminal Fc group with the electrode. The limited access of solvent and counter-ions to the shielded Fc⁺ moiety is due to a relatively buried nature of the latter in the films. Peptide films containing the Fc-phosphate groups are characterized by slow and sluggish ET rates in 3–13 s⁻¹ range (Table 1). Compared to films prepared with N-Fc-peptides which exhibit larger k_ET values (∼ 10³ s⁻¹),^32–34 the rates for Fc-phosphorylated peptides on Au surface are dramatically reduced. Slower ET rates observed in these systems may be attributed to the presence of the negatively charged Fc-phosphate groups on the surface. In addition, the lower solvation energy of the Fc group³⁵ and the orientation of molecules on the surface may contribute to the effect.³⁶ Moreover, blocking and back-filling of the peptide films hinder the ET process at the electrode and contribute to the slower ET rates.

Following the kinase-catalyzed phosphorylation reaction the surface characterization of films revealed a slight rectification of the current response from [Fe(CN)₆]^3−/4−redox couple which is enabled by the presence of ferrocene groups attached to the target peptides on the surface. In addition, EIS experiments were performed to elucidate the film resistance following the Fc-phosphorylation reaction. The impedance spectrum shows an increase in the film resistance after kinase-catalyzed reaction which is in agreement with recent report on the CK2 kinase-catalyzed phosphorylation on the surface. The authors suggested that the negatively charged phosphate groups attached to the surface increase the electron transfer resistance of the film.¹⁹ Hence, the negatively charged phosphates in the film are repelling the negatively charged [Fe(CN)₆]^3−/4−redox couple and impeding the charge transfer overall.

3.2 Surface studies by TOF-SIMS and XPS

The surface characterization of the biosensing interface before and after the kinase-catalyzed Fc-phosphorylation was achieved using surface sensitive TOF-SIMS and XPS methods. TOF-SIMS is a useful method for probing of different chemical groups present on surfaces.²⁵ Hence, the positive and negative MS modes were used to investigate target ions of interest.

Shown in Fig. 3 are negative secondary ion mass spectra for the films based on NHS-ester (A), substrate peptide (B) and films prepared in the absence of Src kinase (control). These films are characterized by a relatively low [PO₂]⁻ (62.96) and [PO₃]⁻ (78.94) ion intensities as described in Table 2. In contrast, films prepared in the presence of Fc-ATP and Src kinase (C) contained the highest phosphate group intensities characteristic of the kinase phosphorylation. The iron content in peptide films was investigated by the positive secondary ion mode as the qualitative measure of kinase activity. Peptide films treated with Fc-ATP and Src kinase protein (C) are characterized by high Fe⁺ (55.92) ion fragment intensities. On the basis of TOF-SIMS data, the relatively high Fe⁺ ion content in the peptide films can be correlated to the surface-assisted phosphorylation reaction in the presence of protein kinase. The presence of this ion fragment in the films in the absence of protein kinase is due to the residual Fc-ATP following the incomplete washing step for the purpose of TOF-SIMS experiments.

Table 2 XPS and TOF-SIMS analysis of films on Au with (A) NHS-ester, (B) peptide substrate, and (C) after Fc-phosphorylation reaction. The control spectra correspond to Au surfaces prepared in the absence of Src. The binding energies (eV) and % peak areas were extracted from XPS survey scans

XPS binding energies (% area) in films
Films
Core level	A	B	C	control
O 1s	532.2 (6.5)	531.5 (8.0)	531.5 (8.5)	532.2 (7.7)
N 1s	399.9 (1.8)	399.9 (1.6)	399.9 (3.1)	400.6 (1.6)
C 1s	285.1 (36.3)	285.1 (35.8)	285.2 (44.2)	285.1 (37.4)
S 2p	162.5 (2.7)	162.6 (1.5)	161.9 (1.3)	162.6 (2.0)
Fe 2p	0	0	707.9 (0.4)	0

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TOS-SIMS ion intensity in films
Films
Ion fragment (m/z)	A	B	C	control
PO2^− (62.96)	10000	9100	180000	43000
PO3^− (78.94)	9700	10000	260000	65000
Fe^+ (55.92)	487	1016	82300	43800

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Fig. 3 Negative and positive secondary ion TOF-SIMS spectra of Au surfaces with (A) NHS-ester, (B) substrate peptide, and (C) following the Src-catalyzed phosphorylation reaction with Fc-ATP as a co-substrate. The control spectra correspond to Au surfaces prepared in the absence of Src.

Next, we carried out XPS studies of the films. XPS survey scans reveal the presence of S, C, O and N atoms in all samples and their respective values are presented in Table 2. All films have a significant S coverage, due to NHS-ester, and the deconvoluted S 2p spectrum gives rise to two doublets. The first doublet at a binding energy of 162.7 eV is assigned to the S 2p_3/2 peak and the second doublet at 164.1 eV was due to S 2p_1/2 peak. The signal for S 2p_3/2 is ascribed to S atoms bound to Au surfaces as thiolate species while the later doublet, S 2p_1/2, represents a physisorbed moiety.^37–39 Two types of the S 2p photoelectrons are present at 1 : 2 area ratio with a peak separation of 1.3 eV. The excess sulfur content, due to adsorbed NHS-ester, does not compromise our electrochemical response. Furthermore, after Fc-phosphorylation the results of high-resolution XPS analysis of the Fe 2p_3/2 region show that Au surface is characterized by Fe (II) binding energy at 708.3 eV which is associated with ferrocene group (Fe 2p_3/2) and was observed only in the presence of Src kinase. The binding energy of Fc group matches well the reported literature values.²⁴ In contrast, in the control experiment performed in the presence of Fc-ATP and in the absence of Src, no signals related to the Fc group or any oxidized species were observed by XPS.

4. Conclusions

In summary, here we demonstrate the usefulness of the bioconjugate Fc-ATP probe for the electrochemical detection of protein kinase activities of Src, Erk1 and Cyclin A dependent CDK2 protein kinases with tyrosine, threonine and serine-based residues, respectively. Overall, the electrochemical response is due to a protein kinase-catalyzed phosphorylation reaction and a transfer of Fc-phosphate group to the target peptide on the surface. After Fc-phosphorylation, all peptide films exhibit slow electron transfer kinetics, which is in contrast to N-Fc-peptide conjugates in which k_ETs are orders of magnitude higher. Presumably, the presence of a negatively charged Fc-phosphate group results in a significant activation barrier for electron transfer from the Fc to the Au surface. Our methodology is well suited to probe variety of Fc-phosphorylation reactions by protein kinases. We are currently exploring a multiplexed approach for screening of biochemical signalling pathways and protein kinase inhibitors.

Acknowledgements

S. Martić is thankful to Ontario Ministry of Research and Innovation and The University of Western Ontario for funding. The authors are also grateful to National Science and Engineering Research Council for strategic funding. We are grateful to Western's Nanofabrication Facility for access to its facilities.

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Footnote

† Electronic supplementary information (ESI) available: electrochemical data for Erk1 and CDK2/Cyclin A and XPS spectra. See DOI: 10.1039/c0an00438c

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