Electrochemical Strategy for Sensing Protein Phosphorylation

Dec 11, 2011 - We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity b...
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Electrochemical Strategy for Sensing Protein Phosphorylation Peng Miao,†,‡ Limin Ning,‡ Xiaoxi Li,‡ Pengfei Li,† and Genxi Li*,†,‡ †

Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, P.R. China Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.R. China

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ABSTRACT: We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr4+ mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr4+ links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH3)6]3+, via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.

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INTRODUCTION Protein kinase-catalyzed phosphorylation is an important biological process in metabolism and plays vital regulatory roles in cell communication. The abnormal regulations of protein kinase may lead to various diseases including cancer, diabetes, and some neurodegenerative diseases.1,2 Therefore, the identification of protein kinase activity as well as its inhibition is not only an important research topic of basic biology, but also a promising approach to early diagnosis and drug discovery.3−6 Many methods have been proposed for the detection of protein kinase-catalyzed phosphorylation with various techniques, including electrochemical,7−11 optical,12−14 and fluorescent approaches.15−17 Also, nanoparticles such as Au, TiO2, and Ag nanoparticles are exploited for the detection of phosphorylation.18,19 Considering the increased interest in the study of phosphorylation, we present a novel electrochemical method in this paper for assay of protein kinase activity based on Zr4+ mediated signal transition and rolling circle amplification (RCA), a simple enzymatic process that generates very long single-stranded DNA under isothermal conditions. 20−27 Compared with other signal amplification methods such as polymerase chain reaction and nanoparticle mediated signal amplification, RCA have many advantages: (1) the reaction can be carried out at a constant temperature, so neither a thermally stable DNA polymerase nor sophisticated instrumentation is required; (2) high specificity can be achieved over DNA single base mismatch; (3) the created long DNA chains can meet the requirement of in situ amplification; (4) the experiment is carried out in pure biological system without the influence of nonbiological materials like nanoparticles. In this strategy, substrate peptide is first modified on a gold electrode. After phosphorylation by PKA, the phosphorylated peptide is linked to the DNA primer probe for RCA by Zr4+.28 Consequently, © 2011 American Chemical Society

RCA can be achieved on the surface of the electrode with the help of template probe and phi29 DNA polymerase. After the binding of the electrochemical speices, [Ru(NH3)6]3+ on the RCA products,29,30 sensitive electrochemical response can be obtained for the detection of phosphorylation and the assay of the protein kinase activity with cascade signal amplification. The detection limit of this method is 0.5 unit/mL, which is similar to that of a recently reported fluorescent method16 and the lowest one using an electrochemical assay.7 Moreover, the linear detection range of this method is improved to be 5 to 500 unit/mL, which is rather wide.

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EXPERIMENTAL PROCEDURES Materials and Chemicals. Substrate peptide LRRASLGGGGC and control peptide WKGEWTGRRGC were synthesized by Shanghai Ketai Biotechnology Co., Ltd. Oligonucleotides named as primer probe (5′AAAAAAAAAAAAAAAAACCCTATAAATACCCTAAC-3′) and padlock probe (5′-TTATAGGGTTAGGGTTAGGGTTAGGGTAT-3′) were synthesized and purified by Shanghai Invitrogen Biotechnology Co., Ltd. Both of them were designed to contain 5′-phosphoryl ends. Fetal calf serum was purchased from NanJing SunShine Biotechnology Co., Ltd. Phi29 DNA polymerase was obtained from New England Biolabs Ltd. cAMP-dependent protein kinase (PKA), T4 DNA ligase, H− 89, ethylenediaminetetraacetic acid (EDTA), mercaptohexanol (MCH), hexaammineruthenium(III) chloride ([Ru(NH3)6]3+), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dNTP, ATP, cAMP, and ZrOCl2 were purchased from Sigma. All other chemicals were of analytical grade and used as received. Water Received: September 21, 2011 Revised: December 1, 2011 Published: December 11, 2011 141

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electrode as the working electrode, a saturated calomel reference electrode (SCE), and a platinum auxiliary electrode. The test solution for EIS was 1 M KNO3 containing 5 mM Fe(CN)63−/4− and the experiments were performed upon application of the biasing potential 0.259 V, applying 5 mV amplitude in the frequency range of 0.1 Hz to 100 kHz. For CV and CC experiments, the test solution was 10 mM TBS containing 50 μM [Ru(NH3)6]3+. The scan rate was 100 mV/s for CV, and the pulse period was 250 ms for CC.

purified with a Milli-Q purification system (Barnstead) was used to prepare Tris-HCl buffer solution (pH 7.5, TBS) and all the other solutions. Preparation of Substrate Peptide Modified Electrode. A gold electrode (3 mm diameter) was first soaked in piranha solution (98% H2SO4/30% H2O2 = 3:1) for 5 min (Caution: Piranha solution dangerously attacks organic matter!) and then rinsed with water. Then, the electrode was polished carefully to a mirror-like surface with P3000 silicon carbide paper and 1 μm, 0.3 μm, and 0.05 μm alumina slurry, respectively. After that, it was sonicated for 5 min in ethanol, and then in water. The pretreated electrode was further soaked in nitric acid (50%) for 30 min, followed by being electrochemically cleaned with 0.5 M H2SO4 to remove any remaining impurities. After being dried with nitrogen, the electrode was incubated with peptide solution (0.2 mM, 20 mM HEPES, containing 10 mM TCEP, pH 6.0) for 12 h at room temperature, followed by a 0.5 h treatment with 0.1 mM MCH. Finally, the electrode was washed first with TBS containing 0.5% Tween to block the adsorption of free enzymes and nonspecific proteins, and then with blank TBS and pure water, respectively. Phosphorylation on the Electrode. PKA stock solution was diluted to a series of concentrations with 10 mM HEPES (pH 6.5) containing 200 μM ATP, 600 μM cAMP, 5 mM TCEP, and 10 mM MgCl2. Then, the substrate peptidemodified electrode was immersed in the above solutions for 30 min at 37 °C, followed by an electrode washing procedure. For the control experiment, the control peptide instead of the substrate peptide was modified on the electrode before the phosphorylation. For the inhibition experiments, different amount of H-89 was dissolved in the PKA reaction solution to check the inhibition of H-89 to PKA during this study. Linkage of Peptide with Primer Probe Mediated by Zr4+ and the Execution of RCA. The peptide modified electrode which had been pretreated by PKA was first immersed in a Zr4+ solution (0.2 mM, TBS) at room temperature for 1 h, so that Zr4+ could bind to the phosphorylation site of the peptide by electrostatic interaction. Based on the same mechanism, primer probes were further bound with Zr4+ by incubating the modified electrode in 1 μM primer probe at room temperature for 1 h. Consequently, the peptide and the primer probe were linked mediated by Zr4+. For hybridization of primer probe and padlock probe, the above electrode was incubated with 0.1 mL phosphate buffered saline (pH 7.4) containing 1 μM padlock probe for 0.5 h at 37 °C. In this process, the linear padlock probe transforms into circle template after hybridization with primer probe. The nick was linked by T4 DNA ligase (6 unit/mL) prepared in 50 mM TBS containing 10 mM MgCl2, 10 mM DTT, and 1 mM ATP at 37 °C, the duration of which was about 1 h. The achievement of RCA also required phi29 DNA polymerase (50 unit/mL), which was prepared in 50 mM TBS containing 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 0.2 mg/mL BSA. dNTP was also added in the reaction solution with the concentration of 0.5 mM. After 1 h RCA reaction at 37 °C, phi29 DNA polymerase was inactivated by heating the reaction solution at 95 °C for 5 min. Electrochemical Measurements. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and chronocoulometry (CC) were performed on an electrochemical analyzer (CHI660B, CH Instruments) by using three-electrode system consisting of the modified gold

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RESULTS AND DISCUSSION The strategy in this work to monitor phosphorylation by electrochemical technique coupled with cascade signal amplification has been illustrated in Figure 1. First, the gold

Figure 1. Schematic illustration of the strategy for monitoring the activity of protein kinase.

electrode is modified with the substrate peptide through its cysteine end. After the phosphorylation of the peptide by PKA as the model kinase in this work to phosphorylate the serine in the substrate peptide, Zr4+ is attached to the phosphorylation sites due to the steady interaction between Zr4+ and the phosphate groups. Then, primer probe with 5′-phosphoryl end will also interact with Zr4+ with the same mechanism; thus, the peptide and the primer probe are linked by Zr4+. After that, the linear padlock probe, designed to match primer probe in a head to tail fashion, hybridizes with the primer probe, followed by ligation with T4 DNA ligase. Consequently, the formed circular padlock probe, coupled with primer probe and phi29 DNA polymerase, can then generate a long DNA strand based on the RCA reaction. Therefore, since [Ru(NH3)6]3+, the electrochemical species, may bind to the anionic phosphate of the DNA strands, cascade electrochemical signals are obtained, and sensitive electrochemical methods for the detection of phosphorylation and for the assay of PKA activity can be proposed. Electrochemistry at the Gold Electrodes. Nyquist plot of impedance of the electrode has been obtained after each treatment of the gold electrode. In the impedance spectra, the linear part at lower frequencies corresponds to diffusion and the semicircle portion at higher frequencies relates to the interfacial charge transfer resistance. In Figure 2, the bare electrode contains no semicircle portion, and after the immobilization of substrate peptide, a semicircle can be observed indicating the increased interfacial charge transfer resistance. After phosphor142

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surface. As shown in Figure 4, the redox charges of [Ru(NH3)6]3+ increase with PKA and reach the saturation

Figure 2. Nyquist plot of impedance of the electrode obtained after each treatment, from bottom to top: the bare gold electrode; the immobilization of substrate peptide; phosphorylation on the peptide by 50 unit/mL PKA; the capture of primer probe by Zr4+; the execution of RCA.

Figure 4. Chronocoulometry curves for the modified electrode. The peptide on the electrode was phosphorylated by (a−i) 0, 5, 20, 50, 200, 250, 400, 450, and 500 unit/mL PKA.

ylation on the peptide, the negatively charged phosphorylation sites electrostatically repel the redox species, Fe(CN)63−/4−. Moreover, the latter primers captured and long DNA strands generated are also negatively charged, which can further inhibit interfacial charge transfer. So, following the experiment process, the semicircle diameter becomes larger and larger (Figure 2). We have also employed CV to characterize the electrochemistry of the modified electrode with the electrochemical species [Ru(NH3)6]3+ which may bind to the long DNA chains (RCA product) on the electrode surface. Figure 3 shows that

condition when the PKA concentration is higher than 500 unit/ mL. Anson plots can then be obtained by plotting charge versus t1/2. The linear part of the Anson plot can be extrapolated back to time zero to obtain the intercept for the plot, which can then be used to determine the concentration of PKA (Figure 5). The

Figure 5. Calibration curve corresponding to the increased redox charge of [Ru(NH3)6]3+ for variable concentration of PKA from 0 to 1000 unit/mL. Error bars represent standard deviations of measurements (n = 3). The inset shows a linear relationship between the charge and the concentration of PKA from 5 to 500 unit/mL.

Figure 3. Cyclic voltammograms obtained at substrate peptide modified electrode, pretreated by (a) 0 unit/mL, (b) 20 unit/mL, and (c) 400 unit/mL PKA. Scan rate: 100 mV/s.

results shown in the inset of Figure 5 have revealed a good linear relationship between the charge and the concentration of PKA. The linear fitting equation is y = 0.69657 + 0.00204x, where y is the charge, ×10−6 C; x is the concentration of PKA; n = 3; r = 0.9967. The linear range is 5 to 500 unit/mL. The detection limit is calculated to be 0.5 unit/mL (S/N = 3), which is rather low, compared with some previous reports. In the control experiment, no obvious increase of the electrochemical signal can be observed, and in the inhibition experiments, with the increase of H−89, the redox charge of [Ru(NH3)6]3+ decreases significantly, demonstrating the specificity of this method and the potential to monitor the inhibition of phosphorylation in the future (Figure 6).

3+

two pairs of peaks can be obtained. While [Ru(NH3)6] diffused to MCH contributes to the peak pair at −0.2 V, [Ru(NH3)6]3+ electrostatically bound to the phosphate backbone of DNA contributes to the peak pair at −0.35 V. Moreover, the peak currents at −0.35 V increases with the concentration of PKA, indicating more RCA reaction occurs with more PKA-catalyzed phosphorylation; thus, a new approach to monitor phosphoylation may be developed. Quantitative Detection of PKA by Chronocoulometry. CC technique can provide an accurate approach to measure the redox charges of [Ru(NH3)6]3+ confined at the electrode 143

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(3) Cohen, P. (2002) Protein kinases - the major drug targets of the twenty-first century? Nat. Rev. Drug Discovery 1, 309−315. (4) Himmel, S., Wolff, S., Becker, S., Lee, D., and Griesinger, C. (2010) Detection and identification of protein-phosphorylation sites in histidines through HNP correlation patterns. Angew. Chem., Int. Ed. 49, 8971−8974. (5) Montminy, M. (1997) Transcriptional regulation by cyclic AMP. Annu. Rev. Biochem. 66, 807−822. (6) Domanski, D., Murphy, L. C., and Borchers, C. H. (2000) Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways. Anal. Chem. 82, 5610−5620. (7) Xu, X. H., Nie, Z., Chen, J. H., Fu, Y. C., Li, W., Shen, Q. P., and Yao, S. Z. (2009) A DNA-based electrochemical strategy for label-free monitoring the activity and inhibition of protein kinase. Chem. Commun., 6946−6948. (8) Song, H. F., Kerman, K., and Kraatz, H. B. (2008) Electrochemical detection of kinase-catalyzed phosphorylation using ferrocene-conjugated ATP. Chem. Commun., 502−504. (9) Wieckowska, A., Li, D., Gill, R., and Willner, I. (2008) Following protein kinase acivity by electrochemical means and contact angle measurements. Chem. Commun., 2376−2378. (10) Wang, J., Shen, M., Cao, Y., and Li, G. X. (2010) Switchable ″On-Off″ electrochemical technique for detection of phosphorylation. Biosens. Bioelectron. 26, 638−642. (11) Freeman, R., Gill, R., and Willner, I. (2007) Following a protein kinase activity using a field-effect transistor device. Chem. Commun., 3450−3452. (12) Allen, J. J., Li, M. Q., Brinkworth, C. S., Paulson, J. L., Wang, D., Hubner, A., Chou, W. H., Davis, R. J., Burlingame, A. L., Messing, R. O., Katayama, C. D., Hedrick, S. M., and Shokat, K. M. (2007) A semisynthetic epitope for kinase substrates. Nat. Methods 4, 511−516. (13) Green, K. D., and Pflum, M. K. H. (2007) Kinase-catalyzed biotinylation for phosphoprotein detection. J. Am. Chem. Soc. 129, 10− 11. (14) Wang, Z. X., Levy, R., Fernig, D. G., and Brust, M. (2006) Kinase-catalyzed modification of gold nanoparticles: A new approach to colorimetric kinase activity screening. J. Am. Chem. Soc. 128, 2214− 2215. (15) Sahoo, H., Hennig, A., Florea, M., Roth, D., Enderle, T., and Nau, W. M. (2007) Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection. J. Am. Chem. Soc. 129, 15927−15934. (16) Xu, X. H., Liu, X., Nie, Z., Pan, Y. L., Guo, M. L., and Yao, S. Z. (2011) Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots. Anal. Chem. 83, 52−59. (17) Freeman, R., Finder, T., Gill, R., and Willner, I. (2010) Probing protein kinase (CK2) and alkaline phosphatase with CdSe/ZnS quantum dots. Nano Lett. 10, 2192−2196. (18) Ji, J., Yang, H., Liu, Y., Chen, H., Kong, J., and Liu, B. (2009) TiO2-assisted silver enhanced biosensor for kinase activity profiling. Chem. Commun., 1508−1510. (19) Kerman, K., and Kraatz, H. B. (2009) Electrochemical detection of protein tyrosine kinase-catalysed phosphorylation using gold nanoparticles. Biosens. Bioelectron. 24, 1484−1489. (20) Fujii, R., Kitaoka, M., and Hayashi, K. (2006) Error-prone rolling circle amplification: the simplest random mutagenesis protocol. Nat. Protoc. 1, 2493−2497. (21) Cheng, Y. Q., Zhang, X., Li, Z. P., Jiao, X. X., Wang, Y. C., and Zhang, Y. L. (2009) Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew. Chem., Int. Ed. 48, 3268−3272. (22) Hu, J. A., and Zhang, C. Y. (2010) Sensitive detection of nucleic acids with rolling circle amplification and surface-enhanced raman scattering spectroscopy. Anal. Chem. 82, 8991−8997.

Figure 6. Calculated chronocoulometric results of the assays to detect 500 unit/mL PKA with different amount of H-89 as the inhibitor.

PKA Assay in Biological Fluids. To further demonstrate the application of the proposed method in biological fluids, a certain amount of PKA is added into fetal calf serum and the concentration of PKA in the serum is then measured with our proposed method. Table 1 shows that the relative errors of two Table 1. Detection of PKA with the Samples Prepared with Serum Samples

PKA added in the serum (unit/mL)

Results of detection with the proposed method (unit/mL)

Relative error (%)

1 2

20 50

21.21 45.79

6.05 8.42

different concentration detections are all within 10%, which promises that this method is utilizable for the detection of PKA in biological fluids such as serum.

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CONCLUSIONS In conclusion, we have proposed an electrochemical method to monitor phosphorylation and to assay the activity of PKA. Due to the linkage by Zr4+ between the phosphorylated peptide and DNA probe, and the signal amplification by RCA reaction, highly sensitive detection of PKA can be achieved. This work may not only offer new prospects for monitoring phosphorylation and protein kinase activities, but also propose a new aspect of the application of RCA for protein studies.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 25 83593596. Fax: +86 25 83592510. E-mail: [email protected] (G. Li).

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ACKNOWLEDGMENTS This work is supported by the National Science Fund for Distinguished Young Scholars (Grant No. 20925520), and the Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50108).

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REFERENCES

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