Simultaneous Determination of Protein Kinase A and Casein Kinase II

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Simultaneous Determination of Protein Kinase A and Casein Kinase II by Dual-Color Peptide Biomineralized Metal Nanoclusters Li Zhang, Wei Song, Ru-Ping Liang, and Jian-Ding Qiu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02522 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Analytical Chemistry

Simultaneous Determination of Protein Kinase A and Casein Kinase II by Dual-Color Peptide Biomineralized Metal Nanoclusters Li Zhang, Wei Song, Ru-Ping Liang, and Jian-Ding Qiu* College of Chemistry, Nanchang University, Nanchang 330031, China *Phone: +86-791-8396-9518. Fax: +86-791-8396-9518 *E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

We design two artificial substrate peptides to synthesize blue-emissive Cu nanoclusters and redemissive Au nanoclusters, respectively. In addition to the biomineralization function, these two peptides retain the biological activity to be phosphorylated by protein kinase and digested by carboxypeptidase Y. In the absence of protein kinase, the peptides capped on the nanoclusters suffer consecutive exocleavage by carboxypeptidase Y, resulting in oxidation and thus fluorescence quenching of the nanoclusters due to the losing of peptide protection. In the presence of protein kinase A and casein kinase II, the phosphorylation modification on corresponding substrate peptides protects the peptides against carboxypeptidase Y digestion and then the fluorescence of the nanoclusters can be retained. Since a single excitation wavelength can excite the both nanoclusters, blue- and red- emissive signals can be collected at the same time and then the quantitative determination of protein kinase A and casein kinase II can be achieved simultaneously.


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INTRODUCTION Protein kinase-catalyzed phosphorylation is an essential post-translational modification and is of great importance in adjusting cellular biological processes including cell cycle progression, proliferation, differentiation, signal transduction, and apoptosis.1,2 The protein kinase superfamily contains more than 500 types of kinases, and among them Ser/Thr kinases can be categorized into basophilic kinases, proline-directed kinases, and acidophilic/phosphate directed kinases based on their different preferences on sequences around phosphorylated residues.3,4 Abnormal expression of protein kinase activity has been linked to the onset and development of numerous diseases, such as Alzheimer’s disease5,6 and cancer.7 Accordingly, convenient and cost-effective kinase assays are vital for the inchoate diagnosis of cancers and the identification of specific inhibitors as potential new drugs. Current methods for protein phosphorylation analyses include the use of phospho-specific antibodies,8 phosphate binding proteins,9 radioactive labeling,10 and mass spectrometry.11 For instance, a miniaturized format of [γ32

P]ATP-based phosphocellulose paper binding assay has been successfully proposed for high

throughput assessing kinase activities with good reproducibility and reliability.12 Nevertheless, most of such assays rely on the use of specialized biological reagents (such as antibody or protein binding domain) or need radiolabeling, while the mass spectrometry suffers expensive instrumentation and routine analysis of protein kinase using this method is often impractical. Most recently, the nanoparticle-based homogeneous colorimetric,13,14 electrochemical,15-17 and fluorescent strategies18-20 for protein kinase analysis have gained great attention due to their simple procedures and the relative high sensitivity and selectivity. Regrettably, most of the proposed methods for the phosphorylation analysis deal with individual protein kinase activity. Now, owing to the challenges in phosphoproteomics and kinomics to explore the regulation of


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complex kinase networks, versatile assays for simultaneous protein kinase determination are in urgent need.4 Metal nanoclusters are collections of two to tens of atoms with radii less than the Fermi wavelength of the electron.21 They have a discrete electronic state and exhibit strong sizedependent fluorescence over the region from ultraviolet to near infared,22,23 which facilitates their applications in chemical/biological sensing,24-28 bioimaging,29-31 and single-molecule studies.32,33 Metal nanoclusters have been studied for decades and many soft templates such as oligonucleotides,34,35 dendrimers,23,36 protein,37-40 and peptides22,30,41-43 have been employed for facile synthesis of nanoclusters in water solution. Particularly, artificial peptides with special amino acid sequences have been utilized to biomineralize noble metal nanoclusters for further label-free sensing of the enzymes involved in protein post-translational modifications and their inhibitors.18,44 For the majority of the peptide-templated synthetic approaches, however, additional reductants like NaBH4 were necessary, which may possess side effects in biological applications. Moreover, almost all the peptide-based methods focus on the noble metals, there have been no reported biomimetic approaches for precise producing other non-ferrous metal nanoclusters such as copper using peptides, which is not conducive to the applications of metal nanoclusters as optical probes in simultaneous determination of multiple targets. In this paper, we present a versatile nanocluster-based strategy to realize simultaneous protein kinase determination, which has been demonstrated by employing cAMP-dependent protein kinase A (PKA) and casein kinase II (CK2) as representatives of basophilic kinases and acidophilic/phosphate directed kinases, respectively. We have designed two artificial substrate peptides with the amino acid sequences of CCYLRRASLG (P1) and CCYRRRADDSD5 (P2) to biomineralize Cu nanoclusters (P1-CuNCs) and Au nanoclusters (P2-AuNCs), respectively. In


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addition to the biomineralization function, these two peptides possess biological activity to be phosphorylated by protein kinase and cleaved by carboxypeptidase Y (CPY). In other words, the peptides contain two domains, the domain 1 is CCY, where the phenolic groups of Y can easily reduce Cu2+/Au3+ to Cu0/Au0 under the conditions of alkalinity without additional reductants, and the sulfhydryl groups of C can efficiently capture the nanoclusters. Domain 2 is the specific substrates (LRRASLG and RRRADDSD5 for PKA and CK2, respectively) where the phosphorylation occurs at the S residues in the presence of corresponding kinases and ATP. Without the treatment of protein kinases, the peptides tethering on CuNCs or AuNCs can be digested by CPY into free amino acids concomitant with oxidation of Cu(0) or Au(0) to nonfluorescent Cu(I)/Cu(II) or Au(I) due to the losing of peptide protection.45 In the presence of PKA, phosphorylation of P1 occurs and the generated phosphate groups can escape from the CPY digestion, preventing the fluorescence quenching of CuNCs.4,46 Likewise, the treatment of CK2 leads to phosphorylation of P2 thus prevents the fluorescence quenching of AuNCs. Since the different-colored fluorescence emissions of CuNCs and AuNCs can be collected simultaneously by the same wavelength excitation free from spectra overlapping, simultaneous determination of PKA and CK2 is achieved by employing the different emissive nanoclusters (Scheme 1). Moreover, semiquantitative detection of protein kinases is realized with naked eyes under UV lamp irradiation.


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Scheme 1. Schematic illumination of the different-colored peptide-NCs biosensor for simultaneous detection of PKA and CK2 based on the blocking effect of phosphate groups on CPY digestion. EXPERIMENTAL SECTION Reagents

and

Chemicals.

The cysteine-terminated peptides CCYLRRASLG and

CCYRRRADDSD5 were obtained from GL Biochem (Shanghai, China). The protein kinase A (PKA) and casein kinase II (CK2) were purchased from New England Biolabs (NEB, Beijing). Carboxypeptidase Y (CPY) and adenosine 5′-triphosphate disodium salt (ATP) were obtained from Sigma-Aldrich (USA). Cu(NO3)2·3H2O and HAuCl4·4H2O were commercially available from Sinopharm Chemical Reagent Co., Ltd (China). Other reagents were analytical reagent grade without further purification. Ultrapure water (18.2 MΩ) was used throughout the experiments. Apparatus. The fluorescence spectra and UV−vis absorption spectra were measured on a F7000

fluorescence

spectrophotometer

(Hitachi,

Japan)


and

a

Shimadzu

UV-2450

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spectrophotometer (Japan), respectively. The morphologies of CuNCs and AuNCs were observed on a JEOL Ltd JEM-2010 transmission electron microscopy (TEM). A VG Multilab 2000X instrument was employed for X-ray photoelectron spectroscopy (XPS) measurements. The fluorescent photos were obtained using a digital camera. Preparations of P1-CuNCs and P2-AuNCs. For blue-emissive P1-CuNCs, under vigorous stirring, 16 µL of 25 mM Cu(NO3)2 aqueous solution was slowly dripped into 376 µL of 1.0 mM peptide 1 (CCYLRRASLG), followed by the addition of 8 µL of 0.5 M NaOH within 30 seconds, adjusting the solution to pH 9. The obtained solution was then sealed and incubated at 37 ºC for 10 h in the darkness under vigorous stirring. For red-emissive P2-AuNCs, under vigorous stirring, 200 µL of 10 mM HAuCl4 aqueous solution was slowly dripped into 180 µL of 1.0 mM peptide 2 (CCYRRRADDSD5). After 2 min incubation, 20 µL of 1.0 M NaOH was supplemented under vigorous stirring to make the pH approach 12. The obtained solution was then sealed and incubated at 37 ºC for 12 h in the darkness under vigorous stirring. The prepared P1-CuNCs and P2-AuNCs were finally concentrated with a 3 kDa centrifugal filter unit, and the free peptides were removed. Detection of PKA and CK2. 60 µL of 1 mM P1-CuNCs solution and 60 µL of 5 mM P2AuNCs solution were mixed with 80 µL of 20 mM Tris-HCl buffer (pH 7.5, 50 mM KCl, 10 mM MgCl2), 20 µL of PKA with different concentrations, 20 µL of CK2 with different concentrations, and 20 µL of 1.0 mM ATP solution. Ultrapure water was supplemented to reach a final volume of 360 µL. The mixture solution was then incubated for 40 min at 37 ºC for phosphorylation. Finally, 40 µL of 200 U mL-1 CPY was added, followed by incubation at 25 ºC for 40 min to facilitate the hydrolyzing of unphosphorylated peptide-NCs. Afterwards, the fluorescence spectra of the solutions were collected with the excitation wavelength of 370 nm.


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RESULTS AND DISCUSSION Characterizations of Metal NCs. The optical properties of P1-CuNCs and P2-AuNCs were firstly characterized. In accordance with previous studies on CuNCs,47,48 the P1-CuNCs show a distinct absorption band at about 325 nm (Figure S1a in the Supporting Information), which is ascribed to the interband electronic transitions of the CuNCs due to the discrete energy levels,49 another broad absorption band at about 280 nm can be attributed to the peptide transition where a fraction of the P1 peptide phenolic groups have been converted to phenoxide at pH ~9 (Figure S1a).22 The P2-AuNCs demonstrate two weak absorption peaks at 245 nm and 295 nm respectively (Figure S1b in the Supporting Information), indicating that phenolic peptide moieties of P2 have been completely converted to phenoxide at pH ~12.22 It is worth noting that a gradient absorption from 300 to 800 nm can be observed without obvious peaks belonging to AuNCs, which is similar to the absorption characteristics of previously reported peptidetemplated AuNCs.18,43 Both P1-CuNCs and P2-AuNCs display a maximum excitation peak at 370 nm (Figure S1a and S1b in the Supporting Information), and when excited at 370 nm, the P1-CuNCs show bright fluorescence emission at 450 nm, while the P2-AuNCs show strong fluorescence emission at 620 nm (Figure 1a and Figure S1). The transparent solution of P1CuNCs exhibit bright blue fluorescence while the brownish solution of AuNCs demonstrate bright red fluorescence under a 365 nm UV lamp (inset of Figure 1a). When the blue-emissive P1-CuNCs are mixed with the red-emissive P2-AuNCs, the obtained mixture shows well resolved dual emission bands under a single wavelength excitation at 370 nm (Figure 1a), favourably for their potential applications as dual-colored probes in simultaneous target sensing. To optimize the conditions of the synthesis process, the effect of precursor concentrations on the fluorescence intensity of NCs was studied. It is found that the fluorescence intensity of the P1-


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CuNCs enhances with the Cu2+ concentrations and arrives a platform at 1 mM (Figure S2 in the Supporting Information). Similarly, an optimal Au3+ concentration of 5 mM is chosen for the preparation of P2-AuNCs. The typical TEM images show the monodisperse P1-CuNCs with an average diameter of 1.2 nm as well as the spherical P2-AuNCs with an average diameter of 2.0 nm (Figure 1b and 1c).

Figure 1. (a) Fluorescence characterizations of P1-CuNCs (curve 1), P2-AuNCs (curve 2) as well as the mixture of P1-CuNCs and P2-AuNCs (curve 3) at 370 nm excitation. The insets show the photographs of aqueous solution of P1-CuNCs (left) and P2-AuNCs (right) obtained under visible light and 365 nm UV irradiation, respectively. TEM images of P1-CuNCs (b) and P2AuNCs (c). The insets show the diameter distribution of P1-CuNCs and P2-AuNCs, respectively.

Sensing mechanism. CPY is utilized in this assay to identify the kinase catalytic events since the kinase activities are susceptible to phosphorylation on peptides.46,50 As is well known, CPY is a representative exopeptidase that can hydrolyze peptides into free amino acids from the carboxyl-terminus (C-terminus).45 When the peptide-NCs are treated with protein kinase, the phosphorylated peptides tethering on NCs escape from the CPY cleavage,4,51,52 retaining the basic structure of peptide-NCs and avoiding the fluorescence quenching of NCs. To confirm the blocking effect and verify the suppression mechanism of phosphorylation on CPY cleavage, in-


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depth chemical states of the peptide-NCs in the presence and absence of protein kinases with ATP and CPY were further investigated through XPS. As we know, Cu atoms at zero oxidation state can be distinguished from Cu at higher oxidation states from the XPS spectrum by a shift in the Cu 2p3/2 core level peak to higher binding energies when the oxidation state increases from Cu(0) to Cu(I) or Cu(II).53 As expected, in the presence of PKA, P1-CuNCs show two intense peaks at 932.4 and 952.4 eV (Figure 2b), which are assigned to Cu 2p3/2 and Cu 2p1/2 of Cu(0), respectively,49,54,55 demonstrating that most of the Cu atoms in the peptide-CuNCs maintain their zero oxidation state. While without PKA, a broader peak of Cu 2p3/2 at a slightly higher binding energy of 932.8 eV with a characteristic satellite peak around 942.9 eV can be clearly observed (Figure 2a), indicating the presence of Cu(I) and Cu(II) as a result of oxidation of P1-CuNCs following the CPY digestion,56,57 which can be further confirmed by the decreased absorption band of CuNCs at about 325 nm after the CPY cleavage (Figure S3a in the Supporting Information).58 In other words, P1 forms a compact coating on the CuNCs to protect CuNCs from contact with the dissolved oxygen in the solution and alleviate oxidation or fluorescence quenching. The CPY digestion of P1 into free amino acids destroys the protective coating and thus facilitates the dissolved oxygen contacting with CuNCs, resulting in their fluorescence quenching and even oxidation into nonfluorescent phases.44 For P2-AuNCs solution, when it is treated with CPY without CK2, the absorption suffers evolution similar to the situation for P1CuNCs, and since there is no obvious absorption peak for AuNCs, only the variation of absorbance can be observed after the CPY cleavage (Figure S3b in the Supporting Information). For XPS characterizations, the Au 4f7/2 for P2-AuNCs could be deconvoluted into two distinct components centered at 83.8 and 85.2 eV assigned to Au(0) and Au(I),37 respectively. Obviously, a large percentage of Au(I) (92%) and a small amount of Au(0) (8%) can be observed (Figure


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2c), suggesting that P2-AuNCs lose the peptide protection after the treatment of CPY without CK2 since the peptides tethering on AuNCs are digested into free amino acids concomitant with the oxidation of AuNCs.44 Therefore, fluorescence quenching of AuNCs can be observed. Interestingly, a much higher percentage of Au(0) (93%) is observed if P2-AuNCs suffer phosphorylation by CK2 before the CPY treatment (Figure 2d), which can be attributed to the generation of phosphates on serine residues to block the CPY digestion and maintain the AuNCs stability. These experimental data reveals the close relationship between the phosphorylationcaused blocking of CPY digestion and the oxidation/fluorescence quenching of peptide-NCs, confirming the sensing mechanism shown in Scheme 1.

Figure 2. XPS spectra of Cu 2p electrons in P1-CuNCs incubated with ATP without (a) or with (b) PKA followed by the treatment of CPY. XPS spectra of Au 4f electrons in P2-AuNCs incubated with ATP without (c) or with (d) CK2 followed by the treatment of CPY.


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Sensing Performance. Firstly, the effect of reaction time of protein kinase catalyzing substrate peptides is studied by stopping the catalyzing reaction at different time intervals and analyzing the fluorescence intensity correspondingly. With increasing phosphorylation time, the fluorescence intensity shows an initial quick increase followed by a slow enhancement for both protein kinases, and no obvious change of the fluorescence intensity could be observed when the reaction is performed after 40 min (Figure S4 in the Supporting Information). Under the optimal phosphorylation time of 40 min, PKA and CK2 were then detected by the proposed strategy, respectively. For PKA, after peptide phosphorylation catalyzed by PKA in the existence of ATP, the CPY cleavage is suppressed and the P1-CuNCs maintain their optical properties. With the increasing concentrations of PKA, the fluorescence of P1-CuNCs demonstrates continuous enhancement (Figure 3a). It is found that the fluorescence intensity of P1-CuNCs increases rapidly with low concentrations of PKA and levels off thereafter (Figure 3b). The inset of Figure 3b reveals that the fluorescence intensity of P1-CuNCs linearly increases with the PKA concentrations over the range of 0.4-3 U mL-1 and the limit of detection (LOD) is about 0.1 U mL-1 (3σ). Likewise, for CK2, Figure 3c and 3d demonstrate that the fluorescence of P2-AuNCs increases linearly with the CK2 concentrations over the range of 0.8-7 U mL-1, and the LOD is calculated as 0.2 U mL-1. The data suggest that the proposed fluorescence sensor is of good responses to PKA and CK2, which is comparable to that of previous reports (Table S1) and has potential applications in kinase-peptide studies and disease diagnosis.


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Figure 3. (a) Fluorescence spectra of P1-CuNCs treated with varying concentrations of PKA (curve 1 to 10: 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 3.0, 6.0, 12.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (b) Fluorescence intensity as a function of PKA concentrations. Inset: linear plot of the fluorescence responses on PKA concentrations. (c) Fluorescence spectra of P2-AuNCs treated with varying concentrations of CK2 (curve 1 to 11: 0, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6, 7.0, 10.0, 20.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (d) Fluorescence intensity as a function of CK2 concentrations. Inset: linear plot of the fluorescence responses on CK2 concentrations.

Since a single excitation wavelength of 370 nm can excite both P1-CuNCs and P2-AuNCs to produce different-colored fluorescence emissions, simultaneous determination of PKA and CK2 by the proposed approach is achieved. Before the simultaneous determination of PKA and CK2, we investigate whether the two protein kinases can be differentiated by the sensing platform. As


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demonstrated in Figure 4a, when single PKA exists in the system, the fluorescence intensity of P1-CuNCs at 450 nm increases while the fluorescence emission of P2-AuNCs at 620 nm almost remains constant. Above results demonstrate a vital point: the fluorescence recovery of P1CuNCs is the result of serine phosphorylation catalyzed by PKA that suppresses the CPY cleavage on P1-CuNCs. Likewise, Figure 4b shows that only the fluorescence signal of P2AuNCs at 620 nm increases if CK2 alone exists in the system, while the fluorescence emission of P1-CuNCs at 450 nm remains constant. It’s clear that the two-colored peptide-NCs can be applied to differentiate these two kinases easily and effectively, and moreover, a mixture of P1CuNCs and P2-AuNCs can serve as ratiometric fluorescence probe in quantification of individual protein kinase with good selectivity and accuracy by eliminating environmental interference and light fluctuation.

Figure 4. (a) Fluorescence spectra of P1-CuNCs and P2-AuNCs mixture solution upon increasing the concentration of PKA (curve 1 to 9: 0, 0.8, 1.2, 1.6, 2.0, 2.4, 3.0, 6.0, 12.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (B) Fluorescence spectra of P1-CuNCs and P2-AuNCs mixture solution upon increasing the concentration of CK2 (curve 1 to 6: 0, 2.4, 3.2, 4.0, 7.0, 20.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). Excitation wavelength = 370 nm.


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The different-colored metal nanocluster probe is then utilized to quantitative determination of PKA and CK2 activity simultaneously. Figure 5 shows that if the blue- and red-emissive peptideNCs are together incubated with the target kinases (i.e., PKA and CK2), the γ-phosphoryl can be transferred from ATP to the free -OH of S residues in the corresponding peptide substrates, and the phosphorylated peptide-NCs can suppress the CPY digestion effectively, resulting in the fluorescence recovery of NCs. With increasing amounts of PKA and CK2, fluorescence signals of both blue- and red-emissive NCs increase (Figure 5a). It is found that the fluorescence intensity of P1-CuNCs at 450 nm increases linearly with the concentrations of PKA with the LOD around 0.4 U mL-1 (3σ) (Figure 5b). Remarkably, the fluorescence response of P1-CuNCs to PKA in the simultaneous detection system is comparable to that in the individual detection system (Figure 3), suggesting negligible interference of P2-AuNCs in the mixture on the PKA detection. For CK2 detection, the fluorescence intensity P2-AuNCs at 620 nm linearly increases with the concentrations of CK2 in the range of 0.8-7 U mL-1 and the LOD is about 0.2 U mL-1 (3σ) (Figure 5c). As a homogeneous and simple method for protein kinase sensing, it can also be visually realized under UV lamp irradiation. The photograph in Figure 6 demonstrates semiquantitative detection of PKA and CK2 under the 365 nm UV irradiation. When P1-CuNCs and PKA exist in solution, blue P1-CuNCs brighten with increasing the concentration of PKA due to the exclusive phosphorylation process on P1-CuNCs catalyzed by PKA (Figure 6a). Similarly, when P2-AuNCs and CK2 exist in solution, the color of red P2-AuNCs brightens with increasing the concentration of CK2 due to the phosphorylation process on P2-AuNCs (Figure 6b). If PKA and CK2 coexist in the solution, fluorescence signals of the two different-colored NCs (P1-CuNCs and P2-AuNCs) increase simultaneously, and mixed color is observed (Figure 6c).


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Figure 5. (a) Fluorescence characterizations of P1-CuNCs and P2-AuNCs mixture solution treated with various amounts of PKA (curve 1 to 8: 0, 1.2, 1.6, 2.0, 2.4, 3.0, 6.0, 12.0 U mL-1) and CK2 (curve 1 to 8: 0, 0.8, 1.6, 3.2, 4.8, 5.6, 7.0, 100.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (b) Fluorescence intensity at 450 nm as a function of PKA concentrations. Inset: linear plot of the fluorescence responses on PKA concentrations. (c) Fluorescence intensity at 620 nm as a function of CK2 concentrations. Inset: linear plot of the fluorescence responses on CK2 concentrations. Excitation wavelengths = 370 nm.

Figure 6. Visual determination of PKA and CK2 under the 365 nm UV irradiation. (a) Photograph of P1-CuNCs treated with increasing concentrations of PKA (from left to right: 0,


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3.0, 6.0, 12.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (b) Photograph of P2-AuNCs treated with increasing concentrations of CK2 (from left to right: 0, 4.0, 7.0, 20.0 U mL-1), followed by the supplementary of CPY (20 U mL-1). (c) Photograph of P1-CuNCs and P2-AuNCs mixture treated with increasing concentrations of PKA and CK2, followed by the supplementary of CPY (20 U mL-1). PKA concentrations were 12.0, 6.0, 3.0, 1.2, and 0 U mL-1, and CK2 were 0, 1.6, 4.0, 7.0, and 20 U mL-1 (from left to right).

CONCLUSION In this manuscript, a selective and simple peptide-NCs-based sensor is proposed to simultaneously monitor the activity of PKA and CK2 in homogeneous solution with dual-colored fluorescence outputs. The specific phosphorylation on the peptide substrate catalyzed by the corresponding protein kinase can escape from the CPY digestion, and maintain the fluorescence signals of peptide-NCs. Since P1-CuNCs and P2-AuNCs are excited by a single excitation light source, and the blue- and red-emissions are collected simultaneously without spectra overlapping, the activity of PKA and CK2 can be detected simultaneously. Compared with previously reported individual kinase determination methods, the present strategy realizes simultaneous analysis of PKA and CK2 with high selectivity. Moreover, the experimental methodology is simple, homogeneous, and nonradioactive, i.e., it doesn’t require immobilization of reaction components or products on papers or other solid phases, and meanwhile it avoids the need for radio-labeled ATP and thus saves the cost of addressing radioactive wastes/materials. Furthermore, rapid and precise quantitative detection of protein kinases is achieved by monitoring the variation of fluorescence spectra while visual semiquantitative sensing of protein kinases can be realized under the 365 nm UV irradiation. Finally, the proposed method is


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universal that can be applied in other protein kinases analysis. It is speculated that by modifying the corresponding peptide substrates with CCY residues and optimizing the NCs synthesis conditions (such as pH values and reagent concentrations), multi-colored fluorescent NCs probes can be facilely prepared to deal with different target protein kinases, facilitating potential multiplex sensing and future kinase network analysis. ASSOCIATED CONTENT Supporting Information Figure S1-S4 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (21265012 and 21265017), the Program for New Century Excellent Talents in University (NCET-13-0848) REFERENCES (1) Hunter, T. Cell 2000, 100, 113-127. (2) Pawson, T. Cell 2004, 116, 191-203. (3) Pinna, L. A.; Ruzzene, M. Biochim. Biophys. Acta 1996, 1314, 191-225.


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For TOC only


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Simultaneous Determination of Protein Kinase A and Casein Kinase II

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