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Apr 15, 2013 - Chao Yin , Ming Wang , Chunyang Lei , Zhen Wang , Pei Li , Yong Li .... Lei Wang , Mengke Wang , Fanping Shi , Ziping Liu , Xingguang S...
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Dual-Readout Fluorescent Assay of Protein Kinase Activity by Use of TiO2‑Coated Magnetic Microspheres Jie Bai, Yunjie Zhao, Zhibin Wang, Chenghui Liu,* Yucong Wang, and Zhengping Li* Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, People’s Republic of China S Supporting Information *

ABSTRACT: A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO2 layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSPbased fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science.

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of the antibodies is both time-consuming and expensive.13 Therefore, many efforts have been devoted to the development of nonradioactive and antibody-free assays for monitoring protein kinase activity with various analytical techniques, such as fluorescence,14 electrochemistry,15,16 electrogenerated chemiluminescence,17 nanoparticle-based approaches,18−21 mass spectrometry, 22−24 surface plasmon resonance,25,26 and quartz-crystal microbalance.27 Unfortunately, a majority of these methods require labor-intensive procedures, sophisticated preparation, or expensive instrumentation. Among these existing protein kinase assays, fluorescence approaches have particular advantages, such as easy readout, high throughput, and low sample volume. Fluorescence-based assays for protein kinase activity generally use a fluorescently labeled substrate peptide specific to the target kinase and subsequently monitor the fluorescence change upon phosphorylation, which requires separation of the fluorescent phosphopeptides from the unphosphorylated ones, for example, by electrophoresis,28,29 resulting in time-consuming and laborintense procedures. To overcome this limitation, fluorescence

rotein phosphorylation by kinases is one of the most important posttranslational modification mechanisms in the process of cellular signal transduction, which plays a critical regulatory role in many fundamental biological processes including apoptosis, cell growth, and cellular signal communications.1 Aberrations in protein kinase activities and abnormal protein phosphorylation states can result in a number of diseases, such as diabetes,2 Alzheimer’s disease,3 and cancer.4 So protein kinases are a group of especially important targets for drug therapy.5 Therefore, sensitive and widely applicable assays for monitoring protein kinase activity are of great significance for further understanding the molecular mechanism of signal transduction, clinical diagnosis, and development of targeted therapy. Conventional methods for monitoring protein kinase activities are radiometric assays, which typically rely on transfer of radioactive phosphate of γ-32P from ATP to the specific substrate peptides, catalyzed by protein kinases.6−9 The radioactive 32P is hazardous and has a short half-life, resulting in difficulties of radioactive waste disposal and the need to replenish stocks frequently. An alternative to radiometric assays is immunoassay by using phosphospecific antibody to recognize the phosphosubstrate produced by kinase reaction.10−12 However, the phosphospecific antibodies can exhibit cross reactivity with nonphosphorylated residues, and the preparation © 2013 American Chemical Society

Received: March 17, 2013 Accepted: April 15, 2013 Published: April 15, 2013 4813

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polarization (FP) methods have been applied to detection of protein kinase activity based on specific combination of fluorescent phosphopeptides with phosphoantibodies30 or phosphopeptide-binding nanoparticles.31 The FP-based approaches can detect kinase activity in homogeneous solution with simple procedures. However, it has been reported that the FP-based detection is very sensitive to fluorescence interference, and it is liable to produce false positives because many cellular components can bind to the fluorescent peptides.32,33 On the basis of fluorescence resonance energy transfer (FRET) from cyan fluorescent protein to yellow fluorescent protein and specific binding interaction between phosphoamino acid binding domain and phosphopeptide, Tsien and co-workers34,35 have developed fluorescently encoded reporters for detection of protein kinase activities. Lawrence, Imperiali, and co-workers36−38 have also reported fluorescent chemosensors for monitoring protein kinase activities, in which the fluorescence signal is generated by increasing Mg2+-binding affinity to the chemosensors upon phosphorylation of the specific substrate peptides. The encoded reporters and chemosensors offer great potential for detection of protein kinase activities in a real-time manner or in living cells. However, these sensors demonstrate very modest fluorescence changes upon phosphorylation of substrate peptides by protein kinase reaction. For the encoded reporters, only 20−50% of phosphorylation-induced changes in the ratios of yellow to cyan emissions can be observed. A 3−5-fold increase in fluorescence intensity upon phosphorylation is the greatest change thus far for the chemosensors. In addition, fabrication of both the encoded reporters and the chemosensor is complicated and they are specific to only one kinase, which limit their wide application. More recently, Hong and co-workers32 and Whitten and co-workers39 reported the fluorescence quenching-based methods for detection of protein kinase activities by using bis(Zn2+-dipicolylamine) complex and fluorescent conjugated polymer, respectively. Although the fluorescence quenching-based methods are rapid, versatile, and homogeneous, they are negative assays that often suffer from the disadvantage that it can be difficult to judge if a reduction in fluorescent signals is due to poor assay performance or the presence of kinase target.40 Therefore, a rapid, versatile, sensitive, and cost-effective method for detection of protein kinase activities is highly desired for high-throughput screening of kinase inhibitors and for disease diagnosis. Recently, magnetic separation with functionalized magnetic nanoparticles or microspheres has offered a rapid, cost-effective, and high-throughput separation platform for bioassays. It has been documented that TiO2 can specifically bind with phosphate groups on protein and substrate peptides.41,42 Herein, we demonstrate in this paper a simple, highly sensitive, robust, and reliable platform for detection of protein kinase activities based on magnetic separation with TiO2-coated Fe3O4/SiO2 magnetic microspheres (TMSPs). The TMSPs show extremely high binding selectivity for phosphopeptides over nonphosphorylated ones, resulting in an ultrahigh signalto-background ratio. Therefore, high detection sensitivity for protein kinases can be achieved and the corresponding detection limit for protein kinase A (PKA) is as low as 0.1 milliunit/μL. Moreover, this method is also successfully applied to detect forskolin/IBMX-stimulated activation of PKA in cell lysates.

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EXPERIMENTAL SECTION

Chemicals and Materials. Fe3O4-encapsulated magnetic SiO2 microspheres (with an average diameter of ∼5 μm) were supplied by BaseLine Chromtech (Tianjin, China). PKAspecific substrate peptide (FITC-LRRASLG, where FITC = fluorescein isothiocyanate) and Akt1-specific substrate (TAMRA-CKRPRAASFAE, where TAMRA = tetramethylrhodamine) were synthesized by GL Biochem Ltd. (Shanghai, China). cAMP-dependent protein kinase (PKA) catalytic subunit was obtained from New England Biolabs. Akt1, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma−Aldrich. H-89 was purchased from EMD Bioscience and ATP was from Sangon Biotech Co., Ltd. (Shanghai, China). All other reagents were of analytical grade and were used as received without further purification. All aqueous solutions were prepared and diluted with ultrapure and sterilized water. Synthesis and Characterization of TiO2-Coated Magnetic Microspheres. TMSPs were prepared by hydrolysis of tetrabutyltitanate [Ti(OC4H9)4, TBOT] on the surface of magnetic SiO2 microspheres (MSPs). Briefly, 0.1 g of MSPs was dispersed in 2 mL of absolute alcohol under ultrasonication, followed by the slow addition of 50 μL of TBOT. Then dilute hydrochloric acid (pH = 3) was added into the mixture gradually with vigorous stirring to control the hydrolysis reaction. Hydrochloric acid was slowly added until obvious homogeneous nucleation and growth of TiO 2 appeared, when a milky white suspension was observed after magnetic separation of the MSPs onto the wall of the tube. The as-prepared TMSPs were magnetically isolated, washed repeatedly with ethanol, and allowed to dry naturally. Then the TMSPs were annealed at 350 °C for 2 h and finally dispersed in pure acetonitrile (ACN) before use. The size and morphology of the TMSPs were characterized by scanning electron microscopy (SEM) (KYKY-2008B, KYKY, China) with accelerating voltage of 25 kV. Energy-dispersive Xray analysis (EDX) was also performed to test the elemental composition of the TMSPs. HeLa Cell Culture and Preparation of Cell Lysate. HeLa cells (6 × 105 cells) were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum at 37 °C in a 5% CO2−95% air incubator. Then the culture medium was replaced by serumfree RPMI 1640, and the cells were incubated for 4 h before stimulation. Subsequently, the cultured cells were treated with different concentrations of forskolin and IBMX in dimethyl sulfoxide (DMSO) to activate intracellular PKA. DMSO (equal volume) instead of forskolin/IBMX solution was added to the medium for unstimulated sample. The cultured cells were washed three times with normal saline after 30 min of stimulation. Then, a commercial kit (BSP022, Sangon, Shanghai) was used to extract active proteins from the cultured HeLa cells. Briefly, the cells were removed by scraping and lysed in lysis buffer containing protease inhibitor, dithiothreitol (DTT), and phenylmethanesulfonyl fluoride (PMSF). The mixture was sonicated three times for 30 s on ice at intervals of 60 s and then centrifuged at 22 000 rpm for 15 min at 4 °C. The resulting cell lysate was aliquoted and stored at −20 °C before use. Total protein concentrations in cell lysates were quantified by the modified Lowry protein assay kit with bovine serum albumin as the standard (SK4041, Sangon, Shanghai). The total protein concentration of cell 4814

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lysate was diluted to 10 μg/mL for the proposed kinase activity assay. Standard Procedures for TiO2-Coated Magnetic Microsphere-Based Protein Kinase A Assay. In a typical 100 μL phosphorylation reaction system for PKA, FITCLRRASLG peptide (1.5 μM) was treated with a certain amount of PKA or cell lysate (total protein concentration 10 μg/mL) at 30 °C for 60 min in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 3.0 μM ATP, and 0.05% Tween 20. After the phosphorylation reaction, the mixture was directly mixed with an equal volume of ACN containing 0.5 mg/mL TMSPs and incubated for 5 min at room temperature to capture the phosphopeptides on TMSPs. The phosphopeptideloaded TMSPs were washed three times with Tris-HCl buffer and finally collected via magnetic separation. Dual readout pathways were carried out to determine the amount of captured fluorescent phosphopeptides on TMSPs as a function of PKA activity. The fluorescence signals were read out by use of either a common fluorescence spectrophotometer or a fluorescence microscope. On one hand, the captured phosphopeptides could be eluted into the solution by incubating the peptide-loaded TMSPs with 200 μL of 0.5% NH3·H2O for 5 min. Then the supernatant was collected for fluorescence intensity measurement on an F-4500 fluorescence spectrophotometer (Hitachi, Japan). On the other hand, the fluorescence signal of phosphopeptide-loaded TMSPs could also be directly monitored under a fluorescence microscope without releasing the fluorescent phosphopeptides from the TMSPs. For the fluorescence imaging analysis, 5 μL of the TMSPs was directly dropped onto a glass slide with a 0.17 mm coverslip, and the fluorescence imaging test was performed on a FV-1000 laser-scanning fluorescence confocal microscope (Olympus, Japan) by using a 488 nm laser, and the fluorescence emissions were collected in the range of 500−600 nm. For PKA inhibitor assay, the experiments were carried out via similar procedures as those for PKA assay stated above, except for the involvement of a fixed PKA concentration of 0.1 unit/ μL and varied concentrations of H-89 (0−10 μM) in the reaction mixture. Simultaneous detection of PKA and Akt1 activities was performed by incubating FITC-LRRASLG and TAMRACKRPRAASFAE (1.5 μM each) with different concentrations of PKA and Akt1 in the assay buffer. Other experimental conditions were the same as those stated for PKA assay.

Scheme 1. Schematic Illustration of the Proposed TMSPBased Dual-Readout Fluorescent Kinase Assaya

a

Note that the pictures are not drawn to exact scale.

beads. To verify the important role of dilute hydrochloric acid for the TMSP preparation, a control experiment was carried out by adding pure water instead of hydrochloric acid into the synthetic system. However, cloudy white precipitate immediately appeared upon the addition of water, indicating the hydrolysis rate was so rapid that heterogeneous and homogeneous nucleation and growth of TiO2 occurred simultaneously. These results clearly indicate that dilute hydrochloric acid plays a crucial role for the heterogeneous growth of TiO2 layer on the TMSPs by controlling the hydrolysis rate of TBOT.43 The morphology of the TMSPs was characterized by SEM; typical SEM images taken with different magnifications are shown in Figure S-1 in Supporting Information. It can be seen that all of the as-obtained TMSPs display high size uniformity with an average diameter of ∼5 μm. The representative energydispersive X-ray analysis (EDX) result of a single microsphere (Figure S-2, Supporting Information) reveals an abundance of Ti, Fe, and Si elements, and the EDX results of several randomly selected microspheres show similar atomic composition ratios of Si/Ti/Fe, clearly confirming the successful coating of TiO2 layer on the magnetic beads. Design Principle of the TiO2-Coated Magnetic Microsphere-Based Kinase Assay. Scheme 1 shows the schematic illustration of the design principle of the TMSP-based kinase assay with protein kinase A as a model target. This proposed assay shares the advantages of facile magnetic separation and extremely high binding selectivity of TMSPs for phosphopeptides over the nonphosphorylated ones. As shown in Scheme 1,

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RESULTS AND DISCUSSION Synthesis and Characterization of TiO2-Coated Magnetic Microspheres. The formation of TiO2 layer on the magnetic SiO2 beads is based on the hydrolysis of TBOT. Dilute hydrochloric acid (pH = 3) was gradually added into the mixture of the magnetic beads and TBOT in absolute alcohol to control the hydrolysis reaction. At the beginning, when a small fraction of hydrochloric acid was added, the hydrolysis rate of TBOT was relatively slow. So after magnetic isolation of the magnetic beads onto the wall of the tube, a clear solution could be observed, indicating that heterogeneous nucleation and growth of TiO2 on the surface of beads preferentially occurred over homogeneous processes. As the hydrochloric acid increased to certain content, the hydrolysis rate of TBOT was accelerated and thus obvious homogeneous nucleation and growth of TiO2 in the solution would appear. Therefore the preparation of TMSPs was completed when a milky white suspension was just observed after separation of the magnetic 4815

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Figure 1. Effect of ACN on the binding specificity between TMSPs and the phosphopeptides. Other experimental conditions were in accordance with the standard procedures stated in the Experimental Section.

through dual readout pathways either by direct fluorescent imaging of the TMSPs or measurement of fluorescence intensity with spectrophotometer after release of the fluorescent phosphopeptides into solution. Effect of Acetonitrile on the Inhibition of Nonspecific Adsorption of Peptides on TiO2-Coated Magnetic Microspheres. According to the design principle of the proposed assay, the specific recognition capability of the TMSPs for capturing PKA-induced phosphopeptides versus nonphosphorylated ones is most important for the sensitive detection of kinase activity. The use of ACN for dispersing TMSPs is found to be crucial to avoid nonspecific adsorption of nonphosphorylated peptides on the TMSPs. So the effect of ACN was first investigated by mixing the PKA reaction system with equal volume of TMSPs dispersed in different components of ACN/H2O, then detaching the captured phosphopeptides from the TMSPs into 0.5% NH3·H2O, and subsequently recording the fluorescence spectra of the solution according to the standard protocols stated in the Experimental Section. As can be seen from Figure 1a, the nonspecific adsorption of unphosphorylated peptides is rather strong when the TMSPs are dispersed in aqueous solutions. However, as shown in Figure 1b, dispersing TMSPs in 100% ACN can completely eliminate the nonspecific binding and further facilitate the binding between phosphopeptides and TMSPs. Figure 1c shows the relationship between the signal-to-blank ratio and the ACN content. One can see from Figure 1c that the signal-toblank ratios are gradually enhanced with increasing ACN content and reach a maximum when 100% ACN is used for TMSP dispersion. These results are in good accordance with previous reports,44,45 in which ACN was also found to be effective in eliminating adsorption of nonphosphorylated proteins/peptides on immobilized metal affinity column (IMAC), because ACN may effectively break up the nonspecific hydrophobic interactions between peptides and metal surface. Therefore, the TMSPs are dispersed in pure ACN throughout this work to ensure high binding specificity between phosphorylated peptides and TMSPs. Dual-Readout Fluorescence Detection of Protein Kinase A Activity. After the highly specific recognition and capture of phosphorylated peptides, easy readout of the accumulated fluorescence signal on the TMSPs is another key issue for this proposed PKA assay. Two readout pathways denoted as FI approach, for measurement of fluorescence intensity after release of the fluorescent phosphopeptides into solution, and as FM approach, for direct imaging of the

Figure 2. (a) Fluorescence spectra of the proposed assay in the presence of different concentrations of PKA by using the FI signal readout pathway. PKA concentrations from bottom to top according to the peak at 519 nm: 0 (control), 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 unit/μL. Other experimental conditions are in accordance with the standard procedures stated in the Experimental Section. (b) Corresponding plot of fluorescence intensity at 519 nm with PKA concentrations.

when the fluorescently labeled substrate peptides are phosphorylated by protein kinase reaction, the fluorescent phosphopeptides can specifically bind on the TiO2 layer of TMSPs through their phosphate groups, while the nonphosphorylated peptides will not, resulting in enrichment of the fluorophores on the TMSP surfaces. The amount of fluorophores accumulated on the surface of TMSPs is proportional to the kinase activities, which can be monitored 4816

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Figure 3. (a−f) Representative fluorescence images of TMSPs under different conditions. PKA concentrations for panels a−f: 0, 0.005, 0.02, 0.05, 0.1, and 0.2 unit/μL. (g, h) Representative fluorescence intensity profiles of individual TMSPs shown in images c and d. (i) Average intensity per TMSP as a function of PKA activity.

fluorescent phosphopeptides on the TMSPs, are used respectively to determine the amount of captured phosphopeptides on TMSPs as a function of PKA activity. On one hand, it can be seen from Figure S-3 (Supporting Information) that captured phosphopeptides can be easily released from the TMSPs into solution after incubation with 0.5% NH3·H2O solution. Therefore, PKA activity can be assessed by measuring the fluorescence intensity (FI approach) of the resulting NH3·H2O solutions. Under the optimized experimental conditions such as NH3·H2O concentration and ATP concentration (see Supporting Information), Figure 2a shows fluorescence spectra of the TMSP-based system in the presence of different concentrations of PKA, and the plot of fluorescence intensity at 519 nm with PKA concentrations is shown in Figure 2b. The fluorescence signals increase significantly with increasing concentrations of PKA from 0.5

milliunit/μL to 0.5 unit/μL, and no obvious signal changes are observed when the PKA concentrations are further elevated higher than 0.5 unit/μL. This phenomenon should result from saturated binding of phosphopeptides on TMSPs at high concentrations of PKA due to the limited accommodation capacity of TMSPs for fluorescent phosphopeptides. As Shults and Imperiali37 have demonstrated, small fluorescence changes (typically less than 5-fold increase) upon phosphorylation of the peptide substrate produced by protein kinase constitute the major disadvantage of fluorescent kinase assays. Just recently, Lipchik and Parker46 have reported a protein kinase assay by combining time-resolved luminescence detection and terbium sensitization with a notable 16fold intensity increase upon phosphorylation, which is the greatest signal change we have noticed before for fluorescent kinase assays. In our work, one can see from Figure 2a that the 4817

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Figure 4. (a) Fluorescence spectra of the TMSP-based system by the FI approach in the presence of different concentrations of H-89. The concentration of PKA is fixed at 0.1 unit/μL. (b) Dose−response curve of H-89 inhibition of PKA activity monitored by the change in fluorescence intensity at 519 nm. (c) Fluorescence images of TMSPs by the FM approach in the presence of different concentrations of H-89 (from left to right: 0, 0.01, 0.05, 0.1, and 2 μM) at a fixed PKA concentration of 0.1 unit/μL.

fluorescence background in the absence of PKA is almost negligible (with a relative intensity of ∼45 at 519 nm), but the fluorescence intensity increases to ∼1900 when 0.2 unit/μL PKA is introduced, exhibiting an ultrahigh 42-fold signal increase. To the best of our knowledge, this value is the highest signal change thus far for fluorescent assays for kinase activity. These results clearly demonstrate the high selectivity of the TMSPs for capturing phosphorylated peptides over nonphosphorylated ones. Therefore, excellent analytical performance of the proposed assay is obtained and the detection limit of PKA is calculated to be 0.1 milliunit/μL (3σ, n = 11). Recently, several methods for detection of PKA activity have been developed by combining various signal amplification techniques such as DNA rolling circle amplification (RCA)47 and noble metal nanoparticle amplification strategies,17,42,48 which have been considered as the most sensitive assays for PKA activity detection despite their sophisticated procedures. The detection limits of these assays for PKA activity detection are in the range of 0.07−0.5 milliunit/μL. Therefore, the sensitivity of the TMSP-based approach is superior or at least comparable to those of the known most sensitive assays. Moreover, this proposed approach is more rapid and simple and can be completed within 30 min after the kinase reaction. On the other hand, the diameters of the TMSPs are on the micrometer scale (∼5 μm), which can be clearly identified under a microscope. Therefore, the fluorescence signal of phosphopeptide-loaded TMSPs can also be directly monitored under a fluorescence microscopy (FM approach). Figure 3 shows representative fluorescence images of TMSPs after incubation with substrate peptides treated with different concentrations of PKA. As can be seen from Figure 3, the more PKA is used, the brighter the TMSP image. That is to say, as the PKA concentrations increased, more substrate peptides would be phosphorylated and then accumulated on the TMSPs,

making the TMSPs much brighter. These results are consistent with those obtained from the FI signal readout approach. Moreover, the intensity profile of each TMSP in the fluorescence images can also be recorded, as typically shown in Figure 3g,h. Therefore, the average intensity per TMSP can be statistically obtained for quantitatively determination of protein kinase activities (Figure 3i). It should be pointed out the fluorescence intensities of TMSPs produced by more than 0.1 unit/μL PKA cannot be quantitatively acquired because they would exceed the maximum value (>4095) that can be measured by the instrument of FV-1000 laser scanning fluorescence confocal microscope. Furthermore, the lowest PKA concentration that can be discerned from the blank control is 0.001 unit/μL for the FM approach, indicating that the sensitivity is slightly lower than that of FI approach. Potential Applications for Inhibitor Screening. To test its potential applications in kinase inhibitor screening, the TMSP-based method is further applied in the inhibition assay. The experiments are performed under a fixed PKA concentration in the presence of different concentrations of H-89, a potent and cell-permeable inhibitor of PKA. As can be seen from Figure 4a (FI approach), with increasing concentration of H-89, the fluorescence intensities of the eluting solutions of TMSPs decrease gradually, indicating the inhibition of PKA activity and low levels of peptide phosphorylation. The relationship between fluorescence intensity at 519 nm and H-89 concentration is plotted in Figure 4b, from which the IC50 value (the half-maximal inhibitory concentration) is determined to be 39.5 nM, which is well consistent with that reported in the literature.49 Additionally, the inhibition assay is also evaluated by the FM signal readout approach. As shown in Figure 4c, the higher the concentration of H-89 involved, the darker the TMSPs are in the fluorescence images, further confirming the inhibition effect 4818

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Figure 5. (a) Fluorescence spectra of the TMSP-based system in the presence of different concentrations of Akt1 by the FI readout approach. Akt1 concentrations from bottom to top: 0 (control), 0.00005, 0.0002, 0.001, and 0.003 μg/μL. The excitation wavelength is 543 nm. (b) Multiplex and target-selective analysis of PKA and Akt1 by measuring the fluorescence intensities of FITC (λex 488 nm/λem 520 nm) and TAMRA (λex 543 nm/λem 576 nm) in the presence of no kinase (control), 0.01 unit/μL PKA, 0.0015 μg/μL Akt1, and 0.01 unit/μL PKA + 0.0015 μg/μL Akt1, respectively. (c) Fluorescence images of TMSPs in the presence of no kinase (control), 0.01 unit/μL PKA, 0.0015 μg/μL Akt1, and 0.01 unit/μL PKA + 0.0015 μg/μL Akt1, respectively. The FITC channel is excited at 488 nm and fluorescence emissions are collected in the range 500−540 nm; the excitation wavelength of TAMRA channel is 543 nm, and fluorescence emissions are collected in the range 555−600 nm.

of H-89 on the PKA activity. These results clearly suggest that the TMSP-based assay is of great potential in screening kinase inhibitors. Simultaneous Detection of Protein Kinase A and Akt1. In order to validate the universality of the TMSP-based assay, the activity of Akt1, another randomly chosen kinase, is detected by the proposed method. TAMRA-CKRPRAASFAE is used as the specific substrate peptide of Akt1. As shown in Figure 5a, the fluorescence intensities increase gradually with increasing concentrations of Akt1, clearly indicating that this new strategy can be easily extended for other kinase activities and inhibition assays. Furthermore, intracellular signaling pathways are now an area of intense investigation for better understanding of many fundamental biochemical processes, which are generally associated with several protein kinases. Therefore, simultaneous quantitative analysis of multiple kinase activities is of great significance for the study of intracellular signaling pathways.

Toward this goal, we select PKA and Akt1 as targets to demonstrate the multiplexing capability of our method. The specific substrate peptides of PKA (LRRASLG) and Akt1 (CKRPRAASFAE) are labeled with fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TAMRA), respectively. The reaction systems containing FITC-LRRASLG, TAMRACKRPRAASFAE, reaction buffer, and ATP are treated with PKA, Akt1, or a mixture of PKA and Akt1, respectively. As shown in Figure 5b, the fluorescence signals corresponding to those fluorescent peptides are detected only when their corresponding kinase is present, and the composition of kinases can be read directly from the fluorescence emission at different channels. The fluorescence images shown in Figure 5c demonstrate that the presence of PKA or Akt1 can also be clearly identified under a fluorescent microscope. One can see from Figure 5c that the signal response to specific kinase is not affected by the presence of other kinases, indicating that the 4819

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signal-to-background ratio, which is known to be the highest change thus far for fluorescent assays for kinase activity. Therefore, due to these excellent properties of multifunctional TMSP beads, high sensitivity and rapid detection of protein kinase activity and inhibition are achieved in this work. Furthermore, this proposed approach is also successfully applied to simultaneous detection of multiplex kinases, showing great potential for kinase-related drug discovery and kinase analysis in signal-transduction pathways.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text and four figures showing characterization of TMSPs, effect of NH3·H2O on the release of TMSP-captured phosphopeptides, and effect of ATP concentration on detection of PKA activity. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. Fluorescence spectra of the TMSP-based system via the FI readout approach for detection of cell PKA activities stimulated by different concentrations of forskolin/IBMX. The total protein concentrations of HeLa cell lysates are all 10 μg/mL.

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (C.L.), [email protected] (Z.L.). Notes

The authors declare no competing financial interest.

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TMSP-based method is suitable for simultaneous detection of multiple kinases. Detection of Drug-Stimulated Activation of PKA in Cell Lysates. Protein kinases play crucial roles in intracellular signaling pathways and their activities are highly regulated in a cell. Therefore, a practical kinase assay should be able to work in complex biological samples, particularly in cell lysates. We further investigated whether the TMSP-based method could be applied to detect PKA activity in cell lysates. As is well-known, forskolin is capable of stimulating adenylyl cyclase and IBMX is an effective inhibitor of phosphodiesterase. Therefore, the stimulation of human cells with the combination of forskolin/ IBMX can efficiently increase the intracellular levels of cAMP, leading to the activation of cAMP-dependent PKA. In this work, HeLa cells were stimulated with different concentrations of forskolin and IBMX for PKA activation, and the PKA activities in cell lysates were evaluated by the proposed TMSPbased FI approach. As can be seen from Figure 6, the cell lysate without drug stimulation induced only negligible fluorescence signal compared with the blank control. However, samples of the stimulated HeLa cell lysates all generate obviously increased fluorescence signals, and the fluorescence intensities increase with increasing concentrations of forskolin/IBMX. Therefore, the drug-triggered activation of PKA in cells can be clearly detected via the TMSP-based method, indicating that this proposed approach works well in complex biological media and is feasible for in vitro detection of cell kinase activities.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (20925519, 91127035, 21105020), Program for Changjiang Scholars and Innovative Research Team in University (IRT 1124), Doctoral Fund of Ministry of Education of China (20111301130001), and the Natural Science Foundation of Hebei Province (B2011201175, B2012201108).

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CONCLUSIONS In conclusion, a simple and highly sensitive protein kinase and inhibition assay is developed based on the specific recognition utility of TMSPs for kinase-induced phosphopeptides. This versatile TMSP-based approach has several distinct advantages. First, the use of micrometer-sized magnetic beads greatly increases the surface area for accommodation of TiO2 layer as well as the phosphopeptides. Second, the magnetic characteristic of TMSPs facilitates simple concentration and washing steps within the kinase assay. Third, the TMSPs show extremely high selectivity for capturing phosphopeptides over nonphosphorylated ones, resulting in an ultrahigh fluorescence 4820

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Analytical Chemistry

Article

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