Label-Free, Versatile, Real-Time, and High-Throughput Monitoring of Tyrosine Phosphorylation Based on Reversible Configuration Freeze

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLES13 Jul 2022Label-Free, Versatile, Real-Time, and High-Throughput Monitoring of Tyrosine Phosphorylation Based on Reversible Configuration Freeze Yongxin Chang, Miao Guo, Mengyuan Song, Wenjing Sun, Dongdong Wang, Minmin Li, Jixia Wang, Yahui Zhang, Haijuan Qin and Guangyan Qing Yongxin Chang CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning University of Chinese Academy of Sciences, Beijing 100049 Google Scholar More articles by this author , Miao Guo CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Mengyuan Song CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Wenjing Sun CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Dongdong Wang CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Minmin Li CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Jixia Wang CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Yahui Zhang CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning Google Scholar More articles by this author , Haijuan Qin Research Centre of Modern Analytical Technology, Tianjin University of Science and Technology, Tianjin 300457 Google Scholar More articles by this author and Guangyan Qing *Corresponding author: E-mail Address: [email protected] CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, Liaoning College of Chemistry and Chemical Engineering, Wuhan Textile University, Wuhan 430200, Hubei Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202202070 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Tyrosine Phosphorylation (pTyr) is a critical and ubiquitous regulation mechanism in biology that plays a central role in controlling intracellular signaling networks. Precise recognition and specific detection of pTyr peptides have been of great importance for both discoveries of disease biomarkers and screening of therapeutic drugs, especially cancers. Here we report a label-free, versatile, real-time, and high-throughput detection strategy for phosphopeptide (PP) based on reversible configuration freeze of a unique hemicyanine-labeled 2-(2′-hydroxyphenyl)-4-methyloxazole (H-HPMO). By taking advantage of the “OFF–ON” transition of fluorescence, H-HPMO–Cu2+ complex displays a highly sensitive and selective response to PPs with modified sites on serine, threonine, and tyrosine. Specific recognition of Tyr PPs is achieved by performing a simple logic gate operation and introducing Ca2+ interference as an input. This PP detection approach is universal for various peptide sequences and displays high potential in large-scale kinase inhibitor screening, which will promote the development of targeted anticancer drugs. Download figure Download PowerPoint Introduction Phosphorylation of proteins is one of the most fundamental, common, and important biological processes in metabolism,1 which controls numerous intracellular signal transductions and regulates various cell activities, including cell growth, differentiation, and apoptosis.2–5 Protein kinases are one class of enzymes that modify other proteins by chemically adding the terminal γ-phosphate group of adenosine triphosphate (ATP) to serine (Ser), threonine (Thr), or tyrosine (Tyr) residue.6 Solid evidence has demonstrated that aberrant protein kinase activities can disrupt the protein phosphorylation networks, which results in various human diseases. For example, proliferative disorders, such as cancers, are often observed to have excess protein phosphorylation due to genetic mutations or other mechanisms.7,8 In eukaryotes, phosphorylation mainly occurs on Ser, Thr, or Tyr hydroxyl residues in protein substrates or peptides.9 Among them, tyrosine phosphorylation (pTyr, 1.8%) is far less common than serine (pSer, 86.4%) or threonine phosphorylation (pThr, 11.8%), but pTyr is closely related to the occurrence of various cancers.10 According to a 2009 report by Hitosugi and coworkers,11 most cancers result from the aberrant phosphorylation of Tyr by overactive kinases. Since the discovery of imatinib, a small molecule inhibitor that targets chronic myeloid leukemia in 2001, a new era of targeted cancer therapy began.12,13 As of November 2021, there were approximately 71 small-molecule inhibitors of protein kinases approved by the Food and Drug Administration (FDA).14,15 These drugs are predominantly multitargeted receptor tyrosine kinase inhibitors approved for the treatment of cancers.16 For example, the gene mutation of HER2 promotes the occurrence of lung cancer,17 and Tucatinib, a new drug approved by the FDA in 2020,18 is an oral Tyr kinase inhibitor with high specificity for human epidermal growth factor receptor 2 (HER2).19 The core link of targeted drug development is the large-scale screening of potential drug candidates, including the two-way selection of chemical compounds and pharmaceutical targets20,21 (Figure 1a). Drug screening is a long process and requires a huge amount of funding,22 which greatly limits the development of new drugs. Therefore, it is extremely important to establish precise, efficient, low-cost, and convenient inhibitor screening methods in the fields of drug discovery and targeted therapy of cancers.23 Figure 1 | Design concept for real-time monitoring Tyr phosphorylation. (a) Significance of kinase inhibitor screening for the development of cancer-targeted drugs, in which monitoring method for the kinase activity is critically important. (b) Recognition of phosphopeptide (PP) based on reversible configuration freeze of H-HPMO molecule by Cu2+. (c) Illustration of Ca2+-modulated logic gate operation for specific recognition of tyrosine PP. Download figure Download PowerPoint Developing cancer-targeted drugs often involves the monitoring of kinase activity,24 and mainstream methods include radiometric assay using radioactive ATP with 32P and immunoassay using a specific antibody to be directed against phosphorylated residues.25,26 These assay methods have made great advances in assessing protein kinase activity (PKA).27,28 However, using radioactive 32P for the detection of PKA will produce a lot of radioactive waste and cause serious environmental pollution. Long exposure time for analysis and precautious handling procedures for radioactive compounds severely limit its routine application in laboratories. A radioactivity-based assay is also unsuitable for physiological concentrations of ATP and poses a potential risk to human health. In addition, the half-life of radioactive ATP is very short, only 14 days (32P) or 25 days (33P), which is necessary to frequently order new isotopes. Besides, the energies of the radioisotopes are very high, which could produce a very obvious cross-interference phenomenon in high-throughput screening. Immunoassay with specific antibodies needs to consume various antibody reagents.29 The synthesis, separation, and purification of these antibodies require complicated procedures and expensive reagents, which largely increase the cost of PKA detection. Besides, fluorescent30–34/luminescent35–38 sensors have been developed toward specific sequences of phosphopeptides (PPs),39,40 in which complicated fluorophore synthesis and labeling on the peptide and the following purification process, are usually inevitable. In the past two decades, huge amounts of funds have been invested in the evaluation of PKA. However, hundreds of thousands of new compounds, particularly those extracted from natural products, still need to be evaluated. If the cost of drug screening could be reduced substantially, the development of cancer-targeted drugs would be accelerated. Here we report a label-free, versatile, real-time, highly efficient, and high-throughput monitoring strategy for pTyr based on a reversible configuration freezing of a 2-(2′-hydroxyphenyl)-4-methyloxazole (H-HPMO) molecule (Figure 1b). The addition of Cu2+ freezes the configuration of an H-HPMO and completely quenches its fluorescence. In contrast, the binding of phosphorylated peptide (PP) with Cu2+ triggers the recovery of the free rotation of H-HPMO, accompanied by a remarkable “OFF–ON” fluorescence transition. This effect is only observed on H-HPMO–Cu2+ but not on other Cu2+–complexes. Based on this mechanism, this system displays a highly sensitive and selective fluorescent response toward Tyr PPs superior to nonmodified peptides (NMPs), ATP, adenosine diphosphate (ADP), phosphate, and other interference substances, which largely facilitates the pTyr real-time monitoring test. Meanwhile, this strategy is versatile for the detection of Ser, Thr, and Tyr PPs as well as PPs with single, double, and triple phosphorylation sites, displaying a remarkable advantage in large-scale kinase inhibitor screening. Tests based on a small library containing 63 compounds extracted from the Chinese Medicine Science Research Center (Dalian) validated the high efficiency of our method for kinase–inhibitor screening. Reliable half-maximal inhibitory concentration (IC50) values could be obtained, while the cost of the sensor molecule was lower than $1 for 100 tests, and the total screening cost was less than 1/10 of the radioactive 32P ATP labeling method. Importantly, the specific pTyr detection can be achieved by implementing a basic logic gate function by introducing Ca2+ as an additional input, as illustrated in Figure 1c. Based on this principle, pTyr can be discriminated from pSer through the simple logic gate operation in a dual-site phosphorylation assay. This strategy matches the future tendency of multisite phosphorylation analysis closely and will benefit the multiple PKA evaluation. We are confident that this label-free method will have broad, practical applications in screening small molecule kinase inhibitors. The high efficient detection, low screening cost, and high throughput in combination have the potential to propel the development of target anticancer drugs remarkably. Experimental Methods Materials and instruments A detailed description of synthesis and characterization of H-HPMO Supplemental Information Scheme S1 and reference compounds R1-R6 can be found in the Supplemental Information. UV–vis and fluorescence titration experiments H-HPMO was dissolved in dimethyl sulfoxide (DMSO) to prepare a 5 mmol·L−1 stock solution. H-HPMO was diluted to the desired concentration with a 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic aci (HEPES) buffer solution (50 mmol·L−1, pH 7.3) before testing. A series of mono-, di-, and tri-Ser PPs (1pS, 2pS, and 3pS), mono-, di-, and tri-Thr PPs (1pT, 2pT, and 3pT), mono-, di-, and tri-Tyr PPs (1pY, 2pY, and 3pY), and NMP were dissolved in ultrapure water to prepare 5 mmol·L−1 standard solution, respectively. A stock solution of cation (0.1 mol·L−1, Na+, K+, Zn2+, Mg2+, Ca2+, Cr3+, Cu2+, and Al3+) and the anions (0.1 mol·L−1, SO42−, NO3−, CO32−, ADP2−, O2−, adenine, and adenosine) were prepared in ultrapure water. ATP solution (0.1 mol·L−1) was prepared with ultrapure water and stored at −20 °C. UV–vis and fluorescence experiments were performed in the HEPES buffer solution at room temperature. An equimolar mixed H-HPMO and Cu2+ (33 μmol·L−1), various mono-, di-, or tri PPs, and NMP solution were added into the above buffer solution, and the spectral data were recorded after 5 min. The fluorescence spectra were recorded from 500 to 800 nm with an excitation wavelength of 500 nm. All parameters remained constant during the data collection at different PPs. Calculation of limit of detection The limit of detection (LOD) was calculated based on fluorescence titration and calculated with the formula (LOD = 3σ/K), where σ is the standard deviation for replicating detections of blank solutions, and K is the slope of the calibration curve.41 The fluorescence spectra of H-HPMO–Cu2+ was measured eleven times to get the standard deviation (σ). The fluorescence intensity of H-HPMO–Cu2+ at 598 nm was plotted at a concentration of 2pY in the range of 0–0.02 mmol·L−1 (R2 = 0.996) to obtain the slope (K). 31P NMR titration experiment To investigate the binding between the H-HPMO–Cu2+ component and the phenylphosphate group (PhOPO32−), a 31P NMR titration experiment was performed to investigate the binding details. PhOPO32− anion was prepared according to the reference, and tetrabutylammonium (Bu4N+) worked as cation.42 The PhOPO32− solution and its mixture with Cu2+ or H-HPMO–Cu2+ (5.00 mmol·L−1) were prepared, respectively. d6-DMSO was selected as the solvent to guarantee the solubility of H-HPMO at a high concentration. All chemical shift changes of active hydrogen protons were recorded on a BRUKER AVANCE III 400–MHz spectrometer (Karlsruhe, Germany) and analyzed by MestReNova (Spain) software. Electron paramagnetic resonance test Electron paramagnetic resonance (EPR) spectra were recorded using a Bruker A200 (Karlsruhe, Germany) spectrometer. First, H-HPMO (5.4 mg, 1 mmol) was added to a solution of Cu (NO3)2 (1.9 mg, 1 mmol) in DMSO (0.5 mL). The mixture was stirred for 1 min at room temperature. Then, 0.5 mL of the mixture was transferred via syringe into a standard 4 mm quartz dry EPR tube and quickly placed in a sample chamber. For the titration of PhOPO32− to the H-HPMO–Cu2+ mixture, the PhOPO32− (9 mg, 2 mmol) was added to the above mixture of H-HPMO and Cu2+, using the same operation for the EPR test. Theoretical calculation All theoretical calculations were implemented by the density functional theory (DFT) method on the Gaussian 09 program. The 6-31G** basis set was assigned to the main group element. Geometry optimization of the ground state structures was carried out with DFT at the B3LYP level. The optimizations of H-HPMO and H-HPMO–Cu2+ were determined based on the energy-minimized structures. Kinase activity monitoring A stock solution of the peptide EE-13 (EAIYAAPAAYIAE, 5 mmol·L−1) was prepared prior to the day of the assay, which was stored at −8 °C. Cu(NO3)2 stock solution of 0.01 mol·L−1, MgCl2 stock solution of 0.1 mol·L−1, NaCl stock solution of 0.1 mol·L−1, and ATP (0.1 mol·L−1) were prepared and stored at −20 °C. Tris–HCl buffer solution (25 mmol·L−1) was prepared and adjusted to pH 7.4 with HCl solution (0.1 mol·L−1). First, 120 μL stock solution of peptide substrate EE-13 standard stock solutions were added to 2255 μL of Tris–HCl buffer solution. Then 15 μL ATP2− (0.1 mol·L−1), 300 μL MgCl2 (0.1 mol·L−1), and 300 μL NaCl (0.1 mol·L−1) were added to the above substrate solution. 10 μL kinase stock solution was added with final concentrations of 100 nmol·L−1 to start the reaction. Following incubation at 37 °C for 5 min, then 200 μL reaction solutions were added to 96-well plates separately. At the same time, a mixed solution of H-HPMO and Cu2+ (33 μmol·L−1) was quickly added, and then the changes in fluorescence were recorded. Each experiment was conducted three times to get the final result. IC50 determinations of c-Abl inhibitor A reaction mixture (200 μL) was prepared to contain 25 mmol·L−1 Tris–HCl (pH 7.4), 0.20 mmol·L−1 EE-13 peptide, 0.5 mmol·L−1 ATP, 10 mmol·L−1 MgCl2, 10 mmol·L−1 NaCl, 33 μmol·L−1 H-HPMO–Cu2+, and 100 nmol·L−1 c-Abl. In the experiment, imatinib standard solution with different concentrations ranging from 1 × 10−9 to 1 × 10−4 mol·L−1 were added separately to the above reaction mixture. Then, the changes in the fluorescent intensity were recorded over 120 min. For the IC50 value, the inhibition rate was plotted as a function of the inhibitor concentration and fitted to a 4-parameter logistic equation using the nonlinear regression procedure in SigmaPlot (version 10.0, SYSTAT software). Results and Discussion Responses of H-HPMO–Cu2+ complex to PPs First, the response of H-HPMO to different metal ions was examined. As shown in Figure 2a, H-HPMO has a strong fluorescent emission peak at 598 nm (the excitation wavelength: 500 nm) at room temperature. After adding equimolar Cu2+ (NO3− worked as an anion), remarkable fluorescence quenching was observed, and bright red fluorescence of H-HPMO turned into purple (Figure 2a inset). Under the same conditions, the addition of 5 equiv of other physiologically relevant metal ions, such as Na+, K+, Mg2+, Ca2+, Zn2+, Al3+, and Cr3+ did not cause any evidential change in the fluorescence. This result is consistent with an interesting phenomenon we observed earlier. The configuration of H-HPMO could be specifically frozen by Cu2+. Detailed mechanism analysis by cryogenic hydrogen nuclear magnetic resonance (1H NMR) revealed that intensive coordinate bonds could be formed between Cu2+ and phenolic hydroxyl, oxazole, and methoxyl groups of H-HPMO, which restricted the free rotation of these groups and blocked charge transfer, resulting in a “frozen” configuration,41 as illustrated in Figure 1b, left panel. Figure 2 | Satisfactory selectivity of H-HPMO–Cu2+ toward PPs. (a) Fluorescence spectra of H-HPMO before and after mixing with different cations. Inset shows the colors of the mixtures on an 8-hole ceramic colorimetric plate. (b) Peptide sequences of a NMP and various PPs, the phosphate-modified amino acids are indicated by red characters and their corresponding chemical structures. (c and d) Fluorescence spectra of H-HPMO–Cu2+ (33 μmol·L−1) in HEPES buffer solution (50 mmol·L−1, pH 7.30) at 20 °C before and after the addition of 2.5 equiv of NMP, 1pS, 1pT, or 1pY (c), or NMP, 2pS, 2pT, or 2pY (d), respectively. (e) Fluorescence titration of H-HPMO–Cu2+ (33 μmol·L−1) after the addition of different equivalents of 2pY. Inset shows a 2pY concentration-dependent increase ratio of the fluorescent intensity of H-HPMO–Cu2+, and the red line represents a linear fitting curve. (f) Comparison of fluorescent intensity (at 598 nm) of H-HPMO–Cu2+ after addition of various anions with different concentrations in a Tris buffer containing 10 mmol·L−1 MgCl2, 10 mmol·L−1 NaCl. Inset shows the colors of the mixtures. (g, h) Fluorescence spectra (g) and comparison of the fluorescent intensity (h) of H-HPMO–Cu2+ before and after the addition of 2pY (66 μmol·L−1) in the HEPES buffer solutions, this solution were mixed with tryptic digests of 12.5- or 25.0-fold (molar ratio relative to 2pY) of BSA, respectively, working as interference for the 2pY detection. The excitation wavelength was 500 nm, and the fluorescent intensity was monitored at 598 nm. (i) Comparison of the fluorescent intensity of H-HPMO–Cu2+ (33 μmol·L−1) added to tryptic digest of HeLa cell lysate, cell treated with sodium pervanadate for 0 and 15 min, respectively. Inset of (i) is a characterization of the global pTyr level of HeLa cells by Western blotting. Download figure Download PowerPoint As the product of the phosphorylation reaction, PP detection plays a central role in the PKA evaluation.43 Then the responses of H-HPMO–Cu2+ to various PPs were investigated whereupon a group of PPs with analogous sequences but with different phosphorylated residues or and different of groups mono-, or and was as the target (Figure Then, fluorescence spectra change of H-HPMO–Cu2+ (33 μmol·L−1) after the addition of various PPs or NMP solutions was recorded. The results that in the of 1pS, 1pT, and μmol·L−1), fluorescence of H-HPMO–Cu2+ were observed (Figure and the fluorescence by toward 1pS, 1pT, and 1pY were approximately and Information Figure By the addition of NMP μmol·L−1) did not any evidential change in the fluorescence. This result indicated that H-HPMO–Cu2+ could from was turned to Figure displays the fluorescence changes of H-HPMO–Cu2+ treated by various which obvious fluorescence To the between and 1pY and 2pY were selected as As shown in Information Figure fluorescent increase demonstrated that 2pY a remarkable change than that of and the increase ratio μmol·L−1 of 2pY was added to By this was for to and 2pT, their fluorescent was only than that by and Information Figure In addition to 2pY, H-HPMO–Cu2+ also remarkable fluorescent in response to the addition of or Information Figure their increase were than that of 2pY. We that between the Cu2+ and the phenolic of which to an additional This demonstrated that H-HPMO–Cu2+ was a versatile sensor for various PPs with Ser, Thr, or Tyr Then, 2pY was as an to the response of H-HPMO–Cu2+ to PPs. As shown in Figure the complex fluorescence with the increase of 2pY concentration and 2.5 equiv of 2pY was A linear between the fluorescent intensity at 598 nm and the 2pY concentration could be in a range from 0 to 20 μmol·L−1 with an of of Figure The was calculated to be nmol·L−1 based on The above data indicated the high potential of H-HPMO–Cu2+ in the detection of 2pY. changes in pH values the detection of PPs, and at different pH values were The results that H-HPMO–Cu2+ was to produce a fluorescence response to 2pY physiological (pH However, (pH or basic (pH solutions will to a in the fluorescence change which has a effect on the detection of PPs Information Figure the response of H-HPMO–Cu2+ to was also As shown in Information Figure in the of and μmol·L−1), fluorescence of H-HPMO–Cu2+ (33 μmol·L−1) were This that H-HPMO–Cu2+ could also be for the recognition of The strong of H-HPMO–Cu2+ To the in the PKA buffer (25 mmol·L−1, pH containing MgCl2 mmol·L−1) and NaCl mmol·L−1) was as the working solution, and the interference effect of various anions or that be in the PKA test was evaluated. Figure and Information Figure that H-HPMO–Cu2+ could not to various anions or substances, including ADP2−, NO3−, SO42−, adenine, and these were added in By the addition of 2pY remarkable fluorescent while the solution from purple to red (Figure which that H-HPMO–Cu2+ can be to the phosphorylation reaction by in the following the of H-HPMO–Cu2+ to PPs in complicated was evaluated. a nonmodified tryptic digests were which contain than 100 NMP sequences and are shown in Information First, H-HPMO was to with tryptic digests of (molar in HEPES buffer solution. The addition of equimolar Cu2+ the fluorescence of H-HPMO line in Figure Then 2 equiv of 2pY was added to the mixture. is to that the fluorescent intensity from to as shown by the red line in Figure Satisfactory was 25.0-fold tryptic digests were added to the H-HPMO solution (Figure The addition of 25.0-fold tryptic digests to an increase in the fluorescence line in Figure which be by the binding of peptides with Under this the addition of 2pY a remarkable fluorescence recovery from to This indicated that H-HPMO–Cu2+ could strong interference from with high and 2pY in the protein In addition, peptides containing also Cu2+ to an increase in the fluorescence. different sequences of peptides containing residues were As shown in Information Figure excess μmol·L−1) was added any fluorescence However, with the addition of 2pY μmol·L−1), a fluorescence was observed, which was the same as that of the solution the This indicated that the of numerous peptides did not with the detection of 2pY by with the protein sample detection of pTyr peptides from cells is cells were treated with sodium pervanadate of the activities of tyrosine which the pTyr level in the by the result of Figure Then the total proteins were extracted and into peptides by As shown in Figure H-HPMO–Cu2+ different responses to two peptide and cells treated with pervanadate for 15 and the fluorescent intensity from to with the increase of the pervanadate treatment These results that H-HPMO–Cu2+ can be for pTyr peptides in a peptide toward 2pY Since H-HPMO–Cu2+ strong binding to PPs and could PPs from the other Cu2+ could be to PPs. Therefore, of were (Figure and Information Scheme and As shown in Figure red and Information Figure (33 μmol·L−1) strong with Cu2+ in HEPES buffer solution, accompanied by remarkable fluorescent of 2pY or NMP μmol·L−1) to the above solution to the recovery of fluorescence. However, evidential between 2pY and NMP was which indicated that NMP would with 2pY detection. Figure | toward 2pY. (a) Chemical structures of reference compounds that are of Cu2+. Comparison of fluorescent intensity of the compounds (33 μmol·L−1) before and after addition of Cu2+, as well as that of the complex after addition of 2.5 equiv of 2pY or NMP The fluorescent are

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