Quantitative Mass Spectrometry Combined with Separation and

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Quantitative Mass Spectrometry Combined with Separation and Enrichment of Phosphopeptides by Titania Coated Magnetic Mesoporous Silica Microspheres for Screening of Protein Kinase Inhibitors Liyun Ji,† Jian-Hong Wu,‡ Qun Luo,† Xianchan Li,† Wei Zheng,† Guijin Zhai,† Fuyi Wang,*,† Shuang Lü,† Yu-Qi Feng,*,‡ Jianan Liu,† and Shaoxiang Xiong† †

Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Beijing Centre for Mass Spectrometry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China ‡ College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, PR China S Supporting Information *

ABSTRACT: We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO2/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r2 being 0.99. The IC50 values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/timeeffective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.

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scattering of the compounds or matrix and quenching.5 This creates a great demand for alternative methods to screen PKIs. One possible alternative for determination of enzyme activities and screening of inhibitors against enzymes of interest is mass spectrometry (MS) which allows the detection of even minute mass changes occurring in a substrate during enzymatic conversion.7−12 In contrast to UV-Vis, radioactivity, and fluorescence assays, there are several advantages to use MS for inhibitor screening. First, MS screening of enzyme inhibitors does not require labeling but relies entirely on the mass-to-charge ratio (m/z) of the reporter molecules. This avoids laborious, cumbersome, and costly derivatization and allows the use of native compounds of interest, which does not affect the binding characteristics and kinetics of the compounds

he great success in the discovery of protein kinase inhibitors (PKIs), including gefitinib (a specific inhibitor of the protein tyrosine kinase epidermal growth factor receptor (EGFR)1), imatinib directed against Bcr-Abl and other kinases,1,2 and sorafenib (the first oral multikinase inhibitor targeting Raf signaling pathway),3 as anticancer therapeutics has attracted intensive attention to screen novel PKIs from both synthetic and natural sources. As a consequence, a huge demand has arisen for cost-effective, rapid, and easy operating methods for kinase inhibitor screening. Screening techniques for enzyme inhibitors are mostly based on UV-Vis, radioactivity, and fluorescence detection, for example, enzyme-linked immunosorbent assay (ELISA), radioactive receptor−ligand binding assays, and time-resolved fluorometric assays such as DELFIA, etc.4−6 These approaches are usually accompanied with some disadvantages, for instance time/cost-consuming, intolerable of high concentrations of inhibitor compounds, and in need of radio-isotope labeling and particularly for the fluorometric assay, interference from autofluorescence or light © 2012 American Chemical Society

Received: November 4, 2011 Accepted: January 26, 2012 Published: January 26, 2012 2284

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have then applied the TiO2-deposited capillary column to screen Abelson tyrosine kinase (Abl) inhibitors.48 However, because of the lower abundance of phosphopeptides in EGFRcatalyzed reaction mixtures, the application of the TiO2deposited capillary column for capture of phosphorylated substrates in EGFR-catalyzed reaction mixtures was not successful. In the present work, therefore, we fabricated a novel type of TiO2 coated magnetic hollow mesoporous silica spheres (TiO2/MHMSS) as a solid phase microextraction (SPME) device to concentrate phosphorylated substrates from EGFR-catalyzed reaction mixtures prior to MALDI-TOF-MS characterization of EGFR inhibitors. To avoid stable isotopic labeling, a phosphopeptide, of which the sequence is similar to a phosphorylated substrate of EGFR, was synthesized as the internal standard. Under the optimal conditions, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides over a broad dynamic range, providing a rational foundation for accurate quantification of the low-abundance phosphorylated substrates. To our knowledge, it is the first reported application of TiO2-based magnetic mesoporous spheres to MS quantification of phosphopeptides and characterization of protein kinase inhibitors.

to the targets, and the assays thus do not require extensive validation.11−16 Second, MS can simultaneously detect a large range of compounds including substrates, products of enzymecatalyzed reactions, and internal standards. This confers MS screenings highly flexibility, and all compounds (substrates, products, inhibitors, and inhibitor/enzyme complexes) in the samples can be used to be the reporters.7 Furthermore, this unique feature of MS assays makes it possible to simultaneously characterize the activities of multiple enzymes and screen inhibitors against different enzymes from a compound library.8,10−20 Third, disadvantages related to fluorescencebased assays, e.g., interferences arising from autofluorescence or light scattering of the compounds or matrix, do not exist for MS assays. This may reduce the number of false positives and negatives.12,17,18 With the potential of high speed of analysis, high detection sensitivity, and relatively high tolerance against salts and biological contaminants, during the past decade, matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) has become an indispensable tool for enzyme inhibitor screening.11,13,14,21,22 For MALDI-TOF-MS quantification, the signal intensity is largely dependent on the analyte-matrix cocrystallization; thus, application of stable isotopic labeling to the internal standards, which are in general the isotopomers of the enzyme-catalyzed reaction products, is normally required for successful quantification because the isotopic pairs response identically in MALDI-MS analysis.23−26 Recently, a general method for the MALDI-TOF-MS-based quantitative characterization of PKIs was developed using synthetic peptide substrates bearing both N-terminal stable isotope tags and C-terminal MS-sensitivity enhancement reagents.27 However, because of the low relative ionization efficiency of phosphopeptides compared to that of most nonphosphorylated peptides, it is advantageous to prepurify the phosphopeptides from complex peptide mixtures for successful MALDI-TOF-MS analysis.28 The separation and enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) has been widely used in phosphorylated proteomic research.29−33 This approach implicates adsorbing phosphopeptides to chelated metal ions (Fe3+, Ga3+) followed by elution using basic aqueous solution.30,33 However, frequently nonphosphorylated peptides, in particular those containing multiple acidic residues, are also absorbed by the Fe- or Ga-based IMAC.33,34 To reduce the unexpected absorption of nonphosphorylated peptides, titanium dioxide (TiO2) and zirconium oxide (ZrO2) have been alternatively used as chromatographic materials for more selective capture of phosphopetides from the enzymatic digests of multiprotein complexes.35−42 Very recently, functionalized mesoporous TiO2,43 ZrO2,44 and CeO245 have been applied as affinity materials for the separation and enrichment of phosphopeptides in the enzymatic digests of multiple proteins. The metal oxides are coated to the magnetic core (Fe3O4) via a silicon thin film43,45 or graphene platform,46 conferring the fabricated mesoporous spheres and networks with the virtue of magnetic separability and enhanced surface area for efficient and rapid capture of phosphopeptides. One of us has recently prepared a TiO2 nanoparticle (NP)deposited capillary column (TiO2/NPDCC) by liquid phase deposition to separate and enrich the phosphopeptides from the tryptic digests of α-casein and bovine serum albumin (BSA).47 In the presence of 10-fold BSA, 50 fmol or lower of phosphopeptides in the digest mixtures can be detected. We

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EXPERIMENTAL SECTION Reagents and Materials. The protein tyrosine kinase EGFR, the epidermal growth factor (EGF), and trifluoroacetic acid (TFA) were purchased from Sigma. Other biological agents including the ELISA kits for EGFR inhibitor screening were purchased from Cell Signaling Technology (USA). Substrate peptides EAIYAAPFAKKK (SP1) and RRLIEDNEYTARG (SP2), phosphorylated substrates EAIpYAAPFAKKK (PS1) and RRLIEDNEpYTARG (PS2), and internal standard phosphopeptides DAIpYAAPFAKKK (IS1) and RRLIDDNEpYTARG (IS2) were purchased from HysBio Ltd. (Beijing, China). Acetonitrile (ACN) was purchased from Tedia, and the chemicals for fabrication of TiO2/MHMSS were purchased from Shanghai General Chemical Reagent Factory (Shanghai, China). The deionized water used in the experiments was prepared by a Milli-Q system (Millipore, Milford, MA). Synthesis of EGFR Inhibitors. Three potential EGFR inhibitors (Chart 1) were synthesized, and the details are described in the Supporting Information. Separation and Enrichment of Phosphopeptides Using TiO2 /MHMSS. The general procedure for the separation and enrichment of phosphorylated product of kinase-catalyzed reactions using TiO2/MHMSS is as follows unless otherwise stated. First, 30 mg of TiO2/MHMSS particles was suspended in 1 mL of deionized water, an aliquot (50 μL) of the peptide mixture of nonphosphorylated substrate, phosphorylated substrate, and internal standard in acidic solution (pH 2.5) or the resulting enzymatic reaction mixture (pH 2.5) was mixed with 5 μL of magnetic material suspension and incubated for 30 min at room temperature. After successive washing with 100 μL of acidic solutions (pH 2.5) twice and 100 μL water once, the trapped phosphopeptide on the magnetic spheres were eluted with 5 μL of 2.5% ammonium hydroxide and the eluent was collected for MALDI-TOF-MS analysis. During the procedure, magnetic material-targeting conjugate was separated from the solution by applying an external magnet. Mass Spectrometry Analysis. MALDI-TOF-MS analysis was performed on an Autoflex III mass spectrometer (Bruker, 2285

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order of HMSS, MHMSS, and TiO2/MHMSS (608, 453, and 369 m2/g), and the pore volume also decreased in the same order (2.05, 1.47, and 0.98 cm3/g) (Figure S1c in the Supporting Information). The saturation magnetization values of MHMSS and TiO2/MHMSS were determined to be 7.9 and 2.8 emu/g, respectively (Figure S2a in the Supporting Information). X-ray photoelectron scanning microscopy (XPS) was carried out to confirm the existence of titania in the TiO2/MHMSS (Figure S2b in the Supporting Information). As a control sample, MHMSS had no peak in the Ti binding energy region. In contrast, TiO2/MHMSS had a peak appear in the Ti2p binding energy region, indicating the presence of titania. Compared with MHMSS, several new diffraction peaks were found in the X-ray diffraction pattern of TiO2/MHMSS, indicating the formation of crystal titania on the surface of silica spheres (Figure S3a in the Supporting Information). The Raman spectra of the TiO2/MHMSS were typical of the anatase TiO2 phase, but the Raman peaks slightly broadened and shifted compared to those of single-crystals (Figure S3b in the Supporting Information). The results all indicate that the TiO2 nanoparticles loaded on MHMSS by the LPD method were well crystallized with the anatase structure. Comparison of TiO2/MHMSS and TiO2/NPDCC. We have previously applied a TiO2 nanoparticle-deposited capillary column (TiO 2 /NPDCC) to separate and enrich the phosphorylated substrate of Abl kinase in the enzyme-catalyzed reaction mixtures for MALDI-MS characterization of Abl inhibitors.48 With a synthetic phosphopeptide, of which the sequence is similar to that of the substrate of Abl, as the internal standard, the signal ratio of the phosphorylated substrate to the internal standard detected by MALDI-TOF-MS is linear to the molar ratio of the two phosphopeptides over the range of 0.3 to 3. The determined IC50 values of Abl inhibitor imatinib and its six analogues are consistent with those determined by ELISA and/or docking analysis.48 However, the application of this type of TiO2-deposited capillary column to capture the phosphorylated substrate of EGFR kinase for MALDI-MS screening of EGFR inhibitors was not satisfactory, perhaps due to the relatively lower abundance of phosphorylated substrates in the EGFR-catalyzed reaction mixtures than in the Abl-catalyzed reaction mixtures or due to the relatively lower efficacy of the TiO2-deposited capillary column for capturing the phosphorylated substrate of EGFR compared to that for capturing the phosphorylated product of Abl kinase. Very recently, we have shown that with the large surface area (369 m2 g−1),43 the fabricated TiO2/MHMSS material has highly efficient phosphopeptide enrichment capacity. Moreover, because of its virtue of magnetic separability, TiO2/MHMSS does not need centrifugation during the enrichment procedure, reducing potential loss of the material-phosphopeptides conjugate during sample preparation. Herein, we compared the efficiency of TiO2/MHMSS capturing phosphopeptides with that of TiO2/ NPDCC using the phosphorylated product EAIpYAAPFAKKK (PS1, 1 μM) of Abl substrate and the internal phosphopeptide standard DAIpYAAPFAKKK (IS1, 1 μM) as model phosphopeptides in the presence of 10-fold nonphosphorylated substrate peptide EAIYAAPFAKKK (SP1). The optimal conditions for the enrichment of PS1 and IS1 using TiO2/ NPDCC48 was applied for the comparative study. An aliquot (50 μL) of the peptide mixtures was either loaded using acidic solution (pH 2.5) into the TiO2 deposited capillary column or incubated with TiO2/MHMSS particles in acidic solution (pH 2.5) for 30 min at room temperature. After washing with 70%

Chart 1. Structures of Potential EGFR Inhibitors Screened in the Present Work

Germany). The instrument was equipped with a delayed ionextraction device and a pulsed nitrogen laser operating at 337 nm. The analysis was performed under positive ion reflector mode with an accelerating voltage of 19 kV and a delayed extraction for 75 ns. Typically, 1200 scans were averaged. The MALDI uses a groundsteel sample target with 384 spots. For the analysis of phosphopeptides, 20 mg mL−1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile/49%H2O/1% phosphoric acid was used as the matrix. An aliquot (3 μL) of collected eluting samples containing phosphopeptides was mixed with the matrix solution in a 1:1 ratio prior to deposition onto the target plate for data collection. The Flexanalysis (version 3.0) software was used for analysis and postprocessing. Enzyme-Linked Immunosorbent Assay. The enzymelinked immunosorbent assay (ELISA) was performed following the instruction provided by the supplier of the assay kits (CST, USA). Also, the plate was read on an ELISA plate reader (SpectraMaxM5, Molecular Devices Corporation, USA) at 450 nm for determination of the OD values. More details for the synthesis of potential inhibitors, the sample preparation, and ELISA measurements are given in the Supporting Information.

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RESULTS AND DISCUSSION Preparation of TiO2/MHMSS. The fabrication strategy of TiO2 coated magnetic hollow mesoporous silica spheres (TiO2/MHMSS) were illustrated in Figure S1a in the Supporting Information. In brief, the hollow mesoporous silica spheres (HMSS) were prepared according to a previously reported method.49 Then, the magnetic particles were introduced into the hollow core of HMSS through a vacuum impregnation of Fe(NO3)3.50 The Fe(NO3)3-loaded power were impregnated in ethylene glycol (EG) up to incipient wetness. The impregnated sample was then subjected to heat treatment under nitrogen. Finally, titania was loaded to the asprepared magnetic hollow mesoporous silica spheres (MHMSS) by the liquid phase deposition (LPD) method47 to form TiO2 coated MHMSS. The N2 adsorption−desorption isotherms and pore size distributions of HMSS, MHMSS, and TiO2/MHMSS were measured. All the samples yielded typical IV adsorption isotherms and H1 hysteresis loops, which are characteristic of a mesoporous material (Figure S1b in the Supporting Information). The surface area decreased gradually in the 2286

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accelerating the enrichment rate and improving the enrichment efficiency of the material in phosphopeptides further. In contrast, when the sample solution flows through the TiO2coated capillary column, some phosphopeptides may not be in contact with the TiO2 nanoparticles on the wall, which may account for the lower enrichment efficiency of TiO2/NPDCC for phosphopeptides as well. Compared to other MALDI-MSbased enzymatic activity assays, for example, self-assembled monolayers for MALDI (SAMDI)-MS,8,10 our MALDI-MS combined the preseparation and enrichment of TiO2/MHMSS has several other advantages, including the absence of covalent binding between TiO2 and the phosphopeptides of interest, simple operation, and ability to handle large volumes of samples, which is very helpful for large-scale phosphoproteomic studies. Next, under the given conditions described above, we used TiO2/MHMSS to capture the phosphorylated product of the EGFR substrate peptide RRLIEDNEYTARG (SP2) in an EGFR-catalyzed reaction mixture. The mass spectrum for the phosphopeptide-concentrated sample is shown in Figure 1b. It can be seen that even in the presence of highly abundant nonphosphorylated peptide SP2 (initial concentration, 36 μM), TiO2/MHMSS can efficiently capture the phosphorylated product from the reaction mixture. However, under the similar conditions, no MS signal was detected for the phosphorylated product after separation and enrichment using TiO2/NPDCC from the enzyme-catalyzed reaction mixture (data not shown), again indicating that TiO2/MHMSS is a more effective material for capturing phosphopetides. Optimization of TiO2/MHMSS Separation and Enrichment. Because of the different ionization efficiency of analytes and/or mass bias of MS analysis, it is essential to have a suitable internal standard for MS quantification of phosphopeptides. Stable isotopic labeling to the internal standards, which are in general the isotopomers of the enzyme-catalyzed reaction products, is widely used for MS quantification because the isotopic pairs respond identically in MALDI-MS analysis.23−26 Nishimura and co-workers used a combined labeling technique, that is, N-terminal stable isotope labeling and C-terminal MSsensitivity enhancement tags,27 to synthesize a series of peptides for the MALDI-TOF-MS-based quantitative characterization of human protein kinase inhibitors. The doubly labeled synthetic peptides are well-known synthetic peptides used as common substrates of serine or tyrosine kinases and service not only as the internal standards upon enzyme-catalyzed phosphorylation but also as MS-sensitivity enhancement reagents. In order to avoid cost-inefficient isotope labeling, we have previously designed and synthesized a phosphopeptide DAIpYAAPFAKKK (IS1) as the internal standard, of which except for the N-terminus residue (D), the rest sequence is the same as that of the phosphorylated product (PS1 = EAIpYAAPFAKKK) of Abl kinase optimal substrate SP1, for MALDI-MS screening of Abl inhibitors combined with separation and enrichment of phosphopeptides using TiO2/ NPDCC.48 These two phosphopeptides have almost identical MS responses, providing a foundation for MS quantification of phosphopeptides with nonisotopic labeling internal standards. Following this success, in the present work, we designed and synthesized a phosphopeptide RRLIDDNEpYTARG (IS2) as the internal standard. The sequence of IS2 is similar to that of the phosphorylated product PS2 (RRLIEDNEpYTARG) of an EGFR substrate SP2 (RRLIEDNEYTARG) with the only

ACN/29.9% H2O/0.1% TFA, the captured peptides on TiO2 were eluted using a 1:1 ACN/H2O mixture containing 0.3% NH3·H2O (pH 10.5) from the capillary column or desorbed using 2.5% NH3·H2O (pH 11.3) from the mesoporous spheres, and the eluent was collected for MALDI-MS analysis. As shown in Figure 1a, the MS signal intensities of SP1 and IS1 captured

Figure 1. (a) Mass spectra for the peptide mixture containing Abl kinase substrate peptide (SP1), the phosphorylated substrate (PS1 at m/z 1417.032), and internal standard phosphopeptide (IS1 at m/z 1402.994) after preseparation and enrichment using (i) the TiO2−NPdeposited capillary column and (ii) TiO2/MHMSS. [SP1]−[PS1]− [IS1] = 10:1:1 μM (black) or 10:0.1:0.1 μM (red). (b) Mass spectrum for an EGFR-catalyzed reaction mixture in the absence of inhibitors after preseparation and enrichment using TiO2/MHMSS. The ion peak at m/z 1592.263 and 1672.696 correspond to the nonphosphorylated substrate SP2 at an initial concentration of 36 μM and its phosphorylated product PS2, respectively.

by TiO2/MHMSS are about 5-fold higher than those enriched by TiO2/NPDCC. Meanwhile, TiO2/MHMSS shows comparable selectivity toward the tested phosphopeptides to TiO2/ NPDCC, and no MS signals corresponding to the nonphosphorylated peptide SP1 were detected for the samples resulting from both TiO2/MHMSS and TiO2/NPDCC enrichment (Figure 1a). Furthermore, even the molar ratio of phosphopeptides PS1 and IS1 to the substrate peptide (10 μM) in the peptide mixtures dropped down to 0.01, and the phosphopeptides are still detectable with sufficient signal intensity after enrichment by TiO2/MHMSS but were hardly detectable when the TiO2-based capillary column was used to capture the phosphopeptides from the mixture (Figure 1a). The relatively lower enrichment efficiency of TiO2/NPDCC for the phosphopetides may be due to the relatively lower TiO2 density and the larger dead volume of the coated capillary column compared to TiO2/MHMSS. For the latter, a higher density of TiO2 can be obtained due to the greater surface area of mesoporous silica. On the other hand, the TiO2/MHMSS material can be dispersed in the sample solution completely, 2287

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sequence difference in the fifth residue, which is D for IS2 and E for PS2. Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides containing hydrophobic and/or acidic amino acid residues can also bind and elute under these conditions due to hydrophobic interactions and/or nonspecific bindings of acidic groups, just as in happens to IMAC columns and tips.29,34,51 These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and increase sample complexity. To eliminate the nonspecific binding of acidic nonphosphorylated peptides to IMAC materials, methyl esterification of carboxylate groups has been used in small and large scales of phosphoproteomic studies.28,34,51,52 However, the estrification is sometimes incomplete, especially in Asp/Glu-rich peptides, and the solubility of esterified peptides is decreased, and the tedious procedure, including attachment of chemical tags and removal of excess reagents, can lead to sample losses. In our previously work, a mixture of acetonitrile (ACN) and water (70%/30%, v/ v) containing 0.1% trifluoroacetic acid (TFA) was used as the washing solvent, dramatically reducing the nonspecific hydrophobic and acidic absorption of nonphosphorylated peptides on TiO2 deposited capillary columns,47,48 as reported for IMAC tips.29 The capturing efficacy of TiO2-based materials is not only dependent on their crystallinity but also affected by the nature of both phosphopeptides and nonphosphorylated peptides in the peptide mixtures. To optimize the application of TiO2/ MHMSS for the separation and enrichment of phosphorylated product (PS2) of EGFR substrate and the related internal standard phosphopeptide IS2, we compared the enriching capacity of TiO2/MHMSS for the two phosphopeptides in the presence of 20-fold nonphosphorylated peptide SP2 using different washing solutions, followed by eluting with 2.5% NH3·H2O. Figure 2a shows the relative signal intensity of substrate peptide SP2 (10 μM), phosphorylated substrate PS2 (0.5 μM), and internal standard phosphopetides IS2 (0.5 μM) obtained by MALDI-MS after separation and enrichment using TiO2/ MHMSS, where the highest signal intensity observed in this comparative study was set as 100%. It can be seen that the signal intensity of SP2 increased with increasing the content of ACN in the washing solution containing 0.1% trifluoroacetic acid (TFA), and the signal intensities of phosphopeptides PS2 and IS2 also gradually changed. When washed with 20% ACN/ 80% H2O mixture, the signal intensities of equimolar PS2 and IS2 are almost equal to each other, and the absolute signal intensities of PS2 and IS2 reached a maximum, indicating that under these washing conditions, TiO2/MHMSS presents the highest selectivity toward phosphorylated peptides and shows little discrimination between PS2 and IS2. TFA has been shown to be more effective than hydrochloric acid, formic acid, and acetic acid in discriminating phosphoamino acids from acidic residues on IMAC29 and TiO2/ NPDCC48 as this strong acid protonates carboxyl groups, while it still dissociates phosphates.29 Also the concentration of TFA in washing solutions is a key factor determining the specificity of TiO2-based materials toward phosphopeptides.48 Here, we show that 0.1% TFA in the washing solution containing 20% ACN allowed an efficient and less discriminatory separation and enrichment of PS2 and IS2 from the peptide mixtures (Figure 2b). Therefore, the mixture of 20% ACN and 80% H2O

Figure 2. Effect of the content of (a) acetonitrile and (b) TFA in the washing solvent on the MS responses of the nonphosphorylated substrate (SP2) and phosphorylated peptides (PS2 and IS2) in aqueous solution (pH 2.5) after preseparation and enrichment using TiO2/MHMSS. All intensity values are the means of three independent samples.

containing 0.1% TFA was used as a washing solvent throughout the rest of the present work. Under these given conditions, we achieved an excellent separation and enrichment of phosphopeptides (PS2 and IS2) from the peptide mixture, evidenced by the MS results of the basic (2.5% ammonium hydroxide) elution of the TiO2-captured phosphopeptides as shown in Figure 3a. It can be seen that with the separation and enrichment by TiO2/MHMSS, the MS signal intensity of both phosphopeptides greatly increased compared to that of the nonphosphorylated substrates. Moreover, the same concentration of phosphopeptides PS2 and IS2 show almost identical MS responses, providing a rational foundation for MS quantification of PS2 with the nonisotope labeled phosphopeptide IS2 as the internal standard. It is notable that although the majority of the high concentration of nonphosphorylated substrate SP2 was removed from the peptide mixture by using TiO2/MHMSS, there was still a little amount of SP2 retained on the material (Figure 1b, Figure 3a (II)). The main reason is that SP2 contains three acidic amino acid residues (2 × Glu and 1 × Asp) which might bind more strongly and nonspecifically to TiO2 than SP1 containing only one acidic amino acid residue Glu. We attempted to eliminate further the nonspecific absorption of acidic groups of SP2 on TiO2 by decreasing the pH of both loading and washing solutions down to 1.0. 2288

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the calibration curve (Figure S5 in the Supporting Information), which showed that the ratio of signal intensities of PS2 to IS2 (0.3 μM) is linearly correlated with the molar ratio of the two phosphopeptides over the range of 0.1−4 with the linear equation as follows: Y = 1.74X − 0.23

(1)

where Y is the ratio of signal intensities of PS2 to IS2 and X the molar ratio of PS2 to IS2, with r2 being 0.99 and RSD (n = 3) ranging from 0.72% to 9.60%. These results indicate that the signal ratios of two phosphopeptides measured by MALDITOF-MS allow accurate quantification of PS2 produced by the EGFR-catalyzed phosphorylation of SP2 and are thus applicable to quantitative characterization of the potencies of EGFR inhibitors synthesized in our laboratory. Screening of EGFR Kinase Inhibitors. The inhibitory potency of the synthesized EGFR kinase inhibitor compounds 1, 2 and 3 (Chart 1), were quantitatively characterized using the developed MS-based assay. The inhibition percentage was calculated using eq 2: %inhibition = (([PS]0 − [PS]i )/[PS]0 ) × 100%

(2)

where [PS]0 represents the concentration of the phosphorylated substrate in enzymatic reaction mixtures in the absence of inhibitors and [PS]i the concentration of PS subject to inhibition of various concentrations of kinase inhibitors. Both [PS]0 and [PS]i were determined by MS with application of eq 1. As shown in Figure 4a, the IC50 values of compounds 1, 2,

Figure 3. (a) Mass spectra for the peptide mixture containing EGFR substrate peptide SP2 at m/z 1592.985, the phosphorylated substrate PS2 at m/z 1673.071, and the internal standard phosphopeptide IS2 at m/z 1658.963 (i) without and (ii) with preseparation and enrichment using TiO2/MHMSS. [SP2]:[PS2]:[IS2] = 10:0.5:0.5 μM. (b) Mass spectra for the EGFR-catalyzed reaction mixtures containing highly abundant nonphosphorylated substrate peptide SP2 and various concentrations of the internal standard IS2 at m/z 1658.667. The ion peak at m/z 1672.733 corresponds to the phosphorylated product of SP2.

However, the result (Figure S4 in the Supporting Information) indicated that the TiO2/MHMSS material shows significant discrimination between the phosphopeptides PS2 and IS2 under these conditions, though less SP2 remained. Because of such a low abundance of nonphosphorylated peptides did not interfere with the detection of the phosphopeptides PS2 and IS2, after enrichment by TiO2/MHMSS, their MS signals were almost as intense as that of SP2 (Figure 3a (II)), so the pH values of both the loading and washing solutions were set at 2.5 throughout the subsequent experiments. Quantification of Phosphorylated Substrate. With the optimal conditions described above, a series of EGFR-catalyzed reaction mixtures containing a different concentration of the internal standard IS2 was analyzed by MS after separation and enrichment of phosphopeptides using TiO2/MHMSS. As shown in Figure 3b, the MS signal intensity of the phosphorylated substrate is similar to that of 0.5 μM of IS2. To keep the MS signal ratio of the phosphopeptides PS2 and IS2 in EGFR-catalyzed reaction mixtures subject to the inhibition of various concentrations of inhibitors in a reasonable range, the concentrations of IS2 in both the standard samples and the enzyme-catalyzed reaction mixtures were chosen as 0.3 μM. The series of standard samples containing 36 μM SP2, 0.3 μM IS2, and various concentrations of PS2 ranging over 0.03− 1.2 μM were then analyzed by MALDI-TOF-MS after separation and enrichment using TiO2/MHMSS to generate

Figure 4. IC50 curves of EGFR inhibitors compounds 1, 2, and 3 generated by (a) MALDI-TOF-MS and (b) by ELISA. 2289

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and 3 against EGFR kinase were determined to be 62.7 ± 12, 7.6 ± 2.7, and 209 ± 37 nM, respectively, consistent with the values detected by ELISA (Figure 4b). The IC50 values against EGFR of the three compounds measured by ELISA are 60.2 ± 6.3, 4.6 ± 0.08, and 283 ± 11 nM, respectively. These results indicate that the developed MS-based quantification approach is a promising alternative to conventional methods for screening protein kinase inhibitors.

CONCLUSIONS With the efficient separation and enrichment of phosphopeptides by titania coated magnetic hollow mesoporous silica spheres (TiO2/MHMSS), we developed an effective MALDITOF-MS method for general quantitative characterization of protein kinase inhibitors. Apart from that, the TiO2/MHMSS material provides a comparable selectivity toward phosphopeptides to nanoparticle-based TiO2 materials, and the large surface area of TiO2/MHMSS provided the foundation for its impressive phosphopeptide enrichment capacity. Moreover, the phosphopeptide conjugates with TiO2 coated on MHMSS does not need centrifugation during the enrichment procedure, reducing potential loss of the material-phosphopeptides conjugate during sample preparation. Combined with the preseparation and enrichment of phosphopeptides by TiO2/ MHMSS, MALDI-MS quantification allows a rapid screening and precise generation of IC50 curves for potential kinase inhibitors, eliminating the use of cost-inefficient and/or radioactive reagents such as monocloned antibodies and radio-isotope labeled ATP. Compared with the conventional MS quantification of phosphopeptides, one more advantage of this new MS approach is the use of a nonisotope labeled phosphopeptide as the internal standard which was shown to give an identical MS response to the phosphorylated substrate under optimal conditions, further reducing the cost of screening and making the MS-based approach an ideal alternative for rapid screening, generation of dose−response curves, and comparative IC50 measurements of protein kinase inhibitors. ASSOCIATED CONTENT

S Supporting Information *

More details of experimental section, characterization data of TiO2/MHMSS materials, and MS calibration curve. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fuyi Wang: e-mail, [email protected]; fax, +86 10 62529069. Yu-Qi Feng: e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NSFC (Grant Nos. 90713020, 20975103, and 21020102039 for F.W. and Grant Nos. 91017013 and 31070327 for Y.-Q.F.), the 973 Program of MOST (Grant No: 2007CB935601), and the Chinese Academy of Sciences (Hundred Talent Program) for support and Professor Peter J. Sadler at the University of Warwick (England) for help on the revision of the manuscript. 2290

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