Site-Specific, Covalent Attachment of Poly(dT)-Modified Peptides To

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Bioconjugate Chem. 2007, 18, 1778–1785

Site-Specific, Covalent Attachment of Poly(dT)-Modified Peptides To Solid Surfaces for Microarrays Naoki Kimura,*,† Takashi Okegawa,† Kiyokazu Yamazaki,† and Koji Matsuoka*,‡ Research and Development Center, Nisshinbo Industries Inc., 1-2-3 Onodai, Midori-ku, Chiba 267-0056, Japan, and Area for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura, Saitama 338-8570, Japan. Received March 13, 2007; Revised Manuscript Received August 30, 2007

The present study reported proof-of-principle for a kinase assay approach that can detect specific peptide phosphorylation events. The method involves attachment of peptides onto commercial aminosilane and polycarbodiimide-coated glass slides, using a newly developed DNattach linker system that consists of a poly(dT) tail (Nisshinbo Industries Inc.), followed by a detection step using fluorescently labeled antiphosphoamino acid antibodies. The linker-modified peptides are efficiently synthesized by Michael addition between maleimidomodified peptides and thiol-containing DNattach. Specific covalent immobilization of the modified peptides onto aminosilane and poly carbodiimide-coated slides is then achieved by short exposure to UV-light. Highly selective and quantitative recognition by standard antiphosphoamino acid antibodies (antiphosphotyrosine and antiphosphoGFAP) and kinases (c-Src and PKA) to the corresponding modified peptides on the microarray spots is demonstrated. Furthermore, we found that this immobilization method provides greater signal-to-noise ratio and better discrimination ability of phosphorylated amino acids than does the conventional immobilization technique. The phosphorylation pattern of target sequences, detected using fluorescently labeled antiphosphoamino acid antibodies, revealed that the linker system preference of the kinase is determined by its activity profile.

INTRODUCTION Kinase-catalyzed phosphorylation of proteins is one of the most important mechanisms for the regulation of cell function. Indeed, it has been estimated that more than one third of all mammalian proteins may be subject to modification by phosphorylation in ViVo (1, 2). To date, however, only a limited number of kinases have been identified and fully characterized. One of the most important features of protein kinases is their substrate specificity, which to a large extent is determined by the primary amino acid sequence around the phosphorylation site of the corresponding target protein. As a result, a number of methods have been developed to identify potential kinase substrates, including combinatorial synthesis of peptide libraries on membranes using SPOT technology, one-bead-one-compound peptide libraries, positional-scaling combinatorial libraries, and peptide libraries using affinity-column selection (3–6). More recently, protein- and peptide-based microarrays have also been developed (7–11). In comparison to SPOT technology, a much higher density of spots can be attained using a protein array, making it possible to simultaneously screen tens of thousands of kinase substrates on the solid surface. Unfortunately, however, current peptide and protein chip strategies have several limitations. Chemical strategies leading to the oriented immobilization of a polypeptide chain are ideal because this preserves binding capacity and improves the control and reproducibility of the overall process. However, unwanted adsorption of soluble proteins often results in competition with the detection of protein–substrate interactions, leading to a noisy background (12). Usually, only a fraction of the immobilized * To whom correspondence should be addressed. Phone: +81-43205-0794. Fax: +81-43-205-0844. E-mail: [email protected] (N.K.). Phone: +81-48-858-3099. E-mail: [email protected] (K.M.). † Nisshinbo Industries Inc. ‡ Saitama University.

proteins are competent to participate in binding interactions because many of them are immobilized in inaccessible orientations or are partially denatured (13). A consequence of these limitations is that most immobilization procedures are not well suited to quantitative assays of protein–substrate interactions. Several immobilization methodologies that involve the modification of peptides prior to spotting onto the solid surface for specific binding events might circumvent these limitations (7–10, 14–16). For example, MacBeath et al. (14) and Lesaicherre et al. (15) independently used slides treated with maleimide and slides treated with glyoxylic acid as platforms, respectively, for the immobilization of N-terminally Cys-containing peptides. Houseman et al. (7) reported the fabrication of a peptide microarray by the Diels–Alder-mediated immobilization of the kinase substrate on a self-assembled monolayer of alkanethiolates on gold. Furthermore, Duburcq et al. (8) fabricated the peptide microarray using semicarbazide slides that permitted the immobilization of glyoxylyl peptides. However, current methodologies are time-consuming (in the order of hours) and often require relatively large amounts of peptide (i.e., concentrations between 100 µM and 5 mM) for the immobilization procedure (7–10, 14–16). In addition, although appropriate for DNA immobilization, additional surface modifications that would make microarray fabrication a laborious and complex procedure are usually required when peptides or very small proteins are printed on commercial DNA microarray slides, and there are only a few commercial slides for fabricating peptide arrays (17). Thus, the increased need for simple, robust, and high throughput assays has led us to develop a novel protocol for preparing peptide and protein microarrays. Over the past few years, we have been developing polycarbodiimide-coated glass and plastic slides for use with DNA microarray substrates (18–20). We found that thymine base(s) of DNA efficiently react with the carbodiimide group under UV irradiation (20). On the basis of these findings, we have further developed the patented DNattach system (Nisshinbo Industries

10.1021/bc070083+ CCC: $37.00  2007 American Chemical Society Published on Web 10/23/2007

Poly(dT)-Based Peptide Microarrays

Inc.), which consists of a poly(dT) tail and spacer group, for immobilizing DNA onto various surfaces including polycarbodiimide, nonmodified metal, and plastic surfaces by UV irradiation (21, 22). Using this system, Takahashi et al. (22) successfully detected synaptogenesis in the developing mouse cerebellum on a gold surface. As a first step toward the goal of developing an inexpensive, simple, and robust diagnostic tool for assaying kinase activity, we have further expanded this technology. Here, we describe a novel, simple, and convenient methodology that uses the DNattach immobilization system to sitespecifically immobilize peptides onto commercial aminosilane and polycarbodiimide-coated glass slides by UV irradiation in a microarray format. We have found that the technology is also appropriate for attaching peptide to aminosilane and polycarbodiimide-coated slides by UV irradiation. In this detailed quantitative study, we show that the immobilization system is a simple and rapid technique compared to conventional technologies. Furthermore, the methodology provides a uniform environment for kinase assays, resulting from site-specific immobilization onto the solid surface and the use of a selective immobilization chemistry to prepare the chips. The phosphorylation pattern of target sequences, detected using fluorescently labeled antiphosphoamino acid antibodies, revealed that the linker system preference of the kinase is determined by its activity profile.

EXPERIMENTAL PROCEDURE Materials and General Methods. β-Alanine and maleic anhydride were purchased from Kanto Chemical Company (Tokyo, Japan). p60c-src kinase was purchased from Oncogene Science (Cambridge, MA). cAMP-dependent protein kinase (PKA) was purchased from New England BioLabs (Ipswich, MA). AG213 was purchased from Calbiochem-Novabiochem International, Inc. (La Jolla, CA). Anti-phosphoGFAP (YC10) and antiphosphotyrosine (PY20) were purchased from Medical & Biochemical Laboratories (Nagano, Japan) and Exalpha Biologicals (Maynard, MA), respectively. NAP5 columns and the Cy3 and Cy5 mAb Labeling Kits were purchased from GE Healthcare Biosciences (Tokyo, Japan). Thiol-modified DNattach was prepared at Nisshinbo Industries Inc. (Chiba, Japan) as follows (23). Poly(dT) (DNattach) modified with a 5′-thiol group and a six-carbon spacer was synthesized on an ABI 3900 DNA synthesizer at a 0.2 µmol scale using the standard phosphoramidite method. The thiol-modified DNattach was then purified on a reverse-phase cartridge following a standard procedure and dried in Vacuo. All of the amino acids of the peptide sequence that were subsequently coupled on a Wang resin, following the standard procedures of Fmoc solid-phase peptide synthesis, were purchased from Operon (Tokyo, Japan). Peptides modified with biotin and the C4 alkyl spacer group at the N-terminus of the amino acid were also synthesized and purified at Operon. All other chemicals were purchased from Wako Pure Chemical (Tokyo, Japan) and used without further purification. Synthesis of 3-(Maleimido) Propionic Acid. 3-Maleimido propionic acid was synthesized according to a previous report with some modifications (24). Briefly, β-alanine (1 g, 11.2 mmol) and maleic anhydride (1.1 g, 11.2 mmol) in acetic acid (40 mL) were stirred at RT for 15 h under a N2 atmosphere. The resulting suspension was then heated to 150 °C for 8 h. The solvent was removed in Vacuo, and the residue was purified by silica gel column chromatography (CHCl3/MeOH ) 50:1), affording the compound (1.1 g, 57%) as a colorless solid. 1H NMR (400 MHz, CDCl3): δ 10.30 (1 H, br), 6.73 (2 H, s), 3.84 (2 H, t, J ) 7.2 Hz), 2.71 (2 H, t, J ) 7.2 Hz). MS (MALDITOF): m/z calcd for C11H10N2O6 [M + H]+, 267.22; found,

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266.7. Alternatively, the title compound is commercially available from Wako Pure Chemical. Synthesis of Maleimide–Peptide. The Fmoc group of the fully protected peptide on the Wang resin (Operon) was first deprotected with 20% piperidine in DMF for 20 min, and 3-(maleimido) propionic acid was then introduced to the N-terminus of the amino acid using 3 equivalents of 3-(maleimido) propionic acid, dichlorohexy carbodiimide, and HOBt in DMF for 1 h. Cleavage and deprotection were effected by reaction with TFA/H2O ) 95:5 or thioanisole/H2O/1,2ethanedithiol/triisopropylsilane/TFA ) 2.5:2.5:2.5:1:91.5 for 2 h. Cold diethyl ether was added to the reaction mixture, and the resulting pellet was purified using a NAP5 column according to the manufacture’s procedure. Synthesis of DNattach Linker–Peptide Conjugate. One nanomole of the thiol-modified DNattach (Nisshinbo Industries Inc.) and 20 nmol of each maleimide–peptide were dissolved in 100 mM phosphate buffer (pH 7.0) (a total 10 µL of solution) and mixed. The reaction mixtures were stirred at RT for 18 h, and progress of the reaction was monitored at 254 nm by HPLC with a µBondasphere C-8 column (Nihon Waters K.K., Tokyo, Japan). A total of 90–95% conjugate was formed as assessed from the relative areas of the thiol-modified DNattach and the conjugate on the HPLC profile. The resulting conjugate was purified by HPLC. We obtained >90% purity of the conjugates as assessed from the relative areas of the conjugate and the other peaks on the HPLC profile. MS (MALDI-TOF): m/z calcd for Peptide 1 [M + 6Na]+, 7579.2; found, 7576.2; m/z calcd for Peptide 2 [M + 3Na]+, 7430.0; found, 7427.4; m/z calcd for Peptide 3 [M + 3Na]+, 7338.3; found 7338.3; m/z calcd for Peptide 4 [M + 3Na]+, 7947.3; found, 7951.8; m/z calcd for Peptide 5 [M + 4Na]+, 7890.2; found, 7894.6; and m/z calcd for Peptide 6 [M + 4Na]+, 7874.2; found, 7768.3. Fabrication of Aminosilanized and PolycarbodiimideCoated Arrays. A polycarbodiimide-coated slide (CarboStation) from Nisshinbo Industries Inc. and a SuparAmine slide from Telechem International, Inc. (Sunnyvale, CA) were used in this study. Both nonmodified and linker-modified peptides were spotted on the slide as spots of ∼250 µm in diameter with a spot-to-spot distance of 600 µm using a customized microarray robot (19). After printing, the arrays were irradiated (total 0–0.3 J/cm2) using a UV Stratalinker 2400 (Stratagene, La Jolla, CA), treated with a blocking solution (3% BSA, 0.2 M NaCl, 0.1 M TrisHCl at pH 7.5, and 0.05% Triton-X100), then washed with TE buffer (pH 7.2) for 5 min, and dried for storage. UV irradiation was carried out within 3 min. Fabrication of Streptavidin-Coated Arrays. Streptavidincoated slides from Greiner Bio-One Co., Ltd. (Tokyo, Japan) were also used in this study. The customized microarray robot was also used to print arrays for the biotinylated peptides (Operon) in phosphate-buffered saline (pH 7.4). After printing, the arrays were incubated at RT for 4 h in a humidified chamber, washed, and treated with the blocking solution according to a previously reported method (11). Kinase Reactions. Protein kinase assays were done according to the manufacturer’s procedures with some modifications. p60c-src Assay. p60c-src was added to the reaction buffer (50 mM HEPES-HCl at pH 7.5, 30 mM MgCl2, 0.1 mM EDTA, and 0.015% polyoxyethyl (23) lauryl ether) and 150 µM ATP (final concentration of p60c-src 0–0.1 U/µL in the reaction buffer in a final volume of 30 µL). The reaction mixture was applied to the slide surface, and the reaction was performed at 37 °C for 240 min. The array was briefly washed with EtOH at RT for 5 min, twice with H2O at RT for 5 min, and then dried.

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Table 1. Peptide Sequences To Be Immobilized onto the Solid Surface peptide Peptide Peptide Peptide Peptide Peptide Peptide

1 2 3 4 5 6

peptide sequence

kinase

remarks

Y-phosphorylated NH2-IY(PO3)GEFKKK-COOH NH2-IYGEFKKK-COOH c-Src NH2-IAGEFKKK-COOH A-substituted NH2-RRRVTS(PO3)AARRS-COOH S-phosphorylated NH2-RRRVTSAARRS-COOH PKA NH2-RRRVTAAARRS-COOH A-substituted

For inhibition experiments, the inhibitor was included in the reaction mixture, and the concentration of DMSO was adjusted to 2%. PKA Assay. PKA was added to the reaction buffer (25 mM Tris-HCl at pH 7.5, 2.5 mM MgCl2) and 200 µM ATP (final concentration of PKA 0–5 U/µL in the reaction buffer in a final volume of 30 µL). The reaction mixture was applied to the slide surface, and the reaction was performed at 30 °C for 60 min. The array was briefly washed with EtOH at RT for 5 min, twice with H2O at RT for 5 min, and then dried. Detection of Phosphorylated Amino Acids Using Fluorescently Labeled Antiphosphoaminoacid Antibodies. Antiphosphotyrosine and anti-phosphoGFAP antibodies were labeled with either Cy3 or Cy5 using Cy3 or Cy5 mAb Labeling Kit (GE Healthcare Biosciences) and then purified using a NAP5 column according to the manufacturer’s procedure. Either fluorescently labeled antiphosphotyrosine or anti-phosphoGFAP antibody, dissolved in PBST containing 1% BSA, was then applied to the array. The array was incubated at RT for 1 h, washed with PBST at RT for 5 min, briefly rinsed with PBS, and then air-dried. Signal Detection and Data Analysis. A ScanArray 4000 (Packard Biochip, Billerica, MA) or FLA-5100 (Fuji Photo Film, Kanagawa, Japan) instrument was used to detect the signals, with equivalent results. Arrays were scanned with variable photo multiplier tube (PMT) settings to obtain optimum signal intensities. The resulting images were used to generate Tiff data files. Data analysis was done using QuantArray (Packard BioChip) or Science Laboratory (Fuji Photo Film) software, and Microsoft Excel (Microsoft Corp., Redmond, WA). Unless stated otherwise, the average signal values were taken from two spots on two slides processed in parallel. The data were further validated by results from more than five experiments.

RESULTS Effect of UV Irradiation on Aminosilane and Polycarbodiimide-Coated Glass Surfaces. The goal of the current study was to verify a new method of peptide immobilization onto a solid surface by evaluating the DNA–peptide conjugates for peptide microarray applications. To this end, we prepared six different DNA–peptide conjugates (Table 1), which were easily obtained by Michael addition between maleimide– peptides and thiol–DNattach (Figure 1). In all cases, derivatization was carried out after chain assembly by following standard protocols, thus yielding 5′-modified DNattach and peptides with the maleimide group linked to the N-terminus (see Materials and General Methods). The amount of peptide attached to the solid support and the accessibility of enzymes or antibodies to the immobilized peptide are critical factors in microarray analysis, as they determine both the sensitivity and the dynamic range of measurement. To investigate how UV irradiation during immobilization of peptide via the poly(dT) tail on the surface affects the binding affinity and discriminative ability of the antibody, we first immobilized two different DNA–peptide conjugates (Peptides 1 and 2, Table 1) on the polycarbodiimidecoated glass slide. Peptides 1 and 2 both include a tyrosine

Figure 1. Synthesis of DNattach-induced peptides. The reaction mixture of thiol-modified DNattach and maleimide-modified peptide was incubated at RT for 18 h.

Figure 2. Signal intensities produced upon the interaction of Cy3labeled antiphosphotyrosine antibody with four different spotted peptides at 0 to 0.3 J/cm2 of UV irradiation. Peptides 1 and 2 were spotted and immobilized on the polycarbodiimide-coated slide. (A) Spots made with each peptide were present in quadruplicate. (B) The mean signal intensities produced in the experiments are plotted vs UV dose. The error bars indicate standard deviation. Peptide sequences are shown in Table 1. All arrays were scanned with the same PMT setting (600 mV).

residue, although only the tyrosine from the former peptide was phosphorylated. The DNA–peptide conjugates were dissolved in 3× SSC at a concentration of 1 µM and applied to the glass slide in quadruplicate. In peptide–antibody interaction experiments, Cy3-labeled antiphosphotyrosine antibody, which specifically interacts with the phosphorylated tyrosine in peptide sequences, was used for detection. Figure 2A shows a typical image of fluorescence signal intensities from these experiments. Irrespective of UV dose, peptide–antibody interaction was specific to the phosphorylated tyrosine residue. No signals were obtained from either the non-UV-irradiated arrays or from the UV-irradiated arrays spotted with nonmodified peptides. These results indicate that immobilization of the linker-modified peptide onto the surface by UV irradiation did not originate from residual peptide molecules that were physically absorbed onto the surface. Signal intensities increased with increasing UV dose, whereas non-

Poly(dT)-Based Peptide Microarrays

Figure 3. Signal intensities produced upon interaction of Cy5-labeled antiphosphotyrosine antibody with two different spotted peptides at 0 and 0.3 J/cm2 of UV irradiation. Modified Peptide 1 (white bar) and Peptide 2 (dark bar) at a concentration of 1 µM were spotted and immobilized on the aminosilane slide. The error bars indicate standard deviation. Peptide sequences are shown in Table 1. All arrays were scanned with the same laser power (100%) and PMT setting (50%).

Figure 4. Fluorescent intensity vs amount of phosphorylated substrate. Varied ratios of phosphorylated/nonphosphorylated c-Src (Peptides 1 and 2) and PKA (Peptides 4 and 5) substrates were arrayed onto the polycarbodiimide-coated (A) and aminosilanized (B) slides and probed with the corresponding Cy5-labeled antiphosphoamino acid antibody. The error bars indicate standard deviation. All arrays were scanned with the same laser power (100%) and PMT setting (50%).

specific signals were at background levels. Using a UV dose of up to 0.3 J/cm2, the signal intensity was 528-fold (Peptide 1) greater for the UV-irradiated array than for the nonirradiated control (Figure 2B). We did not observe an obvious increase in nonspecific signals when the UV dose was increased to 0.3 J/cm2. A similar trend with the aminosilane slides was also observed (Figure 3). These results indicate that the modified peptide is immobilized via the poly(dT) attached to the peptide and that the reaction between the DNA–peptide conjugates and polycarbodiimide (aminosilane) on the glass surface is facilitated by UV irradiation. Detection and Quantification of on-Chip Phosphorylation. We next determined whether the fluorescently labeled antibodies can quantitatively detect phosphorylated peptides on the aminosilanized and polycarbodiimide-coated arrays. Varied ratios of phosphorylated/nonphosphorylated Peptides 1 and 2 (Table 1), with a combined constant concentration (1 µM), were immobilized onto the slide by UV irradiation and then probed with the Cy5-labeled antiphosphotyrosine antibody for 1 h (Figure 4A and B). Figure 4A and B shows that the intensity of the spots increased as the ratio of phosphorylated to nonphosphorylated peptide increased. The graph of fluorescent intensity versus phosphorylated peptide substrate shows a linear correlation for the Peptide 1 ratio, ranging from 0 to 100%, demonstrating the feasibility for on-chip quantification of

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phosphorylated peptides using this methodology. A similar trend was also observed when we immobilized Peptides 4 and 5 onto the slides by UV irradiation and probed with Cy5-labeled antiphosphoGFAP antibody (Figure 4A and B). Thus, the UVirradiated DNA–peptide conjugates formed under these conditions are suitable for peptide microarray applications. Detection Sensitivity. To estimate the detection sensitivity of immunofluorescence detection and laser scanning, modified Peptides 1 and 2 (Table 1) at different concentrations were immobilized onto the aminosilane and polycarbodiimide-coated slides by UV irradiation (0.3 J/cm2) and then probed with the Cy5-labeled antiphosphotyrosine antibody for 1 h (Figure 5A and B). Figure 5A and B shows that the signal intensity was nearly saturated at a concentration of modified Peptide 1 of only 2.5 µM (carbodiimide) and 5 µM (aminosilane), whereas nonspecific signals were minimal across all DNA–peptide conjugate concentrations. The maximum signal intensity was 1.25-fold (Peptide 1) greater for the carbodiimide slide than for the aminosilane slide. The lowest detection limit was around 0.6 µM, where the signal-to-noise was still around 10 for the aminosilane and 238 for the carbodiimide slide, as shown in Figure 5A and B. These differences most likely arise from different surface conditions, such as loading capacity, charge, hydrophobicity, and efficiency of immobilization and interaction. Further investigation was performed using two different modified peptides (Peptides 4 and 5, Table 1) and the Cy5-labeled anti-phosphoGFAP antibody. A similar trend with these peptides was also observed (Figure 5A and B). The performance of different attachment chemistry (i.e., most widely used in streptavidin–biotin interaction) was also assessed by testing the detection sensitivity of phosphopeptide, as shown in Figure 5A and B. To this end, we immobilized a total of four different biotinylated peptides (same sequences as those of Peptides 1 and 2, and Peptides 4 and 5 in Table 1) at different concentrations onto streptavidin-coated glass slides and then probed with corresponding Cy5-labeled antiphosphoamino acid antibody (11). In our hands, however, we could not detect any signals for the phosphorylated Peptide 1 on the streptavidin slide using interaction and scanning conditions (laser power 100%, PMT 50%) identical to those used in our methodology (data not shown), probably due to the fact that the streptavidin-coated slide has limited peptide binding capacity and/or due to the steric interference of the surface on interaction with the antibody where the phosphorylated tyrosine residue in Peptide 1 is located adjacent to the slide surface (Table 1) (25). By contrast, although the streptavidin arrays could discriminate between the phosphorylated Peptide 4 and nonphosphorylated Peptide 5 (the signal intensity for Peptide 4 at a concentration of 5 µM was 17.7-fold greater than that for the Peptide 5 control), quantification revealed lower signals (∼9.1-fold) in comparison to those obtained using the poly(dT)-modified peptides and carbodiimide slides (Figure 6). Thus, we find that DNattach chemistry provides a better substrate for the immobilization of peptides and the interaction with antibody than do conventional biotin–avidine interaction techniques. Characterization of Kinase Activity. In order to further demonstrate the utility of our methodology for the detection of kinase activity in a microarray format, we next investigated the dose-dependent detection of peptide phosphorylation on a chip. Two different nonphosphorylated substrates at a concentration of 10 µM, Peptide 2 modified with poly(dT) as a substrate for c-Src tyrosine kinase and Peptide 3 modified with poly(dT) as a negative control, were spotted and immobilized onto carbodiimide and aminosilane slides by UV irradiation (Table 1). Nonphosphorylated substrates, Peptide 5 modified with poly(dT) as a substrate of PKA serine kinase and Peptide 6 modified

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Figure 5. Signal intensities produced upon the interaction of Cy5-labeled antiphosphoaminoacid antibodies. The modified peptides were dissolved in 3× SSC at a concentration of 0–10 µM and spotted on polycarbodiimide (A) and aminosilane (B) in duplicate (see Materials and General Methods). After immobilization, the arrays were probed with the corresponding Cy5-labeled antiphosphoamino acid antibody. Scanned spots and quantitative analysis of spots over interactions with the corresponding antiphosphoamino acid antibody are shown. The error bars indicate standard deviation. All arrays were scanned with the same laser power (100%) and PMT setting (50%). (Note: the maximum mean signal intensity of each specific signal among the different arrays was normalized to 100 (no units); thus, the results represent the relative intensity of the corresponding antiphosphoamino acid antibody.)

Figure 6. Signal intensities produced upon the interaction of Cy5labeled anti-phosphoGFAP antibody on the streptavidin arrays. The biotinylated peptides were dissolved in PBS at a concentration of 0–20 µM and spotted on streptavidin slides in duplicate (see Materials and General Methods). After immobilization, the arrays were probed with the Cy5-labeled anti-phosphoGFAP antibody. The error bars indicate standard deviation. All arrays were scanned with the same laser power (100%) and PMT setting (50%). (Note: the maximum mean signal intensity of each specific signal among the different arrays was normalized to 100 (no units) as shown in Figure 5; thus, the results represent the relative intensity of the corresponding antiphosphoamino acid antibody.)

with poly(dT) as a negative control, were also spotted and immobilized onto the same slide (Table 1). The arrays were first incubated with either c-Src or PKA kinase at 5 different kinase concentrations for 240 or 60 min and then incubated with the corresponding fluorescently labeled antiphosphoamino acid antibody for 1 h. In both cases, Figure 7A and B shows that fluorescent intensities were directly proportional to the concentration of each kinase, whereas nonspecific signals were minimal across all kinase concentrations. In addition, Figure 7A shows that the signal intensity was nearly saturated at a concentration of c-Src kinase of 0.05 U/µL. The CV of intensities for all elements

Figure 7. Characterization of c-SRC and PKA kinase activities. The modified Peptide 2, Peptide 3, Peptide 5, and Peptide 6 at the same concentration of 10 µM were immobilized on the slides by UV irradiation (0.3 J/cm2) and then incubated with (A) c-Src and (B) PKA kinases for 240 or 60 min, respectively. Detection was performed using the corresponding Cy5-labeled antiphosphoamino acid antibody. All arrays were scanned with the same PMT setting (75% and 55% for c-Src and PKA assays, respectively). The error bars indicate standard deviation.

was between 12% and 14% across the entire signal range. As anticipated, the maximum signal intensity was 1.4-fold (Peptide 2) and 1.3-fold (Peptide 5) greater for the carbodiimide slides than for the aminosilane slides (Figure 7A and B). The signalto-noise ratios for Peptide 2 at a concentration of 0.05 U/µL c-Src kinase were 71.1 (the carbodiimide slide) and 52.5 (the aminsilane slide). In addition, the signal-to-noise ratios for Peptide 5 at a concentration of 5 U/µL PKA kinase were 770

Poly(dT)-Based Peptide Microarrays

Figure 8. Effect of the AG213 inhibitor upon kinase activity. Modified Peptide 2 was immobilized on the slide by UV irradiation (0.3 J/cm2) and then incubated with c-Src, ATP, and inhibitor in serial dilutions for 240 min. Detection was performed using the Cy3-labeled antiphosphotyrosine antibody. The error bars indicate standard deviation. All arrays were scanned with the same laser power (100%) and PMT setting (80%).

(the carbodiimide slide) and 678 (the aminsilane slide). These results indicate that, under this immobilization condition, the linker-modified peptide probes are suitable for peptide microarray application. In order to assess the performance of the different attachment chemistry, again, the biotinytated peptides (Peptides 5 and 6) were spotted and immobilized onto streptavidin slides, and the arrays were incubated with PKA kinase for 60 min and then incubated with the fluorescently labeled anti-phosphoGFAP antibody for 1 h. However, under interaction and scanning conditions (laser power 100%, PMT 55%) identical to those used with the new methodology, we could not detect any signals for the phosphorylation of Peptide 5 (data not shown), probably due to the limited peptide binding capacity on the streptavidin slide and/or due to the steric effect as mentioned above. Again, we find that DNattach chemistry provides a better substrate for the immobilization of peptides and interaction with kinases than do conventional biotin–avidine interaction techniques. Evaluation of AG213 Inhibitor of c-Src on the Peptide Arrays. Finally, we used the peptide microarray to evaluate an inhibitor of c-Src, AG213, on substrates. Mixtures containing the kinase, ATP, and inhibitor at concentrations ranging from 1 nM to 1 mM were applied onto the polycarbodiimide surface. After 4 h at 37 °C, the arrays were probed with Cy3-labeled antiphosphotyrosine antibody. When the kinase reactions were performed in the presence of 0.04 U/µL c-Src kinase, an IC50 value (1.7 µM) close to the published data in solution-phase assays (8 µM) was obtained (Figure 8) (7, 26). The difference observed with this inhibitor may be due to different sources of enzymes or slightly different assay conditions. However, this result is expected since the inhibitor used in this study is known to compete with ATP by binding to the ATP binding site of the kinase (26). These results show the feasibility of our methodology for the characterization of inhibitors.

DISCUSSION The experiments described in this article demonstrate that a combination of modified-peptide and UV irradiation technology can be used to generate an efficient and robust assay for kinases. Nucleases have been studied using peptide–DNA/PNA conjugates by the introduction of a reporter group to investigate DNA–protein interactions (27–31). However, this is the first report to demonstrate the utility of UV irradiation of spotted peptides with a poly(dT) tail on commercial aminosilane and polycarbodiimide-coated slides to detect phosphorylation events. The DNA–peptide conjugates are immobilized by UV irradiation to form covalent bonds between the thymidine residues in DNA and the carbodiimide or the amine group on the surface (20, 32). On the molecular level, although it is not yet clear how the poly(dT) binds to the substrates by UV irradiation, it can be looked on as any other functional group that is required for

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efficient immobilization of samples on surfaces (21, 33, 34). Polycarbodiimide resin was identified by the presence of absorption at 2145 cm-1 in FT-IR assignable to the carbodiimide function. Although the carbodiimide group hydrolyzes very easily at acidic pH around 5 as well as under basic conditions (18–20, 35), the polycarbodiimide used in this study was hydrophobic and stable for 12–24 months. In addition, the polycarbodiimidecoated slides can be stored for 6–12 months at room temperature before use (20). The presence of carbodiimide groups on the surface was confirmed by X-ray photoelectron spectroscopy. In fact, when fluorescently labeled poly(dT) in 3× SSC were printed on the carbodiimide substrate and the slides were UVirradiated after 1, 24, 72, and 168 h at RT and then briefly washed, signal intensity from only the slide after 168 h of incubation indicated less signal intensity (1/10-fold) than those obtained from the slides after 1, 24, and 72 h of incubation (data not shown). These results indicate that decomposition of carbodiimide groups on the surface that is involved with the immobilization procedure would dramatically affect immobilization efficiency after 72 h of incubation. The slides should therefore be UV irradiated within 72 h after printing to circumvent this problem. Thus, using this immobilization system, peptides can be reliably and specifically immobilized onto the solid surface. Furthermore, this new immobilization method provides signal intensities greater than those of conventional biotin–avidine interaction techniques. Here, we have demonstrated that this property can be exploited to measure kinase activity. Although the introduction of an additional spacer unit between the biotin and the peptide might avoid steric hindrance and result in facilitating the signal intensity on the streptavidin slide and/or the use of secondary antibody might also facilitate the signal intensity on the streptavidin slide (25), the technique described in this study provides three major advantages over those of peptide microarrays that have been reported by other researchers. First, the immobilization reaction involving UV irradiation is both simple and quick, providing a regular, homogeneous environment for immobilized peptide ligands. The peptides therefore have equal activity toward soluble enzyme and are well suited to quantitative assays. Second, a low concentration (up to 10 µM) of modified peptide is sufficient for the kinase assays. By contrast, conventional technologies generally require a peptide concentration of up to 5 mM and a period of several hours for immobilization onto a solid surface (7–10, 14–16). Third, using this immobilization method, commercial DNA microarray slides without additional surface modifications can be used for producing peptide microarrays, thereby avoiding making microarray fabrication a laborious and complex procedure. Mukumoto et al. recently reported that nonmodified DNA could be immobilized onto a carbodiimide-coated gold slide through the covalent bond by a reaction of carbodiimide with the imino moiety of the thymine base in DNA (35). Inconsistent with our results, they do not require a UV irradiation procedure for immobilization of DNA. In our studies, however, we could detect only weak signals from Peptide 1 modified with DNattach on non-UV-irradiated slides (Figure 2A and B). These differences may be caused by different surface conditions between our slides treated with polycarbodiimide and their slides treated with the carbodiimide monomer, thereby causing an insufficient amount of immobilized peptides and/or steric effects to detect phosphorylation events on our surface when immobilized peptides on the slides are used in this study without UV irradiation. One potential drawback of this system is that one has to link the DNattach to the peptides individually followed by multiple purification steps. It might make microarray fabrication a laborious and complex procedure when constructing high-

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density peptide microarrays. However, it is possible to synthesize the DNA–peptide conjugates on a solid support and purify the conjugates all together (36), and studies to optimize a solidphase synthesis protocol with DNattach are currently underway. In addition, certain adjustments in the methodology described here will be required if cysteine is to be included in the peptide sequence. The reaction between the free thiol of cysteine and the maleimide group of the peptide conjugate may compete with the reaction between the thiol of DNattach and the maleimide group during the conjugation, yielding side products as a result of Michael additions. To avert the complex mixtures, the conjugate reaction should be carried out using maleimide–peptides with a protected cysteine and followed by unmasking of the thiol group after conjugation. Alternatively, the conjugate reaction may be carried out using 5′-peptide–oligonucleotide conjugates during the conjugation on solid supports as described above (36). In this proof-of-principle study, although we have used a model system consisting of a relatively small number of distinct antiphosphoamino acid antibodies and protein kinases to illustrate the effectiveness of this immobilization methodology, we show that the technique is robust, reliable, and reproducible for generating peptide microarrays. Thus, this method has the potential to provide a key technology for the fabrication of arrays to explore peptides as potential substrates for kinases. This technique might also be applied to high-throughput analysis of various enzyme activity assays and protein expression profiling. In addition, because the linker can be immobilized onto gold surfaces and plastics (21, 22), it should be possible to immobilize the modified peptides onto nonmodified gold or plastic surfaces. We are also currently investigating various biological assays, including those for detecting kinase activity, using this system by employing label-free detection methodologies (i.e., SPR and MALDI-TOF Mass). Furthermore, our research group has successfully synthesized poly(dT)-modified carbohydrates and is currently monitoring lectin assays in a carbohydrate microarray format. The results will be reported elsewhere in the near future.

ACKNOWLEDGMENT We gratefully acknowledge NAI Inc. (Kanagawa, Japan) for their support in proofreading the manuscript and providing valuable suggestions.

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