On-Chip Identification and Interaction Analysis of Gel-Resolved

Yokohama City University, International Graduate School of Arts and Sciences, ... Yokohama 230-0045, Japan, Toyo Kohan Co., Higashi Toyoi, Kudamatsu, ...
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On-Chip Identification and Interaction Analysis of Gel-Resolved Proteins Using a Diamond-like Carbon-Coated Plate Yuko Iwafune,†,# Jian-Zhong Tan,†,# Yoko Ino,† Akiko Okayama,† Yuji Ishigaki,† Koji Saito,† Nobutake Suzuki,† Mikiko Arima,† Mitsuyoshi Oba,‡ Shuichi Kamei,‡ Michifumi Tanga,‡ Takeshi Okada,§ and Hisashi Hirano*,† Yokohama City University, International Graduate School of Arts and Sciences, Suchiro 1-7-29, Tsurumi, Yokohama 230-0045, Japan, Toyo Kohan Co., Higashi Toyoi, Kudamatsu, Yamaguchi 744-8611, Japan, SUS Co., Shinjuku, Tokyo 160-0022 Japan Received February 15, 2007

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or twodimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect proteinprotein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip. Keywords: protein chip • DLC • gel electrophoresis • electroblotting • protein interaction • protein identification

Introduction Protein chips are a useful tool to profile proteins such as disease-associated proteins and to analyze protein-protein interactions including antibody-antigen interactions,1-5 proteinsmall molecule interactions,1,6 and enzyme-substrate7,8 interactions. The most common form of analytical protein chips is antibody microarrays in which antibodies that bind to specific antigens are arrayed on a solid surface. Many research groups directly have arrayed antibodies on the surface of glass or silicon slides to detect antibody-antigen interactions quantitatively.1,4,5,9-11 For example, Sreekumar et al.11 spotted 146 antibodies onto a glass slide to monitor protein expression levels to detect radiation-induced proteins. In addition to antibodies, various purified proteins such as recombinant proteins have been immobilized on the chemically modified surface of protein chips. MacBeath and Schreiber1 covalently immobilized recombinant proteins on the surface of a glass slide treated with aldehyde-containing silane to analyze proteinprotein interactions. Houseman et al.12 constructed a highdensity peptide chip on gold-coated glass slides and analyzed kinase-substrate specificity and the dynamics of kinase reactions. The gold-coated glass slide is available for monitoring interactions by surface plasmon resonance (SPR). They char* To whom correspondence should be addressed. E-mail; hirano@ yokohama-cu.ac.jp, tel., 81-45-508-7439; fax, 81-45-508-7667. † Yokohama City University. # These authors contributed equally to this study. ‡ Toyo Kohan Co. § SUS Co. 10.1021/pr070083j CCC: $37.00

 2007 American Chemical Society

acterized phosphorylation of immobilized peptides using SPR and phosphorimaging. Recently, protein chip technology has been used for largescale analysis of post-translational modifications.12-17 Zhu et al.17 prepared 119 yeast protein kinases and covalently immobilized them on a nanowell chip. The chips were incubated with 33P γ-ATP and 17 different substrates. Proteins on the chip were analyzed for phosphorylated substrates by phosphorimaging. Using this nanowell protein chip, they characterized many new kinase activities including the unexpected tyrosine kinase activity. Gelperin et al.16 analyzed glycoproteins using the same protein chip with immobilized recombinant yeast proteins. To produce the chips, they purified MORF proteins from a “movable ORF” library of 5854 yeast expression plasmids that contained a His-tag. Using this chip with an antibody that binds to specific glycan chains, they identified 109 newly confirmed N-linked glycoproteins and 345 candidate glycoproteins in the yeast proteome. In this way, the protein chip is useful for protein identification, analyzing protein-protein interactions and post-translational modification. Production of high-density protein chips has been achieved using a robotic spotting microarrayer; however, the automatic spotting system is not very versatile. Moreover, to immobilize a large number of proteins on the chip, it is necessary to purify the individual proteins. Purification of a large number of proteins is laborious, time-consuming, and often impossible, which limits the production of highdensity protein chips. To overcome this problem, we developed a diamond-like, carbon-coated stainless steel plate (DLC plate), which was chemically modified with N-hydroxysuccinimide Journal of Proteome Research 2007, 6, 2315-2322

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research articles (NHS) ester. The NHS ester reacts with primary amines on the proteins to covalently attach them to the surface of the DLC plate. Since the DLC plate is conductive, it can be used for electroblotting of proteins from gels following separation by gel electrophoresis, for example, from polyvinylidene difluoride membranes. Two-dimensional electrophoresis (2-DE) using isoelectric focusing in the first dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.18 To produce high-density protein chips in a short period of time, we have resolved proteins using gel electrophoresis instead of chromatography purification, and immobilized gel-resolved proteins on the DLC plate by electroblotting. Moreover, this DLC plate is available for use as a target plate for MALDI-TOF MS. Using the DLC plate and MALDI-TOF MS, we performed protein digestion on the DLC plate and peptide mass fingerprinting (PMF) analysis to identify proteins immobilized on the chip. For proteomic analysis, in-gel digestion of proteins is a widely used procedure. Protein spots in gels are digested with proteases for protein identification on 2-DE gels, and the resultant digests are measured by mass spectrometry. On the basis of the PMF or amino acid sequences obtained from the mass spectra, gel-resolved proteins are identified. In this study, the on-chip digestion method, which involves digestion of proteins on the DLC plate, obtained almost the same sequence coverage as the in-gel digestion method. Also, the interactions between ligand proteins and proteins immobilized on the chip could be detected on the DLC plate. These interacting proteins were identified by MS analysis of the chip. Moreover, we detected glycoproteins on the chip by electroblotting gel-resolved proteins from the gel and identified them by MS analysis. This is the first report that demonstrates the development of a technique whereby gel-resolved proteins are electroblotted onto a chemically modified stainless steel plate to make a protein chip, which is then used in mass spectrometry to analyze the proteins.

Materials and Methods Proteins. Low-molecular-weight marker proteins, peptide markers kit, CyDye reactive Dye, and peptide marker kits were purchased from GE Healthcare (Piscataway, NJ). Phosphorylase b, bovine serum albumin (BSA), carbonic anhydrase, trypsin inhibitor, immunoglobulin G (IgG), insulin, protein A, and calmodulin (CaM) were purchased from Sigma Aldrich (St. Louis, MO). Concanavalin A (Con A) was obtained from Wako Pure Chemical Industries (Osaka, Japan). The 4-kDa hormonelike peptide was purified from soybean according to the method described by Hanada et al.19,20 Calmodulin binding peptide (CBP), KRRWKKNFIAVSAANRFKKISSSGALC, was synthesized at the TORAY Research Center (Kanagawa, Japan). Yeast proteins were extracted from Saccharomyces cerevisiae B-8032. The extract was centrifuged at 40 000g for 20 min, and the resultant supernatant was passed through a 0.22-µm membrane filter. With the use of a pestle and mortar, soybean proteins were extracted from cotyledons into lysis buffer for 2-DE.21 These protein samples were labeled with Cy3 or Cy5 maleimide or NHS ester labeling reagents according to the instructions supplied by GE Healthcare. Detection of Proteins Labeled with CyDye. Cy3-labeled proteins were detected using a Typhoon 9410 (GE Healthcare) 2316

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at an excitation wavelength of 532 nm and emission filter of 580 nm. Similarly, the Cy5-labeled proteins were detected at an excitation wavelength of 633 nm and emission filter of 670 nm. Fluorescence intensity was quantified using ImageQuant (GE Healthcare). Production of the DLC Plate. Diamond-like carbon film was deposited on a stainless steel plate (SUS410, Toyo Kohan Co., Yamaguchi, Japan) by the ion-assisted deposition method.22 Briefly, the plate was set into a reactor evacuated under 8 × 10-3 Pa by a turbo molecular pump and then cleaned by plasma using hydrogen as a feed gas. The flow rate of hydrogen gas was controlled at 40 sccm, and the RF power of self-bias for the plate was 100 W. After the treatment, methane and hydrogen gases were introduced into the reactor to deposit diamond-like carbon film on the plate at ambient temperature. The flow rates of methane and hydrogen gases were 47.5 and 2.5 sccm, respectively. The working pressure in the reactor and the RF power for the plate were kept at 3 Pa and 200 W, respectively. The surface of diamond-like carbon film was chlorinated using chlorine gas and aminated by plasma using ammonium as feed gas at the flow rate of 18 sccm and a working pressure of 3 Pa. To introduce carboxyl groups to the aminated surface of the diamond-like carbon film, the plate was immersed in 1-methyl-2-pyrolidone solution containing 0.14 M succinic anhydride and 0.1 M sodium borate at 25 °C for 10 min. The plate was then rinsed with deionized water and dried under a vacuum. To activate the carboxyl groups attached to the diamond-like carbon film surface, the plate was immersed in an aqueous solution containing 0.2 M N-ethyl-N′-1-(3-dimethylaminopropyl) calbodiimide hydrochloride, 0.2 M N-hydroxysuccinimide, and 0.1 M K2HPO4/KH2PO4 at 25 °C for 20 min. The plate was then rinsed with deionized water and dried under a vacuum at 100 °C. The DLC plate is commercially available from Nihon Parkerizing (Tokyo, Japan). SDS-Gel Electrophoresis. SDS-PAGE was performed using the method described by Laemmli,23 but with some modifications; thin slab gels (80 mm × 60 mm × 0.3 mm) were used, and electrophoresis was carried out at 5 mA. Tricine-SDS-PAGE was performed as described by Scha¨gger and von Jagow24 with some modifications; thin slab gels (80 mm × 60 mm × 0.3 mm) were used, and electrophoresis was carried out at 5 mA. Electroblotting of Gel-Resolved Proteins onto the DLC Plate. After gel electrophoresis, the gel was soaked in 10% (v/ v) methanol for 30 s, and then freshyly prepared 10% methanol for 3 min, and rinsed with deionized water. The proteins were electroblotted onto the DLC plate from the gel in the semi-dry blotting apparatus. We used 1 M borate buffer (pH 8.0) as a blotting solution. Two pieces of filter paper (3MM; Whatman, Florham Park, NJ) were soaked in the blotting solution, and excess solution on the gel or filter paper was removed. The DLC plate was placed on the cathode carbon layer of the semi-dry blotting apparatus and overlaid with the gel and two wet filter papers. After electroblotting was performed for 1 h at 2 V and room temperature, the DLC plate was rinsed with deionized water. Immobilized pH Gradient Electrophoresis. Yeast (50 µg) and soybean proteins (20 µg) were separated using ReadyStrip IPG Strips (7 cm, pH 3-10, NL) and the PROTEAN IEF Cell from Bio-Rad laboratories (Hercules, CA). The running program supplied by the manufacturer was used. Gel-resolved proteins were electroblotted onto the DLC plate using a semi-dry blotting apparatus (SUS, Tokyo, Japan).25

On-Chip Identification of Gel-Resolved Proteins

On-Chip Digests. For protein identification, proteins immobilized on the DLC plate by electroblotting were digested with proteases applied to the chip. One microliter of protease solution containing lysylendopeptidase (Wako Pure Chemical Industries; 1 µg/mL) or trypsin (Promega, Madison, WI; 15 µg/ mL) in 5 mM NH4HCO3 (pH 8.0) was applied to the DLC plate. An on-chip digest was performed for at least 5 h at 37 °C, in a humidified environment in a petri dish. After the digest was performed, matrix solution saturated with R-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid was dispensed onto the chip. The digests were analyzed using MALDI-TOF MS (TofSpec-2E, Micromass, Manchester, U.K.). The proteins immobilized on the chip were identified by PMF using the Mascot server (http://www.matrixscience.com/). Detection of Protein-Protein Interactions. After protein immobilization on the chip, the remaining active groups on the surface were blocked with blocking reagent N102 (NOF corporation, Tokyo, Japan). For interaction analysis with IgG, insulin, or soybean 4-kDa hormone-like peptide, the protein chip was incubated in TBS buffer (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 10 µg/mL IgG, insulin, or the 4-kDa peptide for 1 h. The protein chip was then washed with TBS buffer and rinsed with deionized water. For analysis of the interaction between CaM and CBP, 10 µg/mL CaM in TBSC buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM CaCl2) was used instead of TBS buffer. These protein chips were imaged with Cy3 or Cy5 fluorescence. The image matching was analyzed using ImageQuant. On-chip digest was performed as described in Materials and Methods. The digests were measured using MALDI-TOF MS. Detection of Glycoproteins on the Protein Chip. Cy3labeled soybean proteins (20 µg) were resolved by 2-DE and electroblotted onto the DLC plate. After blocking, the chip was incubated with 10 µg/mL Cy5-labeled Con A in TBS buffer. The chip was scanned for Cy3 and Cy5 fluorescence. On-chip digest was then performed as described in Materials and Methods. The digests were measured using MALDI-TOF MS.

Results and Discussion Production of DLC Plates and Preparation of the Protein Chips. Diamond-like carbon film was deposited on conductive stainless steel plates instead of nonconductive glass or silicon plates by the ion-assisted deposition method. The surface of the DLC plate was then activated with NHS ester (Figure 1A). The resulting chemically modified DLC plate can covalently immobilize proteins. We predicted that since the DLC plate was conductive, gel-resolved proteins could be immobilized on the plate from the gel by electroblotting using a semi-dry blotting apparatus, in order to make high-density protein chips (Figure 1B). We investigated the experimental conditions for electrophoresis and electroblotting to produce the protein chips efficiently. The diamond-like carbon film is similar to diamond film but not identical in molecular structure. The diamondlike carbon film is amorphous and more chemically fragile than the diamond film. Therefore, when proteins were electroblotted from the gel onto the DLC plate using more than 3 V, the diamond-like carbon film deposited on the surface of the DLC plate was easily electrolyzed in aqueous solution (Figure 2A). We found that the electroblotting should be performed quickly at very low voltage. However, a 1-mm thick gel, which is usually used for SDS-PAGE, was not appropriate for electroblotting,

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Figure 1. (A) Chemical modification of the DLC plate with NHS ester. The modified DLC plate bound gel-resolved proteins covalently. (B) Scheme for protein identification and proteinprotein interaction analysis using the DLC plate. Proteins separated by gel electrophoresis are electroblotted onto the DLC plate using a semi-dry blotting apparatus. Digests are generated by on-chip digestion and are measured by MALDI-TOF MS to identify proteins (a). Alternatively, protein or peptide probes are incubated on the chip, and interacting probes are then detected by MALDI-TOF MS (b).

because it is too thick for rapid and efficient transfer of proteins from the gel at 2 V. The blotting efficiency of 0.3-mm thick gels was 50-200 times higher than that of 1-mm thick gels (data not shown). Therefore, thinner gels showed higher protein transfer efficiency. As less than 0.3-mm thick gels were difficult to handle, we used 0.3-mm thick gels in this study. Transfer efficiency increased with longer transfer times. However, we found that the diamond-like carbon film was electrolyzed when electroblotted for longer than 2 h (Figure 2B). Therefore, we employed 1 h blotting time. In general, 25 mM Tris or 40 mM Tris 6-aminocaproic acid is used for Western blotting with a semi-dry blotting apparatus. However, for the electroblotting of proteins onto the DLC plate, 1 M borate buffer was used. Borate buffer had a 2.5-5 times better blotting efficiency than Tris and Tris 6-aminocaproic acid solutions. The reduction of blotting efficiency in the Tris and 6-aminocaproic acid solutions may be due to the amino groups of Tris and 6-aminocaproic acid, because these amino groups can attach to NHS esters on the surface of the DLC plate. We obtained better results with 1 M borate buffer than with 0.1 and 0.5 M borate buffer. The blotting efficiency was further improved Journal of Proteome Research • Vol. 6, No. 6, 2007 2317

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Figure 2. (A) Effect of different blotting voltage on the blotting efficiency from gel to DLC plate. Cy3-labeled proteins were separated by SDS-PAGE using 0.3 mm thick gels and electroblotted from the gels onto the DLC plate at different blotting voltages (1, 2, 3, and 4 V) for 1 h. The blotting buffer contained 1 M borate buffer. (B) Effect of blotting time on the blotting efficiency from gel to DLC plate. Cy3-labeled proteins were electroblotted for different blotting times (0.5, 1, and 2 h) at 2 V. Lane 1, Cy3-labeled avidin 0.3 µg; lane 2, Cy3-labeled avidin 0.15 µg and Cy3-labeled IgG 0.3 µg; lane 3, Cy3-labeled IgG 0.6 µg. Table 1. Experimental Conditions for Electrophoresis and Electroblotting SDS-PAGE

Semi-dry blotting

%T %C Thickness of gel Current Running time Protein chip plate Voltage Blotting time Blotting solution Temperature

10% 2.6% 0.3 mm 5 mA constant 1.5 h SUS 410 (Cr > 11%, Ni, Mo, C) 2 V constant 1h 1M Borate buffer 30 °C constant

when the gel was washed twice for 30 s and 3 min with 10% methanol and deionized water after electrophoresis. To prevent swelling of the gels, 10% methanol was added to the washing solution. To make an estimate of the electroblotting efficiency from the gel onto the DLC plate under the conditions shown in Table 1, Cy3-labeled proteins containing marker proteins were separated by SDS-PAGE using a 0.3-mm thick gel (80 mm × 60 mm × 0.3 mm). After electrophoresis, the gel was soaked in 10% methanol, and proteins were electroblotted from the gel onto the DLC plate. Two pieces of filter paper and the gel were equilibrated in 1 M borate buffer, and excess blotting buffer was removed. The DLC plate was placed on the cathode carbon layer of the semi-dry blotting apparatus and overlaid with the gel and the two wet filter papers. After electroblotting for 1 h at 2 V, the DLC plate was rinsed with deionized water and dried. The Cy3-labeled proteins immobilized on the DLC plate were detected by Cy3 fluorescence. According to the results of the experiments described above, we have determined the best conditions for electrophoresis and electroblotting for making the protein chips (Table 1). Using these conditions, we estimated electroblotting efficiency by comparing the fluorescence intensity in the gel before blotting and on the DLC plate after blotting. The highest yield (71%) 2318

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Figure 3. Identification of yeast proteins on the protein chip. Cy3labeled yeast proteins (50 µg) were separated by 2-DE. Proteins in the gel were detected with CBB (A). Cy3-labeled proteins were electroblotted from the gel (without CBB staining) onto the DLC plate. Proteins on the chip were detected with Cy3 fluorescence (B). Proteins were digested with trypsin on the DLC plate and identified by PMF. The results of protein identification from the protein chip are shown in Table 3. Table 2. Blotting Efficiency of Proteins from Gel onto the DLC Plate protein

MW

blotting

Phosphorylase b Avidin BSA Immunoglobulin Ovalbumin ProteinA Carbonic anhydrase

97k 68k 66k 55k 45k 42k 30k

35% 32% 35% 71% 55% 68% 28%

was obtained for immunoglobulin, and the lowest (28%) was for carbonic anhydrase (Table 2). Identification of Proteins Using the Protein Chip. We developed a method to identify proteins electroblotted onto the DLC plate. We prepared two 2-DE gels in which Cy3-labeled yeast proteins were separated. Proteins in one gel were visualized by Coomassie Brilliant Blue (CBB) staining (Figure 3A). Proteins in the other gel were electroblotted onto the DLC plate and detected on the DLC plate using Cy3 fluorescence (Figure 3B). The blotting efficiency was estimated to be about 50% by comparing Cy3 fluorescence in the gel and on the DLC plate, but this varied depending on the properties of the protein. Lowmolecular-weight proteins were easily eluted from the gels. In addition to molecular weight, the amount of proteins in the spots affected the blotting efficiency. In many cases, smaller amount of proteins showed higher yield in the blotting. After blotting, protease solution was applied to the DLC plate which had been used to detect Cy3 signals to perform on-chip digests of proteins. Matrix solution was then added to the resultant digests on the chip, and the chip was set as a MALDI-TOF MS target plate to measure the mass of digests. All raw mass spectra were processed automatically, and selected monoisotopic masses were searched using the Swiss-Prot database. Twenty protein spots (as shown in Figure 3) were analyzed and successfully identified by PMF using on-chip digestion (Table 3). The sequence coverage of peptides generated by on-chip

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On-Chip Identification of Gel-Resolved Proteins Table 3. Protein Identification Using On-Chip Digests spot no.

entry name

protein name

accesion no.

peptides hits

coverage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

PGK_YEAST PGK_YEAST G3P3_YEAST G3P3_YEAST ENO1_YEAST ADH_YEAST PDC1_YEAST PDC1_YEAST ENO2_YEAST ENO2_YEAST ENO2_YEAST ALF_YEAST ALF_YEAST IPYR_YEAST GBLP_YEAST VDAC1_YEAST PMG1_YEAST HSP26_YEAST TPIS_YEAST TPIS_YEAST

Phosphoglycerate kinase Phosphoglycerate kinase Glyceraldehyde 3-phosphate dehydrogenase 3 Glyceraldehyde 3-phosphate dehydrogenase 3 Enolase 1 Alcohol dehydrogenase 1 Pyruvate decarboxylase isozyme 1 Pyruvate decarboxylase isozyme 1 Enolase 2 Enolase 2 Enolase 2 Fructose-bisphosphate aldolase Fructose-bisphosphate aldolase Inorganic pyrophosphatase Guanine nucleotide-binding protein beta subunit Outer mitochondrial membrane protein porin 1 Phosphoglycerate mutase 1 Heat shock protein 26 Triosephosphate isomerase Triosephosphate isomerase

P00560 P00560 P00359 P00359 P00924 P00330 P06169 P06169 P00925 P00925 P00925 P14540 P14540 P00817 P38011 P04840 P00950 P15992 P00942 P00942

8 8 5 5 12 8 10 6 14 12 6 8 7 8 7 7 6 10 4 5

36% 35% 24% 26% 46% 36% 23% 17% 63% 38% 20% 38% 30% 28% 28% 47% 26% 60% 22% 28%

digests was 15-60%, which was sufficient to identify proteins using the protein database and PMF. The sequence coverage obtained with the on-chip digests and conventional in-gel digests was almost equal, showing that the protein chips produced with the DLC plate are useful for the identification of gel-resolved proteins. Analysis of Protein Interactions. 1. Protein-Protein Interactions. We investigated the possibility of performing proteinprotein interaction analysis on the DLC plate. The NHS ester active group on the surface of DLC plate loses covalent activity after a few hours in solution. This inactivation is advantageous, as it reduces nonspecific binding and contamination during incubation of the ligand proteins. However, without blocking after incubation with the ligand proteins, nonspecific protein binding was observed on the DLC plate due to the zeta electric potential. This nonspecific binding was noncovalent binding and could be suppressed by the addition of a high concentration of organic solvent, such as methanol. However, organic solvents inhibit protein-protein interactions. Therefore, it was necessary to block the protein-unbinding sites on the surface. Since proteinous blocking reagents were not suitable for MS analysis using on-chip digests, we used a synthetic polymer blocking reagent, N102. This reagent could block the proteinunbinding sites and had no effect on measuring the mass of proteins on the DLC plate by MALDI-TOF MS. Four proteins (BSA, carbonic anhydrase, trypsin inhibitor, and Cy5-labeled protein A) were separated by SDS-PAGE (Figure 4A) and electroblotted onto the DLC plate. The efficiency of protein immobilization on the chip was evaluated by monitoring the Cy5 fluorescence intensity of protein A. After blocking, the protein chip was incubated with Cy3-labeled IgG in TBS buffer. The interaction between the protein mixture containing protein A and Cy3-labeled IgG was detected by measuring the Cy3 fluorescence intensity (Figure 4B). Only the position corresponding to protein A showed the Cy3 fluorescence signal of IgG. The protease solution was then deposited on the chip at regular intervals, and proteins immobilized on the chip were digested. The resultant digests were measured by MALDI-TOF MS. Figure 4C shows MS spectra of the digests. The MS spectrum (b) in Figure 4C contained the MS spectra of protein A and IgG digests. There was no signal of digests from IgG in the position of any of the immobilizing proteins

except protein A (Figure 4C). We confirmed that protein A immobilized on the protein chip specifically interacted with IgG. These results show that we can specifically detect proteinprotein interactions on the protein chip and identify the interacting proteins by MS. 2. Protein-Peptide Interaction. The 43-kDa hormone receptor-like protein from soybean binds the 4-kDa hormonelike peptide from soybean (Kd ) 1.80 × 10-8 M) and insulin from mammals.19,20 Soybean cotyledon proteins labeled with Cy3 were resolved by 2-DE and electroblotted onto a DLC plate (Figure 5A). After blocking, the protein chip was incubated with Cy5-labeled 4-kDa peptide and insulin in TBS buffer. The interacting peptides were detected by Cy5 fluorescence (Figure 5B). The spots detected with Cy3 and Cy5 were digested with trypsin on the chip, and the protein chip was subjected to MALDI-TOF MS analysis. We detected paired signals for the 43-kDa protein and insulin (Figure 5C) and for the 43-kDa protein and 4-kDa peptide (Figure 5D). We also applied the DLC plate technology to the identification of proteins that interact with gel-resolved peptides on the chip. We separated synthetic peptides containing the calmodulin-binding motif (calmodulin binding peptide, CBP) using Tricine-SDS-PAGE (Figure 6A). The gel-resolved CBP was electroblotted onto a DLC plate (Figure 6B). The protein chips were incubated with Cy5-labeled calmodulin (CaM) in TBSC buffer with Ca2+ or TBS buffer without Ca2+ (Figure 6C). Cy5labeled CaM interacted with CBP only in presence of Ca2+, indicating that we can detect CaM-CBP interaction in a calcium-dependent manner on the protein chip and demonstrating that Cye-labeling has no effect on the interaction between CBP and CaM (Figure 6C, left panel). The position at which Cy5-labeled CaM was detected was digested with trypsin on the chip, and the digests were measured by MALDI-TOF MS. These spectra showed that the digests contained a mixture of CaM and CBP digests; therefore, the interaction between CaM and CBP was detected by MS (Figure 6D). Detection of Glycoproteins on the Protein Chip. We have attempted to detect the post-translational modification of glycosylated proteins on the protein chip using Concanavalin A (Con A), a kind of lectin that specifically binds to N-linked oligosaccharide chains containing R-mannose groups and R-glucose groups. In this study, we analyzed the glycoproteins Journal of Proteome Research • Vol. 6, No. 6, 2007 2319

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Figure 4. Detection of the interaction between protein A and IgG. BSA (66-kDa) (a), Cy5-labeled protein A (43-kDa) (b), carbonic anhydrase (30-kDa) (c), and trypsin inhibitor (20-kDa) (d) were separated by SDS-PAGE and stained with CBB (A). After electroblotting from the gel (without CBB staining) onto the DLC plate, the chip was incubated with Cy3-labeled IgG in TBS buffer, and the interaction with Cy3-labeled IgG was detected by Cy3 fluorescence (B). These proteins were then digested with lysylendopeptidase on the chip, and digests were measured by MALDITOF MS for identification (C). The mass spectra on (a), (c), and (d) were identified by PMF as BSA, carbonic anhydrase, and trypsin inhibitor. The MS spectrum on (b) contained the signals from protein A and Cy3-labeled IgG interacting with protein A. The signals from protein A are indicated with asterisks. The signals from IgG are indicated with arrows.

in soybean cotyledons using Con A on the DLC plate. The Cy3labeled proteins from soybean cotyledons were resolved by 2-DE and electroblotted onto the DLC plate (Figure 7A). The protein chip was incubated with Cy5-labeled Con A in TBS buffer and imaged by fluorescence (Figure 7B). The signals from Cy5-labeled Con A were detected on two major spots. The spots were digested on the chip with trypsin and then measured by 2320

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Figure 5. Interaction between the 4-kDa peptide or insulin and soybean cotyledon proteins. (A) Detection of Cy3-labeled soybean cotyledon proteins electroblotted onto the DLC plate from 2-DE gel. (B) Detection of Cy5 fluorescence on the chip after incubation with Cy5-labeled insulin. Interacting insulin (arrow) was detected by Cy5 fluorescence scanning of the chip. (C) The mass spectrum of the proteins detected with Cy5-insulin signals. The signals from the 43-kDa protein are indicated with asterisks. The signal from insulin, [M + H] ) 4863.2, is indicated with an arrow. (D) The spectrum shows the interaction between the 43kDa protein and the 4-kDa peptide. The signals from the 43-kDa protein are indicated with asterisks. The signal from the 4-kDa peptide, [M + H] ) 3918.2, is indicated with an arrow.

MALDI-TOF MS (Figure 7C). These proteins (spot 1 and 2) were identified as β-conglycinin β-subunit (BAA23361) and 43kDa receptor-like protein (BAA03681) by PMF using Swiss-Prot and Entrez databases. The β-conglycinin β-subunit has sugar chains that interact with Con A.26 In this study, in addition to β-conglycinin, we showed glycosylation of the 43-kDa receptor-

On-Chip Identification of Gel-Resolved Proteins

Figure 6. (A) Detection of CBP stained with CBB on a TricineSDS-PAGE gel. Lane 1, Cy5-labeled peptide marker; lane 2, Cy5labeled CBP 5 µg; lane 3, non-labeled CBP 5 µg. (B) After electroblotting from the gel (without CBB staining) onto the DLC plate, these peptides were detected on the chip with Cy5. (C) The interaction was detected by Cy3 fluorescence after incubation with Cy3-labeled CaM in the presence of Ca2+ (left panel) or absence of Ca2+ (right panel). (D) The mass spectrum of the position detected by the Cy3 signal after on-chip digestion. The spectrum shows the interaction between CBP and CaM. The signal from CBP is indicated with an asterisk. The signals from CaM are indicated with arrows.

like protein using protein-protein interaction analysis on the protein chip.

Future Prospects In this study, we developed a novel protein chip made of a DLC stainless steel plate with the surface modified with an NHS ester. Using this DLC plate, we can simply and rapidly produce useful protein chips by electroblotting. We established three techniques using MALDI-TOF MS for identification of proteins immobilized on the protein chip, for detection of proteins that interact with immobilized proteins, and for analysis of glycoproteins on the protein chip. At present, these techniques have some problems; for example, proteins immobilized on the DLC plate do not keep their primary structure after electrophoresis under denaturing conditions. In fact, protein A could not bind to IgG when IgG was denatured by SDS-PAGE and electroblotted onto the DLC plate (data not shown), as this protein-protein interaction requires a native folding state. However, we demonstrated that it was possible to detect the interaction between

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Figure 7. (A) Detection of Cy3 fluorescence on the protein chip electroblotted from the 2-DE gel-resolved Cy3-labeled soybean proteins. (B) Detection of Cy5 fluorescence after incubation with Cy5-labeled Con A. (C) Mass spectra of two spots on the chip that interacted with Con A. Spot 1 (a) and spot 2 (b) were identified as β-conglycinin β-subunit (BAA23361) and the 43-kDa receptor-like protein (BAA03681) by PMF, respectively. The signals from Con A are indicated with arrows. The signals from the immobilized protein are indicated with asterisks.

the 43-kDa receptor-like protein and insulin or 4-kDa peptide on the chip, because the exact native conformation of the proteins is not required for these interactions. Therefore, at present, DLC plate electroblotted gel-resolved proteins can only detect interactions that do not require accurate native folding. In the future, the techniques may be improved and applied to analysis of native proteins separated by native gel electrophoresis. The DLC plate is available for protein microarray support using a robotic spotting microarrayer or for spotting by hand. With the use of these methods where proteins are spotted on the DLC plate directly, the immobilized proteins are more likely to remain in their native conformation. If it is unnecessary to resolve proteins by electrophoresis; the DLC plate can simply be used as a protein chip for interaction analysis. Journal of Proteome Research • Vol. 6, No. 6, 2007 2321

research articles The DLC plate has great potential to be used for highthroughput screening of protein interactions. We expect that the techniques will be applied to analyses of interactions not only between proteins, but also between proteins and DNA or drugs.

Acknowledgment. This work was supported partially by grants from the Ministry of Economy, Trade and Industry of Japan, the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Ministry of Agriculture, Forestry, and Fisheries of Japan, and Japan Society for the Promotion of Science (Fellowship to J.Z.T., Suzhou University). We thank Dr. Nazrul Islam for his help in preparing the manuscript. References (1) MacBeath, G.; Schreiber, S. L. Printing proteins as microarrays for high-throughput function determination. Science 2000, 289, 1760-1763. (2) Templin, M. F.; Stoll, D.; Schwenk, J. M.; Potz, O.; Kramer, S.; Joos, T. O. Protein microarrays: promising tools for proteomic research. Proteomics 2003, 3, 2155-2166. (3) Chiari, M.; Cretich, M.; Corti, A.; Damin, F.; Pirri, G.; Longhi, R. Peptide microarrays for the characterization of antigenic regions of human chromogranin A. Proteomics 2005, 5, 3600-3603. (4) Haab, B. B.; Dunham, M. J.; Brown, P. O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. GenomeBiology 2001, 2, Research0004.1-0004.13. (5) Schweitzer, B.; Kingsmore, S. F. Measuring proteins on microarrays. Curr. Opin. Biotechnol. 2002, 13, 14-19. (6) Fang, Y.; Frutos, A. G.; Lahiri, J. Membrane protein microarrays. J. Am. Chem. Soc. 2002, 124, 2394-2395. (7) Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; McCartney, R. R.; Schmidt, M. C.; Rachidi, N.; Lee, S. J.; Mah, A. S.; Meng, L.; Stark, M. J.; Stern, D. F.; De Virgilio, C.; Tyers, M.; Andrews, B.; Gerstein, M.; Schweitzer, B.; Predki, P. F.; Snyder, M. Global analysis of protein phosphorylation in yeast. Nature 2005, 438, 679-684. (8) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Global analysis of protein activities using proteome chips. Science 2001, 293, 2101-2105. (9) Ingvarsson, J.; Lindstedt, M.; Borrebaeck, C. A.; Wingren, C. Onestep fractionation of complex proteomes enables detection of low abundant analytes using antibody-based microarrays. J. Proteome Res. 2006, 5, 170-176. (10) De Wildt, R. M.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Antibody arrays for high-throughput screening of antibodyantigen interactions. Nat. Biotechnol. 2000, 18, 989-994. (11) Sreekumar, A.; Nyati, M. K.; Varambally, S.; Barrette, T. R.; Ghosh, D.; Lawrence, T. S.; Chinnaiyan, A. M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins. Cancer Res. 2001, 61, 7585-7593.

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