Specific Affinity Extraction Method for Small Molecule-Binding Proteins

Use of Ligand Based Models for Protein Domains To Predict Novel Molecular Targets and Applications To Triage Affinity Chromatography Data. Andreas ...
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Anal. Chem. 2006, 78, 4668-4675

Specific Affinity Extraction Method for Small Molecule-Binding Proteins Nariyasu Mano,† Koichi Sato,† and Junichi Goto*,†,‡

Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan, and Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan

Validation of the targets of candidate drugs is critical for rapid and efficient drug discovery and development and for understanding the pharmacological action and potential toxicities of the prospective therapeutic agent. Due to the nonspecific binding of abundant proteins to small molecule-immobilized gels, it is difficult to identify the protein targets of small molecules from crude biological samples by affinity extraction. To address this problem, we have developed an affinity gel for the specific extraction of small molecule-binding proteins. We immobilized small molecules on the agarose gel through a disulfide linker that is cleavable by mild reduction. This system has allowed specific and noncovalent complex formation between the small molecule and the target protein, keeping the effect of the nonspecific abundant proteins adsorbed on both the linker and gel surface to minimum. By preparing this affinity matrix with deoxycholate as a model small molecule, we captured two independent deoxycholate-binding proteins of different affinities from mouse ascites, anti-deoxycholate antibody, and serum albumin. As other proteins were not captured, this affinity extraction method should contribute significantly to the accurate and rapid drug discovery and development. Proteomics requires high-throughput, sensitive methods to analyze drug-target interactions, a key step for understanding both the signal transduction pathways functioning in important intracellular events and the pharmacological actions of drugs. Within cells, proteins exist in high concentrations, forming complexes with other cellular proteins for setting up of the efficient intracellular signaling pathways. Tandem affinity purification using tagged proteins, therefore, allows the rapid purification and identification of binding proteins of the target proteins.1 When an exogenous low-molecular substance, such as a drug, enters a cell, it will interact with the target protein to exert its pharmacological action. Binding of proteins to drugs or physiological ligands often alters the protein’s three-dimensional shape, resulting in the assembly of a different protein complex. Therefore, analysis of the complexes formed by the binding of small molecules to a * To whom correspondence should be addressed. Tel: +81-22-717-7525. Fax: +81-22-717-7545. E-mail: [email protected]. † Tohoku University. ‡ Tohoku University Hospital. (1) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Se´raphin, B. Nat. Biotechnol. 1999, 17, 1030-1032.

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target protein is critical for the analysis of signaling network pathways. Small molecule-immobilized affinity gels have been used primarily for the purification of enzymes and other small moleculebinding proteins. Schreiber and co-workers used this method to identify FK506-binding protein2 and mammalian histone deacetylase 1.3 Exogenous substances, such as drugs, typically have lower affinities for their target proteins than their physiological counterpart ligands. Affinity purification of low abundance target proteins, such as receptors, by use of small molecule-immobilized affinity gels is difficult, because of adsorption of a greater number of nonspecific proteins. Oda et al.4 described a drug target validation strategy using small molecule-immobilized affinity gel, in which two chemical affinity matrixes, one pharmacologically active and the other inactive, were combined with quantitative analysis using isotope-coded affinity tags5 and two-dimensional differential in-gel electrophoresis.6 Although this complicated method can validate the specificity of drug target proteins, it cannot be used to analyze the role of such protein complexes in signaling network pathways mediated by small molecule-binding proteins. Brocklehurst et al. utilized a Sepharose-(glutathione-2-pyridyl disulfide) conjugate, which is now commercially available, to purify fully active papain from dried papaya latex by covalent chromatography using thiol-disulfide interchange.7 These results clearly suggest that reductive cleavage of disulfide bonds with dithiothreitol (DTT) did not influence the three-dimensional structure of the purified protein. Activated thiol affinity chromatography has also been used for the enrichment of phosphopeptides8 and for the selection of catalytic antibodies from a phage-display human combinatorial antibody library.9 Recently, a method for screening peptide-protein interactions was reported to capture proteins by binding to a target peptide linked by a disulfide linker to a biotin moiety; peptide-binding proteins were then isolated by the reduc(2) Harding, M. W.; Galat, A.; Uehling, D. E.; Schreiber, S. L. Nature 1989, 341, 758-760. (3) Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996, 272, 408-411. (4) Oda, Y.; Owa, T.; Sato, T.; Boucher, B.; Daniels, S.; Yamanaka, H.; Shinohara, Y.; Yokoi, A.; Kuromitsu, J.; Nagasu, T. Anal. Chem. 2003, 75, 2159-2165. (5) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (6) Unlu, M.; Morgan, M. E.; Minden, J. S. Electrophoresis 1997, 18, 20712077. (7) Brocklehurst, K.; Carlsson, J.; Kierstan, M. P.; Crook, E. M. Biochem. J. 1973, 133, 573-584. (8) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836. (9) Cesaro-Tadic, S.; Lagos, D.; Honegger, A.; Rickard, J. H.; Partridge, L. J.; Blackburn, G. M.; Plu ¨ ckthun, A. Nat. Biotechnol. 2003, 21, 679-685. 10.1021/ac060213s CCC: $33.50

© 2006 American Chemical Society Published on Web 05/20/2006

tive cleavage of the disulfide linker.10 This report also demonstrated changes in the isolated protein composition upon target peptide phosphorylation using stable isotopic amino acids in cell culture. Here we designed and prepared a novel cleavable affinity gel for purification of protein complexes containing small molecules. In this study, we use deoxycholic acid (DCA), a secondary bile acid similar to the lithocholic acid formed by the action of microbial flora in the intestine, as a model small molecule. DCA may act as a colon-specific tumor promoter in high-risk populations.11,12 Bile acids, the major metabolites of cholesterol, assist in lipolysis and the absorption of fats by forming mixed micelles in the intestinal lumen. These metabolites localize to the enterohepatic circulation due to their efficient hepatic uptake and bind to intra- and intercellular carrier proteins,13-18 membrane transporters,19-21 and receptors.22-24 Recent publications have suggested a number of novel physiological functions for bile acids, which may be mediated by bile acid-protein interactions.25 In this study, we use acsites in mouse administrated anti-DCA monoclonal antibody-generating hybridoma26 as a model biological fluid containing DCA-binding proteins. This antibody recognizes the steroid nucleus of DCA26-28 because it has been generated by use of the hapten-carrier protein complex as an immunogen, whose linkage was an amide bond spanning a carboxyl group at the C-24 position of DCA and the -amino group of lysine residues on the carrier protein.26 EXPERIMENTAL SECTION Reagents and Apparatus. R-Cyano-4-hydroxycinnamic acid (CHCA), the matrix for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), was supplied by Aldrich Chemical Co. (Milwaukee, WI). Sequence-grade (10) Schulze, W. X.; Mann, M. J. Biol. Chem. 2004, 279, 10756-10764. (11) Narisawa, T.; Magadia, N. E.; Weisburger, J. H.; Wynder, E. L. J. Natl. Cancer Inst. 1974, 53, 1093-1097. (12) Reddy, B. S.; Narisawa, T.; Weisburger, J. H.; Wynder, E. L. J. Natl. Cancer Inst. 1976, 56, 441-442. (13) Rudman, D.; Kendall, F. E. J. Clin. Invest. 1957, 36, 538-542. (14) Burke, C. W.; Lewis, B.; Panveliwalla, D.; Tabaqchali, S. Clin. Chim. Acta 1971, 32, 207-214. (15) Roda, A.; Cappelleri, G.; Aldini, R.; Roda, E.; Barbara, L. J. Lipid Res. 1982, 23, 490-495. (16) Sugiyama, Y.; Yamada, T.; Kaplowitz, N. J. Biol. Chem. 1983, 258, 36023607. (17) Stolz, A.; Hammond, L.; Lou, H.; Takikawa, H.; Ronk, M.; Shively, J. E. J. Biol. Chem. 1993, 268, 10448-10457. (18) Sinal, C. J.; Tohkin, M.; Miyata, M.; Ward, J. M.; Lambert, G.; Gonzalez, F. J. Cell 2000, 102, 731-744. (19) Abe, T.; Kakyo, M.; Tokui, T.; Nakagomi, R.; Nishio, T.; Nakai, D.; Nomura, H.; Unno, M.; Suzuki, M.; Naitoh, T.; Matsuno, S.; Yawo, H. J. Biol. Chem. 1999, 274, 17159-17163. (20) Hagenbuch, B.; Stieger, B.; Foguet, M.; Lu ¨ bbert, H.; Meier, P. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10629-10633. (21) Suchy, F. J.; Sippel, C. J.; Ananthanarayanan, M. FASEB J. 1997, 11, 199205. (22) Ananthanarayanan, M.; Balasubramanian, N.; Makishima, M.; Mangelsdorf, D. J.; Suchy, F. J. J. Biol. Chem. 2001, 276, 28857-28865. (23) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R. M.; Luk, A.; Hull, M. V.; Lustig, K. D.; Mangelsdorf, D. J.; Shan, B. Science 1999, 284, 1362-1365. (24) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.; Consler, T. G.; Kliewer, S. A.; Stimmel, J. B.; Willson, T. M.; Zavacki, A. M.; Moore, D. D.; Lehmann, J. M. Science 1999, 284, 1365-1368. (25) Mano, N.; Goto, T.; Uchida, M.; Nishimura, K.; Ando, M.; Kobayashi, N.; Goto, J. J. Lipid Res. 2004, 45, 295-300. (26) Kobayashi, N.; Katayama, H.; Nagata, M.; Goto, J. Anal. Sci. 2000, 16, 1133-1138.

Figure 1. Two procedures for preparation of affinity gels immobilizing small molecules using a cleavable linker.

modified trypsin was obtained from Promega (Madison, WI). Affigel 10 and coomassie brilliant blue (CBB) R-250 were purchased from Bio-Rad Laboratories (Hercules, CA). Protease Inhibitor Mix was purchased from Amersham Bioscience Co. (Piscataway, NJ). Cystamine dihydrochloride, DTT, 4-vinylpyridine, iodoacetamide, p-nitrophenol, sodium dodecyl sulfate, and DCA were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Mouse serum albumin was purchased from Sigma Chemical Co. (St. Louis, MO). DCA p-nitrophenyl ester was prepared by the carbodiimide method. ZipTipC18 cartridges were purchased from Millipore (Milford, MA). Distilled, ion-exchanged water was prepared using a CPW-100 Ultrapure Water System (Advantec Toyo Co., Tokyo, Japan). Additional reagents and solvents were HPLC grade. MALDI-TOF MS analysis was performed using a Voyager DESTR spectrometer (Applied Biosystems, Framingham, MA) equipped with an N2 laser (337 nm). In positive ion mode, we used a matrix solution of fresh saturated CHCA in water/ acetonitrile/trifluoroacetic acid (TFA) (50:50:0.1, v/v/v). Peptide samples dissolved in 0.5 µL of water/acetonitrile/TFA (25:75:0.1, v/v/v) were mixed with 0.5 µL of matrix solution on the MALDI plate. Spectra were calibrated either internally or externally using two monoisotopic [M + H]+ peptide standards, angiotensin I and an adrenocorticotropic hormone (ACTH) 7-38 fragment (American Peptide Co., Inc., Sunnyvale, CA), included in the matrix solution. Circular dichroism (CD) spectra were obtained using a model J-720 instrument (Jasco, Tokyo). Scans were performed at a scan speed of 20 nm/min using a cell with a 10 mm light-path length. Fluorescent spectra were monitored on a model RF-550 fluores(27) Mano, N.; Nagaya, Y.; Saito, S.; Kobayashi, N.; Goto, J. Biochemistry 2004, 43, 2041-2048. (28) Mano, N.; Kasuga, K.; Kobayashi, N.; Goto, J. J. Biol. Chem. 2004, 279, 55034-55041.

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Figure 2. SDS-PAGE analysis of purified DCA-binding proteins: a, molecular markers; b, mouse ascites; c, nonadsorbate of gel-A; d, gel-A wash; e, gel-A eluate; f, nonadsorbate of gel-B; g, gel-B wash; h, gel-B eluate.

cent detector (Simadzu, Kyoto). Using an excitation wavelength of 280 nm, the scan range spanned from 300 to 400 nm. Preparation of DCA-Immobilized Cleavable Affinity Gel. Two methods were used in this study to generate small moleculeimmobilized affinity gel matrixes (Figure 1). Gel-A was prepared as follows. Aff-gel 10 (10 mL), an agarose gel activated as an N-hydroxysuccinimidyl ester, was washed twice with 15 mL of 10 mM acetic acid-sodium acetate buffer (pH 4.5). The gel was then added to 60 mg of DCA cystamine conjugate in 15 mL of 50 mM potassium phosphate buffer (pH 7.4)/tetrahydrofuran (THF) (1: 1, v/v). After gently rotating the mixture at room temperature for 3 h, the gel was treated sequentially with 3 washes each of 30 mL of water/THF (1:1, v/v) and 30 mL of 100 mM KH2PO4-50 mM sodium borate buffer (pH 8). Residual active esters were protected by reaction of the gel with 1 mL of 1 M monoethanolamine in the same buffer for 1 h. After washing three times with 30 mL of 50 mM potassium phosphate buffer (pH 7.5), the gel was stored in 50 mM potassium phosphate buffer (pH 7.5) and supplemented with sodium azide (49:1, v/v, from a 10% solution) at 4 °C until use. The gel-B was prepared by a simpler procedure. Washed activated agarose gel (10 mL) was added to 3 g of cystamine

dihydrochloride solution in 15 mL of 50 mM potassium phosphate buffer (pH 7.4). After gentle rotation at room temperature for 5 h, the gel was washed six times in water (45 mL) and then five times in 40 mL of THF. Triethylamine in THF (30 µg/9 mL) and DCA p-nitrophenyl ester in THF (694 mg/8 mL) were then added to the gel. After mixing overnight at room temperature, we added methanol (45 mL) to the gel to terminate the coupling reaction. After washing six times in 40 mL of methanol and then five times in 40 mL of 50 mM potassium phosphate buffer (pH 7.4), the gel was stored at 4 °C until needed. Effect of the Cleavage of the Disulfide Linker on Protein Conformation. Mouse serum albumin (30 µg/3 mL for CD analysis and 16.65 µg/50 µL for fluorescent analysis), dissolved in 50 mM potassium phosphate buffer (pH 7.4), was incubated in the presence of various amounts of DTT. After 1 h at room temperature, the CD spectra of these solutions were monitored from 190 to 300 nm. In addition, the fluorescent spectra of the solutions were examined at emission wavelengths ranging from 300 to 400 nm when using an excitation wavelength of 280 nm. For MS analysis, the free mercapto groups of the cysteine residues on albumin were protected by reaction with 45.5 µg of iodoacetamide in 10 mM potassium phosphate buffer (pH 7.4). Mouse serum albumin (118 µg/1 mL) was then incubated in the presence of various amounts of DTT for 1 h at room temperature. The reaction mixture was then treated in the dark with 122 µL of 4-vinylpyridine/methanol mixture (1:60, v/v) to label the mercapto groups of cysteine residues in the protein molecule liberated from disulfide bonds by the action of the reducing agents. After sequential dialysis in methanol/water (1:9, v/v) and then 50 mM ammonium bicarbonate to remove small molecules, the protein was digested using trypsin (1.71 pmol) into peptide fragments and analyzed by MALDI-TOF MS. Purification of DCA-Binding Proteins in Mouse Ascites. The DCA-immobilized affinity gel containing the disulfide linker (1 mL) was first equilibrated in 50 mM potassium phosphate buffer (pH 7.4). After making a dilution of 100 µL mouse ascites, which contains anti-DCA antibody, in the mixture of 1 mL of 50 mM potassium phosphate buffer (pH 7.4) and 11 µL of Protease

Figure 3. Effect of molar ratios of DTT to the three-dimensional conformation of mouse serum albumin. (A) CD spectra. (B) Changes of the fluorescent intensity and the maximum emission wavelength. CD conditions: sample concentration, 10 µg/mL; solvent, 50 mM potassium phosphate buffer (pH 7.4); cell length, 10 mm; scan range, 200-250 nm; scan speed, 20 nm/min; resolution, 0.1 nm; bandwidth, 1.0 nm; accumulation, 1. Fluorescent measurement conditions: sample concentration, 333 µg/mL; solvent, 50 mM potassium phosphate buffer (pH 7.4); excitation, 280 nm; scan range, 300-400 nm. 4670

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Figure 5. Changes of peak intensity ratios of 4-vinylpyridinederivatized peptide fragments in the presence of variable amounts of DTT.

Figure 4. MALDI-TOF mass spectra of tryptic digests of mouse serum albumin treated without DTT (A), with a 10-fold molar excess of DTT (B), and with a 1800-fold molar excess of DTT (C). MS conditions: instrument, Voyager DE-STR (reflector mode); matrix, R-CHCA; accelerating voltage, 25 kV; grid voltage, 18 kV; guide wire voltage, 50 V. The py represent 4-vinylpyridine-derivatized peptide fragments.

Inhibitor Mix diluted by 10%, the solution was added to the gel and gently mixed at 4 °C overnight. After three washes in 5 mL of 50 mM potassium phosphate buffer (pH 7.4), the gel was incubated with 1.15 molar excess of DTT over the immobilized small molecule in the same buffer (1 mL) at room temperature for 1 h to reductively cleave the disulfide linker under a nitrogen atmosphere. After collection, the supernatant was dialyzed to remove salts and other contaminating small molecules and then lyophilized. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was performed using a Mini-PROTEAN 3 system (Bio-Rad) equipped with a Power Pac 200 power supply (Bio-Rad). Protein samples were separated by SDS-PAGE on 12% polyacrylamide gels, containing 375 mM Tris-HCl buffer (pH 8.8) and 2.5% sodium dodecyl sulfate (SDS), at a constant voltage of 100 V in an electrophoretic buffer of 0.3% Tris-1.44% glycine, which contained 0.1% SDS.

For nonreductive native-PAGE, protein samples were separated using 12% polyacrylamide gels, containing 375 mM Tris-HCl buffer (pH 8.8) at a constant voltage of 100 V. After electrophoresis in 0.3% Tris-1.44% glycine buffer, gels were stained with 0.1% CBB in water/methanol/acetic acid (73:20:7, v/v/v) for 20 min at room temperature. In-Gel Digestion and Database Search for Identification of Proteins. Excised pieces of CBB-stained, 1-mm thick, 12% polyacrylamide gels, which contained target proteins, were washed twice in 0.2 mL of 50 mM ammonium bicarbonate solution/ acetonitrile (1:1, v/v) for 20 min. These gel fragments were then washed three times in 0.2 mL of acetic acid/acetonitrile/water (1:5:4, v/v/v). After washing an additional three times in 0.2 mL of water, the gel was equilibrated with 0.2 mL of 50 mM ammonium bicarbonate (pH 8.0) for 10 min. Acetonitrile (0.2 mL) was added before drying the samples in a vacuum centrifuge. Two microliters of 50 mM ammonium bicarbonate (pH 8.0) containing 0.01 µg of sequence-grade modified trypsin was then added to the gel. The addition of 50 mM ammonium bicarbonate solution then covered the gel fragments. After a 16 h incubation at 37 °C, tryptic peptide fragments were extracted in 20 µL of water/ acetonitrile/formic acid (45:50:5, v/v/v). The solution was concentrated in vacuo and then redissolved in 10 µL of 0.1% TFA in an aqueous solution. Peptides were purified over a ZipTipC18 column prior to analysis by MALDI-TOF MS. For protein identification, ProFound (http://prowl.rockefeller.edu/ profound_bin/WebProFound.exe) was used as the database search engine. RESULTS AND DISCUSSION Preparation of Affinity Gel and Conditions for Cleavage of the Disulfide Linker. Antibodies are 150 kDa proteins consisting of two heavy chains and two light chains; the juxtaposition of the heavy and light chain variable regions form the highly diverse antigen-binding site. Antibodies used for hapten immunoassays typically have high affinities for antigens ranging between 108 and 1010 M-1.26,29,30 The anti-DCA monoclonal antibody was prepared using bovine serum albumin-DCA conjugates as the immunogen. In this complex, a carboxyl group of a DCA side Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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Table 1. Masses Observed in the MALDI-TOF Mass Spectra As Shown in Figure 4 sequences

computed m/z

untreated

10-fold DDT treated

1800-fold DTT treated

234-242 20-28 500-508 +4VP(500,501) 25-34 35-44 461-469 +4VP(461,462) 362-372 439-452 361-372 299-309 +VP(302,303) 422-434 299-310 +VP(302,303) 348-360 243-257 470-483 +4VP(472) 484-499 +4VP(485) 509-524 +4VP(511) 435-452 585-602 +4VP(591) 342-360 509-527 +4VP(511) 268-286 +4VP(269,270,277) 25-44 243-264 414-434 414-434 +4VP(416) 265-286 +4VP(269,270,277) 414-438

1019.5785 1099.6120 1162.5340 1177.6072 1250.5800 1274.5656 1299.7055 1439.7852 1455.8066 1471.7800 1479.7954 1599.8749 1609.7896 1681.8431 1710.8883 1925.0313 1930.9731 1960.0498 2029.9647 2286.1077 2335.1791 2406.0675 2409.1694 2424.2656 2446.2071 2551.2649 2747.2744 2966.4716

1019.5844 1099.6293

1019.5964 1099.6436

1019.5949

1177.6054 1250.5680

1177.6099 1250.5705

1163.5398 1274.5667 1299.7044 1439.7809 1355.7791

1439.7779 1455.7899

1479.7781

1479.7846

1609.7674 1681.8179

1609.7714 1681.8043

1960.1355

1960.0912

2409.1457 2424.2336 2446.1709

2409.1236 2424.2038 2446.1543

chain and the -amino groups of the lysine residues on bovine serum albumin were bound covalently.26 As this antibody can bind to the steroid nucleus of DCA, specifically rings A, B, and C, the disulfide linker should be conjugated to a carboxyl group of a DCA side chain. In this study, we prepared the affinity gel by two independent procedures to investigate the effect of excess linker on nonspecific interactions. The gel-A was prepared by the reaction of a DCA cystamine conjugate with the N-hydroxysuccinimidyl esters linked to the agarose gel and was immobilized 2.7 µmol of DCA cystamine onto 1 mL of gel. Gel-B preparations were created using the standard method for small molecule immobilization onto a gel, a procedure that introduced excess cystamine linkers to the activated agarose gel. This reaction was followed by reaction of DCA p-nitrophenyl ester with amino groups at the end of the cystamine linkers. Reductive cleavage of disulfide linkers within the gel required a complete reaction without conformation disintegration of the proteins bound to the immobilized small molecule. Disulfide bonds, which link R-helices and β-sheets for proper protein folding, generally exist within molecules. To cleave such disulfide bonds, reducing agents are usually used at 1000-10 000-fold higher concentrations (approximately 1 w/v%) than the target protein molecules in the presence of high concentrations of a denaturant, such as a guanidine.31 In contrast, the concentration of DTT, a reducing agent, used for papain purification following covalent chromatography was only 0.077 w/v% (approximately 5 mM).7 We investigated the effect of DTT final molar ratios (1-, 2.5-, 5-, and (29) Kobayashi, N.; Oiwa, H.; Goto, J. J. Steroid Biochem. Mol. Biol. 1998, 64, 171-177. (30) Okuyama, M.; Kobayashi, N.; Takeda, W.; Anjo, T.; Matsuki, Y.; Goto, J.; Kambegawa, A.; Hori, S. Anal. Chem. 2004, 76, 1948-1956. (31) Crestfield, A. M.; Moore, S.; Stein, W. H. J. Biol. Chem. 1963, 238, 622627.

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1455.8197 1471.7657 1479.7822 1599.8833 1609.7798 1681.8471 1710.8723 1925.0503 1930.9864 1960.0825 2029.9659 2286.0737 2335.1465 2406.0640 2424.2359 2446.1428 2551.3091 2747.3141 2966.5471

10-fold) to the immobilized small molecule, respectively, on the cleavage of the disulfide linkers in gel-A. When more than 5-fold molar excess of DTT was used, the disulfide linkers were completely cleaved at room temperature by 60 min after (data not shown). We extracted the proteins from mouse ascites (1.03 mg proteins), which contains the anti-DCA antibody, by use of both gel-A and gel-B, and analyzed by SDS-PAGE. Although mouse ascites include many proteins in addition to the anti-DCA antibody, we applied the present method to extract DCA-binding proteins after only a single dilution, and the results are illustrated in Figure 2. The nonadsorbate and wash fractions exhibited multiple protein bands observed over a broad range of molecular weights similar to crude mouse ascites. When the gel-A was used for the extraction, many of the protein bands disappeared from the extracted protein population. Three protein bands were clearly detected by CBB staining at approximately 68 kDa (protein 1), 50 kDa (protein 2), and 30 kDa (protein 3). The SDS-PAGE pattern of extracted proteins following purification using gel-B, prepared by a simpler procedure, identified the same protein bands as using gel-A. However, three protein bands recovered by using gel-B were light compared with that using gel-A. When compared nonadsorbate and wash fractions between gel-A and gel-B, the color density of 68 kDa protein differed markedly in appearance and was dark in the case of gel-B. Since this 68 kDa protein is one of the DCA-binding proteins in mouse ascites as described later, this result suggests that gel-A, which was prepared by condensation of a DCA cystamine conjugate with an activated agarose gel, immobilized more DCA, model small molecule ligand, than gel-B. Effect of the Cleavage Reaction on Internal Disulfide Bonds within Protein Molecules. Within cells, proteins exist

Figure 6. Amino acid sequences of heavy chain and light chain variable regions of anti-DCA monoclonal antibody. Table 2. Identification of Proteins Found in Elution Fraction from Gel-Aa (A) Masses Observed in the MALDI-TOF Mass Spectra As Shown in Figure 7 Figure 7B

Figure 7A

Figure 7C

sequence

computed m/z

measured m/z

sequence

computed m/z

measured m/z

sequence

computed m/z

measured m/z

234-242 439-452 361-372 422-434 348-360 243-257 153-168 435-452 342-360 25-44 243-264 414-434

1019.579 1439.785 1455.807 1479.795 1609.790 1681.843 1901.896 1960.049 2286.107 2409.169 2424.265 2446.207

1019.575 1439.765 1455.789 1479.779 1609.768 1681.813 1901.885 1960.035 2286.086 2409.164 2424.247 2446.175

VH 66-74 VH 75-85 VH 75-85ox VH 71-85ox VH 1-19 VH 44-63

1051.554 1307.715 1323.710 1751.911 1926.018 2237.233

1051.563 1307.679 1323.720 1751.879 1925.999 2237.226

VL 64-72 VL 24-56 VL 24-63

951.490 3565.731 4242.133

951.502 3565.715 4242.178

rank

probability

1 2 3

1.0E+00 2.4E-19 5.9E-20

(B) Database Search Results of Protein 1 protein candidates coverage (%) Protein 1 albumin 1 mKIAA0185 protein hypothetical protein XP_144896

21 6 17

pI

kDa

5.8 9.2 6.7

68.67 208.12 67.49

a Database search conditions: database search engine, Profound (http://prowl.rockefeller.edu/profound_bin/WebProFound.exe); database, NCBInr (2005/06/01); taxonomy category, Mus musculus (house mouse); protein mass range, 0-3000 kDa; pI range, 0-14; max missed cut, 3; tolerance, 0.10 Da.

in proximity to interacting proteins to facilitate rapid and efficient signal transduction. Upon addition of exogenous small molecules, such as drugs, to cells, novel protein complexes are often formed around the new three-dimensional structure generated by the binding of the exogenous small molecule to the target protein. Therefore, to understand the molecular mechanism mediating the pharmacological actions of drugs, it is important to analyze the protein complexes, which contain the target protein bound with the small molecule. For the extraction of the small moleculebinding proteins and/or the protein complex itself, cleavage of the disulfide linker must occur without any conformational changes, as alterations of the stereostructure of the target proteins would likely result in complex dissociation. The affinity gel utilized in this study completely released small molecules, which were immobilized onto the gel via disulfide linker, by treatment with more than 5-fold molar excess of DTT over the immobilized small molecule. We, therefore, investigated the effect of reducing agents on cleavage of the disulfide bonds within protein molecules using

mouse serum albumin as a model protein, which has multiple internal disulfide bonds. Examination of CD and fluorescent spectra of mouse serum albumin under various conditions (Figure 3) revealed that the R-helical structure32 was retained under all conditions, even when large excess amounts of DTT were used. However, the fluorescence intensity at each maximum emission wavelength was influenced at more than 20-fold molar excess of DTT, and blue shift of emission wavelength at 350 nm occurred (Figure 3B) when more than 100-fold molar excess of DTT over the protein molecule was used, indicating that the microenvironment around tryptophan residues changed through conformational changes following cleavage of disulfide bonds. Next, we analyzed the cleavage of disulfide bonds in mouse serum albumin by mass spectrometry following two independent protocols designed to protect mercapto groups. The free mercapto groups of mouse serum albumin were protected in advance by (32) Oakley, J. L.; Pascale, J. A.; Coleman, J. E. Biochemistry 1975, 14, 46844691.

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Figure 8. Nonreductive native-PAGE analysis of purified proteins bound to gel-A and the MALDI-TOF mass spectrum of an in-gel tryptic digest of protein 4. (A) a, mouse ascites; b, nonadsorbate to the gelA; c, gel-A wash; d, gel-A eluate. (B) MS conditions: instrument, Voyager DE-STR (reflector mode); matrix, R-CHCA; accelerating voltage, 25 kV; grid voltage, 18 kV; guide wire voltage, 50 V.

Figure 7. MALDI-TOF mass spectra of tryptic digests of protein 1 (A), protein 2 (B), and protein 3 (C) from gel-A eluates. MS conditions: instrument, Voyager DE-STR (reflector mode); matrix, R-CHCA; accelerating voltage, 25 kV; grid voltage, 18 kV; guide wire voltage, 50 V.

iodoacetamide. Pretreated serum albumin was then incubated with varying amounts of DTT. Newly appearing free mercapto groups generated by the cleavage of disulfide bonds were then coupled to 4-vinylpyridine.33 The results are shown in Figures 4 and 5 and Table 1. No peaks derivatized by 4-vinylpyridine could be observed on the mass spectrum of mouse serum albumin treated with DTT at a 10-fold molar excess (Figure 4B), an amount significant to perform the complete cleavage of disulfide linkers as described before. In the mass spectrum of mouse serum albumin treated with more than 20-fold molar excess of DTT, multiple peptide fragment peaks derivatized by 4-vinylpyridine could be observed (Figure 4C), and the peak intensity ratio relative to that of angiotensin I, an internal standard, increased with increasing amounts of DTT (Figure 5). These results suggest that the (33) Sechi, S.; Chait, B. T. Anal. Chem. 1998, 70, 5150-5158.

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cleavage of disulfide linkers within the affinity gel enabled the complete extraction of small molecule-binding proteins without any protein denaturation by use of 5-fold molar excess of DTT over the immobilized small molecule. Specific Extraction of DCA Binding Proteins in Mouse Ascites. We cut separately the gel of three protein bands, extracted by the use of either gel-A or gel-B, into 1 mm on a side. In-gel tryptic digestion generated peptide fragments that were subsequently analyzed by MALDI-TOF MS to identify their parental proteins by peptide mass fingerprinting33-37 or comparison with theoretical sequences identified by DNA sequencing of the single chain variable fragment (Fv) derived from the antiDCA monoclonal antibody (Figure 6).38 The results are shown in Figure 7 and Table 2. Protein 1, detected at 68 kDa, was identified as mouse serum albumin by database searching (Figure 7A). Protein 2, which was observed at 50 kDa, was identified as an anti-DCA antibody heavy chain, identified from a peptide sequence identical to the heavy chain variable region (Figure 7B). Mass mapping of protein 3 (30 kDa) was identical to the theoretical structure of the variable region of the anti-DCA antibody light chain (Figure 7C). As the affinity constants of bile acids to serum (34) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (35) James, P.; Quadroni, M.; Carafoli, E.; Gonnet, G. Biochem. Biophys. Res. Commun. 1993, 195, 58-64. (36) Mann, M.; Højrup, P.; Roepstorff, P. Biol. Mass Spectrom. 1993, 22, 338345. (37) Pappin, D. J. C.; Højrup P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-332. (38) Kobayashi, N.; Ohtoyo, M.; Wada, E.; Kato, Y.; Mano, N.; Goto, J. Steroids 2005, 70, 285-294.

albumin are approximately 104-105 M-1,15 which are almost the same as that of many drugs to serum proteins, this gel system would likely capture the various affinities of multiple drugs-binding proteins. The target model protein, anti-DCA antibody, contains two heavy chains and two light chains, linked by disulfide bonds. However, in SDS-PAGE analyses, antibody chains were detected as two split bands. We must, therefore, verify whether the cleavage of disulfide bonds within the antibody occurred during SDSPAGE analysis not the cleavage reaction of disulfide linker with DTT. We analyzed the proteins extracted from gel-A by nativePAGE under the nonreducing conditions to examine if the disulfide bonds in the antibody remained stable during the extraction operation. The protein bands at both 30 kDa and 50 kDa that corresponded to the light chain and heavy chain, respectively, disappeared, and a higher molecular weight band was detected (Figure 8A). The MALDI-TOF mass spectrum clearly indicated the presence of peptide fragments derived from both the antibody heavy and light chains (Figure 8B), suggesting that disulfide cleavage of antibody occurred during SDS-PAGE. CONCLUSIONS We developed a method for the specific extraction of small molecule-binding proteins, such as drug targets, by an affinity gel possessing a cleavable disulfide linker. This affinity gel system

can specifically capture a target protein of the ligand molecule immobilized at the edge of the linker. In addition, this method allows the release of isolated proteins under mild cleavage conditions without substantial conformation changes. We demonstrated the specific extraction of an anti-DCA antibody and serum albumin, both DCA-binding proteins with different affinities, from mouse ascites using a DCA-immobilized cleavable affinity gel. This system would facilitate the identification of drug targets as well as the analysis of signaling network pathways related to the pharmacological actions of drugs. Abbreviations. CBB, coomassie brilliant blue; CD, circular dichroism; R-CHCA, R-cyano-4-hydroxycinnamic acid; DCA, deoxycholic acid; DTT, dithiothreitol; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TOF MS, time-of-flight mass spectrometry. ACKNOWLEDGMENT This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan.

Received for review February 1, 2006. Accepted April 20, 2006. AC060213S

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