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Technical Notes
Automated Ligand Fishing Using Human Serum Albumin-Coated Magnetic Beads R. Moaddel,*,† M. P. Marszałł,† F. Bighi,† Q. Yang,‡ X. Duan,‡ and I. W. Wainer†
Gerontology Research Center, National Institutes in Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, and PSS Bio Instruments Inc., Gaithersburg, Maryland 20877
Human serum albumin, HSA, was immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known HSA ligands from a mixture containing ligands and nonligands. The separation was accomplished manually and was also automated. The results indicate that an automated “ligand-fishing” technique can be developed using magnetic beads containing an immobilized protein. Drug discovery and drug development have increasingly been focusing on the identification of unknown interaction partners/ complexes from cellular and/or botanical extracts to known targets. The elucidation of these biomolecular interactions can result in new targets for therapeutic treatment or directed control of cell functioning.1 Currently, there are a number of established methods available to characterize biomolecular interactions in realtime, namely, surface plasmon resonance (SPR)1 and bioaffinity chromatography.2 However, in both of these cases it has been predominantly limited to the characterization of an immobilized protein with a set of compounds or compound libraries.3 The screening of cellular or botanical extracts as potential sources for ligands (or complexes) of known or orphan receptors is known as ligand fishing. The success of a ligand fishing experiment is dependent on the ability to detect the bound ligand (or complex).4 Ligand fishing experiments have been carried out by multiple methods including SPR,3,4 circular dichroism, photochemical fishing,5 and using 2D and 3D molecular descriptors.6 Of these, SPR has been the most successful and most extensively studied. * Corresponding author. Address: Ruin Moaddel, National Institute on Aging, National Institutes of Health, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825. Phone: (410)-558-8294. Fax: (410)-558-8409. E-mail:
[email protected]. † National Institutes of Health. ‡ PSS Bio Instruments Inc. (1) Zhukov, A.; Schurenberg, M.; Jansson, O.; Areskoug, D.; Buijs, J. J. Biomol. Tech. 2004, 15, 112-119. (2) Moaddel, R.; Wainer, I. W. Anal. Chim. Acta 2006, 564, 97-105. (3) Baynham, M. T.; Patel, S.; Moaddel, R.; Wainer, I. W. J. Chromatogr., B 2002, 772, 155-161. (4) Catimel, B.; Weinstock, J.; Nerrie, M.; Domagala, T.; Nice, E. C. J. Chromatogr., A 2000, 869, 261-273. (5) Sadakane, Y.; Hatanaka, Y. Anal. Sci. 2006, 22, 209-218. (6) Nettles, J. H.; Jenkins, J. L.; Bender, A.; Deng, Z.; Davies, J. W.; Glick, M. J. Med. Chem. 2006, 49, 6802-6810.
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SPR has been used to isolate ligands isolated from several different sources. Lackmann et al. identified a single ligand from 150 different sources for human EPH-like kinase,7 while Sakano et al. identified a ligand for the hepatoma transmembrane kinase from cell supernatants.8 More recently, Zhukov et al.1 successfully immobilized the calmodulin binding domain (CBD) on a CM5 chip. They demonstrated that they could capture calmodulin from a crude biological mixture (bovine brain extract) and subsequently identify calmodulin by MALDI-MS. Although, SPR is a promising method for ligand fishing, to date it has been predominantly applied to cytosolic proteins or only the extracellular portion of a transmembrane protein. As transmembrane proteins are the major targets in drug discovery and drug development, there is a substantial rate-limiting step for SPR. Bioaffinity chromatography, however, has been used for the characterization of cytosolic proteins and transmembrane proteins including ligand gated ion channels,9 G-protein coupled receptors,10 ATP binding cassette transporters,11 and solute liquid carrier transporters.12 Further, Baynham et al. demonstrated that bioaffinity chromatography could be used to correctly rank a mixture of compounds into three different classes (high binders, moderate binders, and nonbinders) using an R3β4 nicotinic receptor column.3 Although this method could be successfully applied to ligand fishing from cell supernatants or botanical extracts, running crude cellular extracts through the column may prove detrimental to the column. In order to circumvent the problem of running crude extracts through a column, the objective of this research was to adapt the method to magnetic beads. To date, no one has reported the use of magnetic beads for ligand fishing, where a protein is immobilized for the isolation of ligands/complexes from a mixture (7) Lackmann, M.; Bucci, T.; Mann, R. J.; Kravets, L. A.; Viney, E.; Smith, F.; Moritz, R. L.; Carter, W.; Simpson, R. J.; Nicola, N. A.; Mackwell, K.; Nice, E. C.; Wilks, A. F.; Boyd, A. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2523-2527. (8) Sakano, S.; Serizawa, R.; Inada, T.; Iwama, A.; Itoh, A.; Kato, C.; Shimizu, Y.; Shinkai, F.; Shimizu, R.; Kondo, S.; Ohno, M.; Suda, T. Oncogene 1996, 13, 813-822. (9) Moaddel, R.; Wainer, I. W. J. Pharm. Biomed. Anal. 2003, 30, 1715-1724. (10) Beigi, F.; Wainer, I. W. Anal. Chem. 2003, 75, 4480-4485. (11) Moaddel, R.; Bullock, P. L.; Wainer, I. W. J. Chromatogr., B 2004, 799, 255-263. (12) Moaddel, R.; Yamaguchi, R.; Ho, P. C.; Patel, S.; Hsu, C. P.; Subrahmanyam, V.; Wainer, I. W. J. Chromatogr., B 2005, 818, 263-268. 10.1021/ac070268+ CCC: $37.00
© 2007 American Chemical Society Published on Web 06/19/2007
Figure 1. (A and B) The Magtration System 12GC from PSS Bio Instruments Inc. (C) Twelve samples are processed in parallel on the 12GC system. Since four fractions need to be collected for the downstream analysis, four capped 1.5 mL microtubes are used for each sample, which can be easily taken out and labeled after the run. It takes around 1 h for one run.
of compounds or cellular extracts. Herein we report such a method, which can be easily adapted to other cytosolic and membrane proteins. In this report, human serum albumin (HSA) was immobilized onto the surface of magnetic beads, which was subsequently used to “fish out” known binders from nonbinders. This was carried out manually using the Dynal magnetic bead separator and was also automated using the Magtration System 12GC from PSS Bio Instruments Inc. (Figure 1). EXPERIMENTAL SECTION Chemicals. Azidothymidine (AZT), fenoterol hydrobromide, labetolol hydrochloride, naproxen, nicotine, and warfarin were obtained from the Sigma Chemical Co. (St. Louis, MO). Two milliliter eppendorf tubes were purchased from Fisher Scientific (Pittsburgh, PA). One micrometer Bc-Mag, amine terminated magnetic beads (50 mg/mL) were purchased from Bioclone Inc (San Diego, CA). Immobilization of Human Serum Albumin (HSA) on BcMag. HSA was immobilized onto the Bc-Mag beads following the protocol provided by Bioclone Inc with slight modifications. In brief, 5 mL (250 mg) of Bc-Mag beads was placed in a conical tube, to which 15 mL of coupling buffer (pyridine (10 mM, pH 6.0)) was added. The tube was vortexed and then separated using a magnetic separator (Dynal MPC-S, Invitrogen Corp., Carlsbad, CA). The supernatant was removed. To the beads 10 mL of 5% gluteraldehyde solution in coupling buffer was added and vortexed and then gently rotated for 3 h. The tube was then placed in a magnetic separator until the solution was clear, and the supernatant was discarded. The beads were then washed three times with coupling buffer. A solution of HSA (50 mg of HSA in 8 mL of coupling buffer) was added to the activated beads and vigorously shaken. The tube was then shaken for 24 h at room temperature with a gentle
rotation. The tube was then placed in the magnetic separator, and the supernatant was removed. Subsequently, 20 mL of the reaction stop buffer (1 M glutaric acid (aq), pH 8.0) was added to the tube and gently rotated for 30 min. The tube was again placed in the magnetic separator, and the supernatant was removed. The beads were then washed three times with wash buffer (10 mM Tris (pH 7.4) containing 0.15 M NaCl, 0.1% BSA, 1 mM EDTA, 0.1% sodium azide) and then stored with ammonium acetate buffer (10 mM, pH 7.4). Manual Ligand Fishing. An amount of 1 mL of the HSAMgB (50 mg) was placed into a 2 mL eppendorf tube. To the beads, a 6 µL solution containing 1 µM azidothymidine, 1 µM fenoterol hydrobromide, 1 µM labetalol hydrochloride, 1 µM naproxen, 1 µM nicotine, and 1 µM warfarin was added. The tube was mixed by vigorously shaking for 15 min and placed into the magnetic separator for 5 min. The supernatant (∼1 mL) was carefully removed and saved (A1). The HSA-MgB was washed twice with 1 mL of ammonium acetate buffer (10 mM, pH 7.4) by vigorously shaking for 2 min and removing the supernatant after they were placed on the magnetic separator for 5 min (A2 and A3). The third wash was performed in 1 mL of ammonium acetate buffer (10 mM, pH 7.4) containing 10% ACN for 2 min. The supernatant was removed and saved (A4). Automated Ligand Fishing Using the Magtration System 12GC. A tip is picked up by the robot from rack 4 (R4). Beads in 1 mL of ammonium acetate buffer (10 mM, pH 7.4) in well 1 are first mixed 50 times and then separated in the tip. The tip with the beads is brought to well 11 with a tube inserted. The beads are resuspended in the 994 µL solution of ammonium acetate buffer (10 mM, pH 7.4) with a 6 µL solution containing 1 µM azidothymidine, 1 µM fenoterol hydrobromide, 1 µM labetalol hydrochloride, 1 µM naproxen, 1 µM nicotine, and 1 µM warfarin and incubated for 15 min. The magnet is attached to the tip and Analytical Chemistry, Vol. 79, No. 14, July 15, 2007
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Figure 2. A direct comparison of the manual ligand fishing results and the automated ligand fishing results for A1 and A4 versus B1 and B4.
the beads are separated within 5 min. The supernatant is removed and saved (B1). The tip with the beads is brought to the tube in R3. The beads are washed for 2 min with 1 mL of ammonium acetate buffer (10 mM, pH 7.4). Then the beads are separated within 5 min. The supernatant is removed and saved (B2). The tip with the beads is brought to the tube in R2. The beads are again washed for 2 min with 1 mL of ammonium acetate buffer (10 mM, pH 7.4). Then the beads are separated within 5 min, and the supernatant is removed and saved (B3). The tip with the beads is brought to the tube in R1. The beads are washed a third time for 2 min with 1 mL of ammonium acetate buffer (10 mM, pH 7.4) containing 10% acetonitrile. Then the beads are separated within 5 min, and the supernatant was removed and saved (B4). The tip with the beads is brought to well 2. The beads are resuspended with 1 mL of ammonium acetate buffer (10 mM, pH 7.4) and left in solution until the next experiment. The fractions are collected from the tube in well 11, R3, R2, and R1 and are labeled as B1, B2, B3, and B4. RESULTS AND DISCUSSION The immobilization of HSA was carried out on the surface of Bioclone Inc magnetic beads using the provided protocol. The magnetic beads were initially endcapped with glycine, as suggested by Bioclone. Two batches of magnetic beads were synthesized: an HSA labeled magnetic bead as well as a glycine coated magnetic bead to be used as a control. Complete binding of all the tested ligands was seen both on the HSA labeled magnetic bead and on the glycine coated magnetic bead. The compounds could not be washed off, even in the presence of 10% acetonitrile for 30 min. In order to address the issue of nonspecific interactions, the magnetic beads were coated with glutaric acid. On the glutaric acid coated beads, 14% of the compounds were retained on average, with labetolol having the least amount of interactions with only 8.8% nonspecifically bound and naproxen having the most at 21% nonspecifically bound. For this reason, 5416
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the HSA was endcapped with glutaric acid, removing almost all the nonspecific interactions seen with the glycine encapped beads. The amount of HSA immobilized on the magnetic beads was measured using a Micro BCA protein assay kit from Pierce. It was determined that, on average, 875 ( 233 µg of HSA was immobilized per mL of magnetic beads (equivalent to 17.5 µg of HSA per mg of magnetic support). After successful immobilization of HSA onto the magnetic beads, the beads were incubated in a sample mixture containing six compounds, three known binders for HSA (warfarin, AZT, and naproxen) and three known nonbinders (nicotine, fenoterol, and labetolol) (Figure 2). The magnetic beads were then isolated using a Dynal magnetic separator. The supernatant was collected and termed A1. Each of the subsequent washes was labeled A2 and A3, and the final wash (10% acetonitrile) was termed A4. It was clearly demonstrated that the majority of the known ligands for HSA were retained until the final wash A4 (warfarin, AZT, and naproxen; 51%, 60%, and 74%, respectively), while for the nonbinders they were predominantly not retained in A1 (nicotine, fenoterol, labetolol; 47%, 71%, and 52%, respectively). The method was then automated using the Magtration System 12GC PSS Bio Instruments Inc. (Figure 2), which can run up to 12 samples in parallel. In our studies, since four fractions need to be collected for the downstream analysis, four capped 1.5 mL microtubes were used for each sample, which were subsequently analyzed by MS. The known binder ligands were successfully separated from the known nonbinders on the Magtration System (Figure 2). Similar results were obtained between the manual and automated protocol, with the exception of AZT, where a slightly larger amount of AZT remained in B1 relative to A1, resulting in a smaller amount retained in B4 versus A4. This is not unexpected, as the automated system mixes more frequently than the manual protocol resulting in a higher likeliness that less AZT would be retained in the initial incubation.
In this study, HSA was immobilized onto the surface of magnetic beads and used for ligand fishing and was fully automated. To our knowledge, there has only been one other report on the use of magnetic beads for fishing. Meyer et al. immobilized a known ligand of the superoxide dismutase (SOD) enzyme onto a magnetic bead and used the functionalized magnetic bead to recover preparative amounts of SOD from crude sweet whey;13 they termed this method high-gradient magnetic fishing. Other uses for the magnetic beads are predominantly for protein purifications. For example, protein A or protein G is immobilized covalently onto the surface of magnetic beads, which is subsequently used to isolate expressed proteins from cellular extracts.14-16 In our case, however, we have immobilized the (13) Meyer, A.; Hansen, D. B.; Gomes, C. S.; Hobley, T. J.; Thomas, O. R.; Franzreb, M. Biotechnol. Prog. 2005, 21, 244-254. (14) Heddini, A.; Treutiger, C. J.; Wahlgren, M. Am. J. Trop. Med. Hyg. 1998, 59, 663-666. (15) Widjojoatmodjo, M. N.; Fluit, A. C.; Torensma, R.; Verhoef, J. Immunol. Methods 1993, 165, 11-19. (16) Ljungquist, C.; Lunderberg, J.; Rasmussen, A. M.; Hornes, E.; Uhlen, M. DNA Cell Biol. 1993, 12, 191-197.
protein (HSA) and are attempting to identify ligands from a mixture of compounds. The results of our study demonstrate that a protein immobilized onto a magnetic bead can be used for ligand fishing. In our study, the HSA functionalized magnetic beads correctly isolated the three known binders from a mixture of six compounds. In addition, it is also the first study to demonstrate the versatility of the Magtration System 12 GC, which fully automated the ligand-fishing expedition. This study can now be extended to the identification of protein-ligand and protein-protein interactions by using a protein functionalized magnetic bead and fishing in cellular and/or botanical extracts. ACKNOWLEDGMENT This research was supported (in part) by the Intramural Research Program of the NIH, National Institute on Aging. Received for review February 8, 2007. Accepted May 8, 2007. AC070268+
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