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Langmuir 2008, 24, 11784-11789
Surface Plasmon Resonance Spectroscopy-Based High-Throughput Screening of Ligands for Use in Affinity and Displacement Chromatography Srinavya Vutukuru and Ravi S. Kane* Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 ReceiVed July 18, 2008. ReVised Manuscript ReceiVed August 11, 2008 We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinsslysozyme and cytochrome csonto these “multimodal” surfaces and to select promising “affinity” ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design “affinity displacers”ssmall molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures.
Introduction The objective of this work was to develop an approach to identify novel ligands for use in the chromatographic separation of proteins. Specifically, we used a combination of surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) to identify ligands for use in affinity and displacement chromatographic processes. SAMs provide simple and flexible model surfaces for correlating the molecular scale structure of surfaces to the extent of protein adsorption.1-7 Furthermore, SAMs of alkanethiolates on gold are thin organic layers that enable protein-surface interactions to be monitored by SPR spectroscopysa powerful and sensitive technique that enables the label-free detection of binding events at surfaces.8-12 Therefore, the combination of SAMs and SPR provides a convenient system for the high-throughput screening of ligands for use in chromatographic processes. Whereas ion-exchange chromatography is a chromatographic process that is widely used for the concentration and purification of proteins,13,14 the use of classical ion-exchange resins is not * Corresponding author. E-mail:
[email protected]. (1) Bain, C. D.; Whitesides, G. M. Angew. Chem., Int. Ed. 1989, 28, 506–512. (2) Barrett, D. A.; Power, G. M.; Hussain, M. A.; Pitfield, I. D.; Shaw, P. N.; Davies, M. C. J. Sep. Sci. 2005, 28, 483–491. (3) Lahiri, J.; Isaacs, L.; Grzybowski, B.; Carbeck, J. D.; Whitesides, G. M. Langmuir 1999, 15, 7186–7198. (4) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87–96. (5) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1103–1169. (6) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55–78. (7) Vutukuru, S.; Bethi, S. R.; Kane, R. S. Langmuir 2006, 22, 10152–10156. (8) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383– 4385. (9) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642–3648. (10) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939–4947. (11) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. Langmuir 1997, 13, 2749– 2755. (12) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388–2391.
always optimal.15,16 There has therefore been an increasing interest in designing “multimodal” stationary phases, involving ligands that interact with the target molecules through multiple modes,15,16 e.g., electrostatic interactions that can dominate at low salt concentrations and hydrophobic interactions that can contribute to protein-ligand interactions even at high salt concentrations.7 Multimodal media are characterized by selectivities different from conventional ion-exchange media, thereby opening up new opportunities for separating proteins which have similar retention times on classical ion exchangers. We describe the use of SAMs to design and screen multimodal surfacesssurfaces that present ligands of interest in a background of charged carboxylic acid groups. Furthmore, we demonstrate that results from such screens can provide ligands for use in two important chromatographic processes: affinity chromatography, in which the stationary phase is functionalized with a ligand that binds reversibly to the protein of interest with high selectivity,17-19 and displacement chromatography, in which a ligand (displacer) added to the mobile phase can change the migration time of a protein of interest. Whereas screening techniques based on combinatorial chemistry and quantitative structure-property relationship (QSPR) modeling have also been developed,20-26 the identification of functional ligands remains a challenge. The combination of SPR spectroscopy and SAMs represents a powerful approach for the rapid identification of ligands for the purification of proteins of interest.
Experimental Section Materials. All materials and reagents were used as-received. Lysozyme (chicken egg white; L6876) and cytochrome c (equine (13) Yamamoto, S.; Nakinishi, K.; Matsuno, R. Ion-exchange chromatography of proteins , 2nd ed.; Marcel Dekker: New York, 1998; p 43. (14) Mazza, C.; Kundu, A.; Cramer, S. M. Biotechnol. Tech. 1998, 12, 137– 141. (15) Johansson, B. L.; Belew, M.; Eriksson, S.; Glad, G.; Lind, O.; Maloisel, J. L.; Norrman, N. J. Chromatogr. A 2003, 1016, 21–33. (16) Johansson, B. L.; Belew, M.; Eriksson, S.; Glad, G.; Lind, O.; Maloisel, J. L.; Norrman, N. J. Chromatogr. A 2003, 1016, 35–49. (17) Narayanan, S. R. J. Chromatogr. A 1994, 658, 237–258. (18) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598– 599. (19) Cuatrecasas, P. J. Biol. Chem. 1970, 245, 3059.
10.1021/la8023088 CCC: $40.75 2008 American Chemical Society Published on Web 09/13/2008
SPR Spectroscopy-Based Screening of Ligands heart; C2506) were purchased from Sigma (St. Louis, MO). 11Mercaptoundecanoic acid, 3,4,5-trimethoxybenzylamine, 2-aminomethyl-18-crown-6, tyramine, 3-methoxybenzylamine, trifluoroacetic anhydride, triethylamine, 1-methyl-2-pyrrolidinone (NMP), dichloromethane, dimethylformamide, and dimethyl sulfoxide were purchased from Aldrich (Allentown, PA). 1H,1H-Pentadecafluorooctylamine, 4-(2-aminoethyl)benzenesulfonyl fluoride, 2-(4-chlorophenyl)ethylamine, and 2,2,3,3,3-pentafluoropropylamine were purchased from Matrix Scientific (Columbia, SC). D-Glucamine, 8-amino-1-octanol, and amylamine were purchased from TCI America (Portland, OR). Glass coverslips (0.16-0.19 mm thick, No. 1.5) were obtained from Fisher Scientific (Agawam, MA). UltraLink Biosupport and GelCode Blue stain reagent were purchased from Pierce Biotechnology, Inc. (Rockford, IL). The cation-exchange media SP Sepharose High Performance was purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). 4-12% TrisGlycine Gels were purchased from Invitrogen (Carlsbad, CA). Preparation of Multimodal SAMs. Prior to gold evaporation, glass coverslips were cleaned by rinsing with benzene and methanol, followed by sonication in acetone. Gold substrates for contact angle measurements were prepared by electron-beam evaporation of 5 nm of titanium followed by 100 nm of gold onto the coverslips. Multimodal SAMs were prepared by the reaction of organic amines with SAMs presenting interchain carboxylic anhydrides as described by Yan et al.27 Briefly, the gold-coated coverslips were first incubated in a 2 mM solution of 11-mercaptoundecanoic acid for 30 min, rinsed with ethanol, and dried under a stream of nitrogen. These substrates were then placed in a freshly prepared solution of 0.1 M trifluoroacetic anhydride and 0.2 M triethylamine in anhydrous dimethylformamide at room temperature for 20 min without stirring. The substrates were rinsed thoroughly with dichloromethane and dried under a stream of nitrogen. The resulting SAMs presenting interchain carboxylic anhydride groups were immersed immediately into a 10 mM solution of the appropriate amine in NMP. D-Glucamine (1, Table 1) was not soluble in NMP; the reaction with this amine was carried out in deionized water. The multimodal SAMs were removed from the amine solution, rinsed with ethanol, and dried under a stream of nitrogen. Commercially available untreated gold sensor chips from Biacore (SIA kit Au) were used for SPR experiments. Contact Angle Measurements. The contact angle of water on SAMs presenting multimodal ligands was measured using a goniometer. Ten microliters of water droplets was dispensed from a Matrix Technologies Microelectrapipette, and the reported contact angle values are the average of three measurements taken at three different locations on two independent SAMs. Surface Plasmon Resonance Spectroscopy. SPR experiments were performed on a Biacore 3000 instrument. For experiments under “low-salt” conditions, 20 mM phosphate buffer at pH 6.8 was used and for experiments under “high-salt” conditions, 20 mM phosphate buffer at pH 6.8 with 300 mM NaCl added was used. All the buffers and samples were filtered and degassed prior to use. The SPR protocol for measuring adsorption of protein consists of the following: (i) flowing a solution of sodium dodecyl sulfate (SDS, 0.5% w/v) for 3 min followed by buffer for 20 min, (ii) flowing a solution of protein (2 mg/mL) in buffer for 10 min and allowing buffer to flow over the surface for 5 min, and (iii) washing the surface with SDS for 5 min. We used a flow-rate of 10 µL/min for the SPR experiments.
Langmuir, Vol. 24, No. 20, 2008 11785 Batch Experiments. Affinity Experiments. The affinity ligands were attached to UltraLink Biosupport resin by the coupling technique recommended by the manufacturers. A 500 µL solution of 10 mM ligand in coupling buffer (0.8 M sodium citrate, 0.1 M sodium bicarbonate, pH 8.6) was added to 25 mg of support. The sample was briefly vortexed at medium speed to suspend the beads and allowed to equilibrate at room temperature for 1 h in a shaker at 150 rpm. The samples were centrifuged at 1200g for 10 min to pellet the beads. The supernatant was removed carefully to retain the beads in the centrifuge tube. The unreacted sites on the support were blocked by adding 2 M quench solution (3.0 M ethanolamine, 0.1 M TrisHCl buffer, pH 9.0) to 200 µL of swelled beads. The samples were briefly vortexed and allowed to equilibrate for 2.5 h at room temperature in a shaker at 150 rpm. The samples were centrifuged at 1200g for 10 min to pellet the beads. The supernatant was removed and the beads were resuspended in 2 mL of phosphate buffered saline (PBS). The samples were vortexed at medium speed to resuspend beads in PBS and the sample was allowed to equilibrate in a shaker at 150 rpm for 15 min. The samples were centrifuged at 1200g for 10 min and the supernatant was removed. The wash step with PBS was repeated once more. 1H,1H-Pentadecafluorooctylamine (5, Table 1) was not soluble in coupling buffer; the immobilization was carried out in dimethyl sulfoxide. The ligandfunctionalized beads were resuspended in sample buffer (50 mM phosphate, pH 8.0) and stored at 4 °C. Ten microliters of the suspension were added to separate Eppendorf tubes. Fifty microliters of 5 mg/mL lysozyme or cytochrome c were then added to the Eppendorf tubes and allowed to equilibrate for 5 h in a shaker at 150 rpm. The percentage of protein adsorbed onto each immobilized support was determined by analyzing the supernatant using SDSPAGE as described below. Displacement Experiments. Batch displacement experiments were carried out separately for each protein. The bulk stationary phase (SP Sepharose High Performance; 100 µL) was washed once with deionized water and twice with buffer (50 mM phosphate, pH 8.0), and the media were equilibrated with buffer for 2 h in a shaker at 150 rpm. After removal of the supernatant, 600 µL of 5 mg/mL lysozyme or cytochrome c were added to 50 µL of washed stationary phase and allowed to equilibrate for 5 h in a shaker at 150 rpm. The supernatant was removed and analyzed by SDS-PAGE to determine the amount of protein adsorbed on the stationary phase. Ten microliter aliquots of stationary phase with adsorbed protein were added to separate Eppendorf tubes. One hundred and twenty microliters of amines (10 mM solutions in phosphate buffer, pH 8) were then added to each tube and allowed to equilibrate for 5 h in a shaker at 150 rpm. The supernatant was removed and analyzed by SDSPAGE to determine the percentage of protein displaced by each amine. SDS-PAGE Analysis. Four to 12% Tris-Glycine Gels were used to estimate the amount of protein in the supernatant. Before electrophoresis, the samples were boiled for 30 min. The electrophoresis was carried out at a constant voltage of 150 V in a Vertical Electrophoresis System, 10 × 10 cm (Fisher Scientific). Gels were stained by the GelCode Blue Coomassie stain reagent following the manufacturers’ standard protocol. Briefly, the gels were washed three times with deionized water for 15 min followed by staining for 1 h. NIH ImageJ software (http://rsb.info.nih.gov/ij) was used for analyzing the stained gels.
Results and Discussion (20) Mazza, C. B.; Rege, K.; Breneman, C. M.; Sukumar, N.; Dordick, J. S.; Cramer, S. M. Biotechnol. Bioeng. 2002, 80, 60–72. (21) Rege, K.; Hu, S. H.; Moore, J. A.; Dordick, J. S.; Cramer, S. M. J. Am. Chem. Soc. 2004, 126, 12306–12315. (22) Rege, K.; Ladiwala, A.; Cramer, S. M. Anal. Chem. 2005, 77, 6818–6827. (23) Rege, K.; Ladiwala, A.; Tugcu, N.; Breneman, C. M.; Cramer, S. M. J. Chromatogr. A 2004, 1033, 19–28. (24) Romig, T. S.; Bell, C.; Drolet, D. W. J. Chromatogr. B 1999, 731, 275– 284. (25) Scheich, C.; Sievert, V.; Bussow, K. BMC Biotechnol. 2003, 3. (26) Tugcu, N.; Ladiwala, A.; Breneman, C. M.; Cramer, S. M. Anal. Chem. 2003, 75, 5806–5816. (27) Yan, L.; Marzolin, C.; Terfort, A.; Whitesides, G. M. Langmuir 1997, 13, 6704–6712.
Strategy for Ligand Display. One approach for designing a surface presenting a ligand of interest involves synthesizing ω-functionalized alkanethiols (presenting the ligand of interest) and using it to form a SAM. The synthesis of alkanethiols, however, is a laborious and time-intensive process, thereby greatly limiting the throughput of a screen. To enable the rapid generation and screening of ligand-functionalized surfaces, we therefore used a method developed by Yan et al.,27 based on the reaction of an active intermediate - an interchain carboxylic anhydride - with an amine-functionalized ligand. This reaction enables the
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Vutukuru and Kane Table 1. Characterization of Multimodal Surfaces
a Log P values represent the values calculated for highlighted head groups. The values of log P were calculated with the Chemdraw Ultra software package using Crippen’s fragmentation method. b Contact angle of water under air. c Reported value is the mean of three readings taken on two different SAMs; uncertainty is the standard deviation of the readings.
rapid generation of “multimodal” SAMs presenting a 1:1 mixture of the ligand of interest and carboxylic acid groups (Scheme 1). Because a large number of amine-functionalized ligands are commercially available, the method represents an attractive alternative to the synthesis of individual alkanethiolates.27,28 Synthesis and Characterization of Multimodal Surfaces. Formation of SAMs. SAMs presenting carboxylic acid functional groups were formed by immersing gold-coated glass coverslips in 2 mM aqueous solutions of the 11-mercaptoundecanoic acid for 30 min. These surfaces were treated with trifluoroacetic anhydride to give SAMs terminating with interchain carboxylic anhydride groups (Scheme 1). These SAMs were then allowed to react with amines (RNH2) to give mixed SAMs that presented
approximately 1:1 mixture of CONHR and -CO2H/CO2-groups. Table 1 shows the chemical structures of the amine-functionalized ligands used in this study. Characterization of SAMs. We characterized the multimodal SAMs using contact angle measurements and by octan-1-ol partition coefficients (Table 1).1,7 The contact angle of water in air was determined by goniometry and we used commercially available software (ChemDrawUltra) to determine the octan1-ol/water partition coefficients (P) of the head groups of the (28) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927–6936.
SPR Spectroscopy-Based Screening of Ligands
Langmuir, Vol. 24, No. 20, 2008 11787
Scheme 1. Schematic Representation of the Procedure Used To Form Multimodal SAMs
ligand-functionalized alkanethiolate (Table 1).29,30 The values of contact angle and partition coefficient are useful indicators of the hydrophobicity of a group, with greater values corresponding to more hydrophobic surfaces. Protein Adsorption on SAMs Presenting Multimodal Ligands. Our goal was to demonstrate a strategy to identify ligands that discriminate between proteins of interest. We therefore used SPR spectroscopy to determine the extent of adsorption of two model proteinsslysozyme and cytochrome cson SAMs presenting ligands of interest. We used lysozyme and cytochrome c in this work because they are well-characterized proteins (lysozyme: MW ) 14 kDa, pI ) 11.1; cytochrome c: MW ) 12.5 kDa, pI ) 10.1)7 and coelute on cation-exchange media under linear gradient conditions at pH 6.8. We first compared the extent of adsorption of the two proteins on SAMs formed using ligands 1-5. As seen from Table 1, these ligands vary significantly in the values of the octan-1-ol/ water partition coefficient; this value is one indicator of hydrophobicity, with a greater value corresponding to a greater hydrophobicity. We hypothesized that secondary interactions based on hydrophobicity7 may facilitate the discrimination of these two proteins which have similar retention times on a classical ion-exchange resin. We observed no significant difference in the amount of lysozyme and cytochrome c adsorbed onto SAMs presenting ligands 1-5 at low salt concentrations, conditions under which electrostatic interactions are predominant (Supporting Information Figure 1). However, there was a significant difference in the amount of protein adsorbed onto the SAMs under high-salt conditions when hydrophobic interactions are expected to be important (Figure 1). The amount of cytochrome c adsorbed under high-salt conditions was lower than the amount of lysozyme adsorbed on all surfaces tested (Figure 1). For the more hydrophobic protein, lysozyme, the amount of protein adsorbed on SAMs presenting ligands 1-5 varied linearly with the value of log P for the ligand (Figure 1). Interestingly, the amount of cytochrome c adsorbed did not vary linearly with the value of log P and was significantly lower on SAMs presenting ligands 3 and 4sligands characterized by intermediate values of log P (0.52 and 0.79, respectively). This result would have been difficult to predict a priori. Ligands 3 and 4 thus showed considerable promise in terms of their ability to discriminate between lysozyme and cytochrome c. Our results also indicated that the value of log P was by itself insufficient to enable prediction of the extent of cytochrome c adsorption on a surface. We were intrigued by the observed lower extent of cytochrome c adsorption on SAMs presenting ligands 3 and 4 than that on (29) Ghose, A. K.; Crippen, G. M. J. Comput. Chem. 1986, 7, 565–577. (30) Gombar, V. K.; Enslein, K. J. Chem. Inf. Comput. Sci. 1996, 36, 1127– 1134. (31) Kwon, Y.; Han, Z. Z.; Karatan, E.; Mrksich, M.; Kay, B. K. Anal. Chem. 2004, 76, 5713–5720.
the other SAMs. Whereas a detailed explanation of the mechanistic basis for this observation is beyond the scope of this study, we carried out some additional experiments to relate the extent of protein adsorption to the chemical structure of the ligand. To that end, we used SPR spectroscopy to determine the extent of adsorption of lysozyme and cytochrome c on mixed SAMs presenting ligands 6-11 (Table 1). Whereas ligands 6-8 had values of log P similar to those of ligands 3 and 4, they were not aromatic (Table 1). Ligands 9 and 10 were aromatic and had values of log P similar to those ligands 3 and 4, but differed in the identity or number of substituents on the benzene ring. Ligand 11 was also aromatic, but had a larger value of log P than ligands 3 and 4. The extent of lysozyme adsorption varied linearly with the value of log P for all the SAMs tested (Figure 1). However, experiments with SAMs presenting ligands 6-11 once again indicated that log P was a poor predictor of the extent of adsorption of cytochrome c. Moreover, for the set of ligands tested, the presence of aromatic groups seemed to be necessary but not sufficient to achieve greater discrimination (i.e., a greater difference in the extent of adsorption of the two proteins). Importantly, the results from Figure 1 illustrate the applicability of the combination of SPR spectroscopy and SAMs to identify ligands (e.g., 3, 4, and 11, Table 1) that discriminate between proteins of interest.
Figure 1. Effect of ligand chemistry on the adsorption of lysozyme and cytochrome c to SAMs. Amount (RU, 1000 RU ≈ 1 ng/mm2)31 of lysozyme (circles) and cytochrome c (triangles) adsorbed under highsalt conditions as a function of log P. The values represent the mean of four measurements on two independent SAMs with standard error
