Online Magnetic Bead Dynamic Protein-Affinity Selection Coupled to

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Anal. Chem. 2009, 81, 4263–4270

Online Magnetic Bead Dynamic Protein-Affinity Selection Coupled to LC-MS for the Screening of Pharmacologically Active Compounds N. Jonker, A. Kretschmer, J. Kool, A. Fernandez, D. Kloos, J. G. Krabbe, H. Lingeman,* and H. Irth BioMolecular Analysis Group, Department of Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, proteinanalyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trappingdevice.Bioactiveligandsboundtotheprotein-Co(II)PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%. The discovery and development of new chemical entities is an ongoing process. To accomplish the time-consuming task of the screening of pharmaceutical libraries, often consisting of (multi)millions of compounds, a large variety of methodologies is available nowadays. However, in order to cover an even greater variety of different target molecules, different classes of proteins, and thus different types of potential ligands, there is a constant demand for new, more selective, and faster screening approaches. At present, most screening methodologies applied by the pharmaceutical industry are fluorescence-based and conducted either off-line or at-line with multiwell plate technologies.1-3 * Corresponding author. (1) Inglese, J.; Johnson, R. L.; Simeonov, A.; Xia, M. H.; Zheng, W.; Austin, C. P.; Auld, D. S. Nat. Chem. Biol. 2007, 3, 466–479. (2) Li, Z. Y.; Mehdi, S.; Patel, I.; Kawooya, J.; Judkins, M.; Zhang, W. H.; Diener, K.; Lozada, A.; Dunnington, D. J. Biomol. Screening 2000, 5, 31–37. 10.1021/ac9000755 CCC: $40.75  2009 American Chemical Society Published on Web 05/07/2009

Although these methodologies provide both high sensitivity and selectivity, they suffer from the inherent disadvantages of fluorescence-based techniques. First, a suitable fluorescent reporter molecule must be developed, which might be a costly and timeconsuming process. Second and more importantly, a fluorescence signal only indicates activity of a potential hit compound, whereas it does not yield any chemical information about the identity of this compound. Therefore, false positives due to library impurities or degradation products are frequently observed.4 In recent years, a number of approaches have been developed using mass spectrometric detection in order to simultaneously generate structural and biological data and to avoid false positives and negatives. Using pulsed-ultrafiltration, for example, the complex of target protein and bound ligand is separated from the nonbound fraction by transport through a membrane.5 This provides an enrichment of the analytes but suffers from a relatively high background signal of nonbound compounds remaining in the sample. Nonspecific binding of the protein or the ligand can also impede the performance. In other approaches immobilized proteins are used to achieve a separation between nonbinders and the binding ligands. Zonal elution or frontal-affinity methods6 are a fast way to obtain affinity rankings of compounds in mixtures, but coeluting compounds and the required buffers usually make mass spectrometric detection rather challenging.7 An alternative methodology is to immobilize a protein on a suitable support material, capture the ligands with sufficient affinity, wash away the nonbound fraction, and subsequently elute the bound ligands, either by competition or by denaturation of the protein.8 This provides a highly selective isolation of affinity compounds. Protein immobilization can be performed either covalently, which has been demonstrated on magnetic particles before,9-13 or noncovalently (e.g., by His-tagging). Immobilization utilizing His-tags has mostly (3) Parker, G. J.; Law, T. L.; Lenoch, F. J.; Bolger, R. E. J. Biomol. Screening 2000, 5, 77–88. (4) Yan, B.; Fang, L. L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.; Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547–559. (5) Van Breemen, R. B.; Huang, C. R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y. Z.; Venton, D. L. Anal. Chem. 1997, 69, 2159–2164. (6) Hage, D. S. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 768, 3–30. (7) Ng, W.; Dai, J. R.; Slon-Usakiewicz, J. J.; Redden, P. R.; Pasternak, A.; Reid, N. J. Biomol. Screening 2007, 12, 167–174. (8) Ferrance, J. P. J. Chromatogr., A 2007, 1165, 86–92. (9) Schlosser, G.; Vekey, K.; Malorni, A.; Pocsfalvi, G. Rapid Commun. Mass Spectrom. 2005, 19, 3307–3314.

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been performed on nonmagnetic silica material, immobilizing a wide range of targets.14-16 Surface-functionalized paramagnetic particles are widely employed in various protein purification processes. Although they are originally designed to enhance and simplify the protein isolation and purification process, their field of application has significantly widened. Hydrophobic surfaces like C8 and C18 are used for peptide isolation and proteome profiling.17,18 The interaction of DNA-binding drugs was studied with the help of immobilized oligonucleotides on magnetic particles.9 Antibodymodified magnetic beads have been used for selective analyte isolation and subsequent detection19 as well as for on-bead enzyme-linked immunosorbent assays (ELISA)20,21 and the selective isolation of stem cells.22 In a similar way, immobilized proteins have been used for ligand fishing. This has been shown for many proteins, such as the heat shock protein 90 R12 and human estrogen receptor.10 The handling of magnetic particles has been accomplished manually23 as well as with automated liquid handling systems,13 sequential injection methods,24 and microfluidic devices.25 Finally, an interesting application has been shown by using immobilized enzymes on magnetic particles, enabling the screening of enzyme inhibitors.11 The majority of the methods described so far, using magnetic beads, possess similar limitations. Most importantly, covalent immobilization of the protein may alter the binding properties of the protein and, thus, the efficacy of the screening method. Second, many of these systems are difficult to automate, suffer from limited reproducibility, and are rather laborious. In this study an online methodology is reported using cobalt(II)-coated magnetic beads which are able to bind any His-tagged protein after an in-solution incubation with a possible ligand. The beads are coupled online to a dual solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) system, thus combining the convenience of magnetic immobilization with the (10) Choi, Y.; van Breemen, R. B. Comb. Chem. High Throughput Screening 2008, 11, 1–6. (11) Hu, F.; Zhang, H.; Lin, H.; Deng, C.; Zhang, X. J. Am. Soc. Mass Spectrom. 2008, 19, 865–873. (12) Marszałł, M. P.; Moaddel, R.; Kole, S.; Gandhari, M.; Bernier, M.; Wainer, I. W. Anal. Chem. 2008, 80, 7571–7575. (13) Moaddel, R.; Marszall, M. P.; Bighi, F.; Yang, Q.; Duan, X.; Wainer, I. W. Anal. Chem. 2007, 79, 5414–5417. (14) Luckarift, H. R.; Johnson, G. R.; Spain, J. C. J. Chromatogr., B 2006, 843, 310–316. (15) Moaddel, R.; Lu, L. L.; Baynham, M.; Wainer, I. W. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 768, 41–53. (16) Moaddel, R.; Price, G. B.; Juteau, J. M.; Leffak, M.; Wainer, I. W. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 820, 197–203. (17) Yao, N.; Chen, H. M.; Lin, H. Q.; Deng, C. H.; Zhang, X. M. J. Chromatogr., A 2008, 1185, 93–101. (18) Baumann, S.; Ceglarek, U.; Fiedler, G. M.; Lembcke, J.; Leichtle, A.; Thiery, J. Clin. Chem. 2005, 51, 973–980. (19) Zhang, R. Q.; Hirakawa, K.; Seto, D.; Soh, N.; Nakano, K.; Masadome, T.; Nagata, K.; Sakamoto, K.; Imato, T. Talanta 2005, 68, 231–238. (20) Kourilov, V.; Steinitz, M. Anal. Biochem. 2002, 311, 166–170. (21) Hayes, M. A.; Polson, N. A.; Phayre, A. N.; Garcia, A. A. Anal. Chem. 2001, 73, 5896–5902. (22) Odabas, S.; Sayar, F.; Gu ¨ ven, G.; Yanikkaya-Demirel, G.; Piskin, E. J. Chromatogr., B 2007, 861, 74–80. (23) Girault, S.; Chassaing, G.; Blais, J. C.; Brunot, A.; Bolbach, G. Anal. Chem. 1996, 68, 2122–2126. (24) Wang, J. H.; Hansen, E. H.; Miro, M. Anal. Chim. Acta 2003, 499, 139– 147. (25) Verpoorte, E. Lab Chip 2003, 3, 60N–68N.

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selectivity of dynamic protein-affinity selection26 and the accuracy of MS detection. EXPERIMENTAL SECTION Materials. ELISA blocking reagent was purchased from Roche Diagnostics (Pensberg, Germany). Glycine · HCl, 17β-estradiol, equol, paracetamol, epibatidine, nicotine, diclofenac, naproxen, sulfadimethoxine, testosterone, trimethoprim, potassium chloride, monosodium dihydrogen phosphate, and disodium monohydrogen phosphate were obtained from Sigma (Schnelldorf, Germany). Diethylstilbestrol, sodium chloride, ammonium monohydrogen carbonate, and warfarin came from Riedel de Haen (Seelze, Germany). Coumestrol was purchased at Fluka (Bachs, Germany). Ethylenediaminetetraacetic acid was received from Acros (Geel, Belgium). Nickel nitrate was purchased from Aldrich (Steinheim, Germany). Methanol and formic acid were obtained from Biosolve (Valkenswaard, The Netherlands). Water was produced by a Milli-Q device of Millipore (Amsterdam, The Netherlands). The binding buffer consisted of 0.5 mg/mL ELISA blocking reagent in 10 mM phosphate-buffered saline (PBS) at pH 7.4. Magnetic Beads. The 1 µm cobalt-loaded Dynabeads TALON were purchased from Dynal, Invitrogen (Breda, The Netherlands) and supplied in an ethanol/water (20:80) solution. According the manufacturer’s recommendation, before use 250 µL of the bead suspension was washed three times with 1.5 mL of binding buffer and finally resuspended in 500 µL of binding buffer, resulting in a concentration of 20 mg/mL. The magnetic bead suspensions were prepared directly before the addition of the beads to the mixture. Trapping of the magnetic beads was achieved in PEEK tubing (i.d. ) 0.25 mm) by a 50 N permanent neodymium magnet (1.4 T) with dimensions of 7 cm × 4 cm × 3.5 cm. The tubing was attached to an in-house built, pneumatically driven, aluminum arm. The movement of the arm was triggered by a contact closure provided by the Symbiosis. Both the arm and tubing could move vertically into two different positions, one allowing contact between the magnet and the tubing, used when trapping the magnetic beads, and one where the magnet is 2.6 cm away from the tubing, used during the elution of the beads (Figure 1). hERr-LBD Preparation. hERR-LBD (ERR) was expressed and purified as reported by Eiler et al.,27 with the difference that the final solution was dissolved in the binding buffer not containing any estradiol. The ERR concentration in the batch was determined to be 841 ± 72 nM. Measurements were corrected for the 1.5% ± 0.2% activity loss per hour caused by degradation of the estrogen receptor at 4 °C. The immobilization efficiency of the receptor on the magnetic particles was determined by varying the absolute amounts of beads and protein in the incubation mixture. Sample Preparation. Samples were prepared in 1.8 mL (32 mm × 11.6 mm) glass autosampler vials by adding 1.2 mL of receptor solution and 150 µL of the compound(s) to be screened at a final concentration of 1 µM. The incubation was performed during 15 min at room temperature, while the mixture was slowly being shaken using an electronically modified vortex (VWR International, Amsterdam, The Netherlands). Subsequently, 60 µL (26) Jonker, N.; Kool, J.; Krabbe, J. G.; Retra, K.; Lingeman, H.; Irth, H. J. Chromatogr., A 2008, 1205, 71–77. (27) Eiler, S.; Gangloff, M.; Duclaud, S.; Moras, D.; Ruff, M. Protein Expression Purif. 2001, 22, 165–173.

Figure 1. Magnetic bead trapping in PEEK tubing. Liquid flow directed from left to right. On the left the bead-protein-ligand complex is injected and immobilized in the magnetic field of the magnet positioned in direct contact with the PEEK tubing. On the right the PEEK tubing and magnetic field are physically removed from each other, resulting in elution of the beads and protein residue.

Figure 2. System settings used for simultaneous magnetic protein immobilization and SPE-LC-MS analysis.

of freshly prepared magnetic bead solution, containing 4.4 µg of beads, was added to the mixture and slowly shaken for another 5 min. The resulting mixture was stored in the Symbiosis Pharma Reliance autosampler at 4 °C. Before every injection the autosampler homogenizes the solution by aspirating and reinjecting 100 µL of the sample into the autosampler vial. System Setup. The protein-affinity SPE system was constructed using the Symbiosis Pharma system (Spark Holland, Emmen, The Netherlands). The setup is shown in Figure 2. The magnetic immobilization module is positioned between the Reliance autosampler and the dual SPE automated cartridge exchange (ACE) module of the Symbiosis Pharma system. This system enables the simultaneous loading of one SPE column and elution of a second SPE column, limiting the measurement time to 9.5 min per sample. The chronology of a measurement series starts

with the previously described sample preparation and injection of 100 µL of the incubation mixture in the system. The injection plug is transported to the magnetic field by using 300 µL of the binding buffer. The magnetic field retains the complex of beads, proteins, and ligands, while the nonbound fraction is washed away with 400 µL of ammonium bicarbonate buffer at pH 7.4. Elution of the bound ligands occurs with 400 µL of glycine · HCl at pH 2.0, denaturating the protein and releasing the ligands so they can be trapped on the C18 SPE cartridge. This cartridge is switched online with the LC-MS unit, and its contents are eluted, separated on the C18 LC column, and detected by mass spectrometry. The magnetic field is removed, and the beads are eluted to waste. Immediately afterward, but still during the SPE-LC-MS run, a second sample is immobilized using the magnetic field and Analytical Chemistry, Vol. 81, No. 11, June 1, 2009

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eluted to a second SPE cartridge, which is analyzed by the SPE-LC-MS system as soon as the previous analysis is completed. LC-MS Detection. The LC-MS system consists of a highpressure gradient LC system (Shimadzu LC20, Kyoto, Japan) with a 7 min linear gradient from 60% (v/v) to 100% (v/v) of methanol in water. The compounds are separated on a Sunfire C18 column (flow rate 200 µL/min, 2.1 mm i.d., 3 µm particles, 50 mm in length, Waters, Milford, MA). All measurements were performed on a Thermo Electron (Breda, The Netherlands) LCQ Deca ion trap mass spectrometer. In case of positive electrospray ionization (ESI(+)) 0.1% (v/v) formic acid was added to both eluents, and in the case of negative atmospheric-pressure ionization (APCI(-)) no formic acid was used. Calibration curves were measured for a set of 12 compounds in both ESI(+) and APCI(-) mode by injecting the analytes in a concentration range of 20 nM to 5 µM. In the APCI(-) mode, a capillary temperature of 225 °C and a vaporizing temperature of 450 °C were used. The capillary voltage was set to -40 V, and the tube lens was set to 20 V. The corona discharge current was set to 10 µA. The nitrogen flow was 15 L/min for the nebulizing gas and 5 L/min for the auxiliary gas. In the ESI(+) mode the capillary temperature and voltage were set to 250 °C and +15 V, respectively. The tube lens offset was 50 V, and the nebulizing and auxiliary gas flows were identical to the APCI(-) mode. RESULTS AND DISCUSSION System Setup. A schematic representation of the magnetic bead dynamic protein-affinity selection system is shown in Figure 2. Off-line sample preparation consists of incubation of the Histagged target protein, His-tagged human estrogen-receptor R ligand-binding domain (hERR-LBD), with a mixture of possible ligands. The binding of these ligands to the receptor takes place in solution, using conditions comparable with native binding conditions. The complex of protein and bound ligand was then retained on cobalt(II)-coated paramagnetic affinity beads (Co(II)PABs) by addition of the magnetic nanobeads to the incubation solution. The solution is injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are eluted online toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily removing the magnetic field (see Figure 1). Important parameters influencing the performance of the magnetic bead trapping method are the amount of beads used, the flow rate of the carrier flow, the dimensions of the tubing, and the protein-bead ratio. The final SPE-LC-MS analysis is a gradient elution system that has previously been described.22 Magnetic Trapping. The magnetic field used to trap the beads is not permanent but induced by a permanent magnet. The attraction force must be larger than the force of the liquid flow on the stagnant particles in order to perform an efficient trapping. Determining the attraction force requires knowledge of a number of parameters, such as the force (q) of the permanent and the induced magnetic dipoles and the magnetic permeability (µ) of the medium separating both dipoles, as given by Coulombs’ law: 4266

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F)

µqm1qm2 4πr2

The force of the permanent magnet can be calculated from its field strength, but for individual magnetic beads or even the total amount of beads injected, this entails complex measurements. A second complication is that the magnetic permeability is hard to define because the medium is anisotropic; consisting of buffer solution, PEEK tubing, and adhesion material between the two magnetic dipoles. The consequence of this anisotropy is that the permeability can only be expressed as a second rate tensor, which makes it impossible to apply in this situation. The fact that all beads have a different spatial orientation and possess their own magnetic dipole moment, influencing each other, explains that the system cannot be modeled easily. As a consequence, a comparison between the attractive force of the two magnetic dipoles and the force of the liquid flow on the beads will have to be established experimentally, by varying the flow rate through the tubing and the inner diameter of the tubing. Flow Rates Optimization. The first experiments were performed using the incubation mixture described in the Experimental Section and using the flow rates specified by the manufacturer of the Symbiosis Pharma system (1 mL/min for wash and elution steps). This included a 2 min wash step and a 4 min elution step. The result was that hardly any bound ligand could be detected. Systematically varying the relevant parameters (e.g., flow rates, wash volumes, buffer concentrations, organic modifier concentrations) revealed that a decrease in flow ratesand simultaneously a decrease of the volume by keeping the total time constantsimproved the signal considerably. The results of all the experiments showed a very sharp signal decrease (cutoff) at a certain flow rate (Figure 3). The three lines represent the experiments in which the flow rate was varied and the total volume per curve was kept constant. The curves differ from each other in the volumes used in the three analysis steps in which the trapped complex is exposed to a liquid flow (transport, wash, and elution). Therefore, also the time these combined steps take varies. In order to compare the curves it is important to know the total flow time these steps take in the experiment where the cutoff appears. For curve 1, the cutoff appears at 230 µL/min, with a total flow time of 318 s, for curve 2, the cutoff appears at 355 µL/min with a total flow time of 180 s, and for curve 3, the cutoff appears at 380 µL/min with a total flow time of 150 s. The inset in Figure 3 shows the results of a duplicate experiment of the first curve, but with a number of data points added between 200 and 350 µL/min. From these data, the observation is confirmed that the decrease in the response is rather defined and fast. This supports the assumption that, above a certain flow rate (200-300 µL/min), the force generated by the liquid flow in the PEEK tubing exceeds the force exerted by the magnetic field on the beads and they are washed out of the tubing, which immediately decreases the response. Furthermore, it shows that the total analysis time influences the maximum flow rate that is applicable, implying that even at low flow rates the beads migrate slowly through the tubing. Both effects imply that the attractive force between the magnets and the force exerted by the liquid flow on the beads are in the same order of magnitude. This means that

Figure 3. Three experiments performed to study the effect of varying flow rates. In the first experiment (9) the flow rate and time of the wash step were varied. In the second experiment (2) the flow rate in the entire system was varied, but kept identical for all separate steps. In the third experiment ([) the flow rate and time of the injection step were varied.

during the actual analysis the magnetic force always must be larger than the liquid-flow force. Tubing Dimensions Optimization. Another possibility to decrease the force generated by the liquid flow on the beads is to increase the inner diameter of the PEEK tubing. However, this will also increase band broadening and result in a longer analysis time. After determining that the immobilization of beads in PEEK tubing with a 0.13 mm i.d. was not possible, three inner diameters were tested: 0.25, 0.50, and 0.75 mm. The first experiment, using the flow rate as optimized for the 0.25 mm i.d. tubing, showed a significant decrease in signal: 22% ± 2% for the 0.5 mm i.d. and 35% ± 1% for the 0.75 i.d. tubing. Increasing the flow rates, to compensate for the larger volume of the tubing, significantly improved the results. However, the responses with the 0.50 and 0.75 mm i.d. tubing were still lower compared with the 0.25 mm i.d. tubing: 8% ± 3% and 13% ± 2%, respectively. These results clearly show that the smallest inner diameter provides the best results; as long as the flow is decreased to a value at which magnetic trapping is still possible. The conclusion is that decreasing the flow is probably the best way to ensure an efficient trapping of the beads. In a final experiment the effect of the decreased flow rates of the wash and elution step on the response is studied. The results showed that decreasing the volume of the wash step did not significantly influence the overall response of the system, as long as the total wash time remained constant. This was also valid for the elution step. In the previously optimized elution step with 2 mL of solvent at a flow rate of 1 mL/min, which is ideal for the column dimensions used, the beads were eluted to the SPE column. Clogging and reproducibility problems were observed, and the pumps automatically shut down because of an increased back pressure after about 20 runs. In order to solve this problem, the flow rate of the elution step was adjusted. It was found that an elution step of 400 µL at 0.2 mL/min provided the same results, indicating that only elution time is important. These results showed that decreasing the flow rate did not influence the results significantly. Therefore, in all further experiments a flow rate of 200 µL/min was used, with a 300 µL binding buffer to transport

Table 1. Schematic Representation of All Test Compounds, Their Abbreviations, the Ionization Mode Used, and Their Limit of Quantitation in the Bioassay (When Applicable) substance

abbr

ionization

LOQ (nM)

estradiol coumestrol diethylstilbestrol equol norethisterone warfarin epibatidine nicotine diclofenac sulfadimethoxine testosterone trimethoprim

E2 CMS DES EQ NET WF EPI NIC DIC SUL TES TMP

APCIESI+ APCIAPCIESI+ ESI+ ESI+ ESI+ APCIESI+ ESI+ ESI+

60 30 30 80 20

the injection plug to the magnetic field, a 400 µL wash step, and a 400 µL elution step. With these settings, over 150 measurements have been performed without any noticeable clogging or reproducibility or pressure problems. In summary it can be stated that per analysis the total volume should not exceed 1.2 mL and that the flow rate should be below 230 µL/min. Pressure Generation. Trapping of the beads in the system resulted in an increase of the back pressure. Usually, the back pressure in an online system is a combination of the back pressure caused by the tubing (mostly negligible) and by the various columns, in this case the SPE cartridge. However, a significant pressure increase was registered when the beads were trapped in the tubing. In order to predict the distribution of the magnetic beads in the PEEK tubing, the Kozeny-Carman equation28,29 can be used. This equation is applicable to any system with particles of a reasonably equal size and shape distributions under Darcian conditionssneglecting both anisotropy and electrochemical interactions. Chromatographic systems generally comply with these conditions, and separation scientists use the Kozeny-Carman (28) Kozeny, J. Wien. Akad. Wiss. 1927, 136, 271. (29) Carman, P. C. J. Soc. Chem. Ind., London, Trans. Commun. 1938, 57, 225.

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Figure 4. Results of individual incubations of all 12 compounds in a test mixture, corrected for nonspecific binding.

equation to calculate the maximum back pressure particles or beads can generate in a system. The equation is represented as ¯ 0µ (1 - )2 150V ∆p ) 2 L Φs Dp2 3 In this equation ∆p is the pressure increase, L is the length of the plug of immobilized magnetic beads in the PEEK tubing, V0 is the average superficial velocity (flow rate in m/s), µ is the viscosity, ΦS is the sphericity of the particles, Dp is the particle diameter, and  is the permeability. The values for L (1.9 × 10-3 m), V0 (6.8 × 10-2 m/s), and µ (8.9 × 10-4 kg/ms) can be calculated, and the values for DP (1.1 × 10-6 m) and ΦS are obtained from the manufacturer. Assuming the beads are packed like a chromatographic column, the best-case scenario, and rather unlikely, the permeability should be around 0.4.30 Applying these values results in a calculated pressure increase of 6.5 bar at a flow rate of 200 µL/min. When using the standard amount of beads and the conditions established in the previous paragraph, the back pressure generated by the system was 1.7 ± 0.4 bar (n ) 10). This corresponds to a permeability of around 0.21, which does not correspond with any known packing material. In spite of the fact that the packing of the beads in PEEK tubing may not be considered to be a homogeneous packed column, it does imply a certain amount of compactness in the packing of the beads, and it is rather unlikely that the beads are spread out like a film on the inside of the PEEK. Probably it is justified to conclude that the beads are indeed immobilized as a plug in a small part of the tubing and that a comparison is allowed with packed beds in SPE cartridges. Protein-Bead Ratio. As mentioned before the two primary processes in system characterization are magnetic trapping and the protein-bead ratio. Studying the amount of protein efficiently immobilized on the beads, preliminary experiments showed that the response was not even close to the maximum response that could be generated with an 841 nM receptor mixture. Experiments (30) De Smet, J.; Gzil, P.; Vervoort, N.; Verelst, H.; Baron, G. V.; Desmet, G. J. Chromatogr., A 2005, 1073, 43–51.

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with varying amounts of magnetic beads and a constant amount of 84.1 pmol of receptor (100 µL of 841 nM) showed that the maximum amount of protein that could be immobilized was 42 ± 7 pmol. Higher amounts of magnetic beads led to aggregation of the beads in solution, which dramatically decreased the response. However, when decreasing the amount of protein in the incubation mixture, the response also reduced proportionally. This implies that a minimum excess of 100% of protein, with respect to binding sites on the beads, is required in order to obtain an optimal immobilization. Unfortunately, the measurements were limited by two parameters: the concentration of the receptor stock could not be increased, and higher amounts of beads could not be studied due to the aggregation effect. With the use of the current conditions, a protein immobilization efficiency of 50% ± 8% was the maximum value that could be achieved. Finally, a decrease of the concentration of bead-protein complex shows a proportional decrease in response (R2 ) 0.98), suggesting that the immobilization efficiency is unchanged as long as the bead concentration is kept below the aggregation threshold. With these optimized settings the relative standard deviation was determined to be 8.8% for an n ) 10 experiment performed over 24 h using the strong binder coumestrol as a ligand. Screening of Test Compounds. In order to show the applicability of the system a set of test compounds (see Table 1) was screened. All compounds were incubated separately and corrected for nonspecific binding with the result of a competition experiment including 5 µM of ethynylestradiol, a synthetic estrogen with a subnanomolar affinity toward ERR (Figure 4). Figure 4 demonstrates the simplicity with which binders and nonbinders can be separated. The first five compounds, β-estradiol (E2), coumestrol (CMS), diethylstilbestrol (DES), equol (EQ), and norethisteron (NET), are estrogenic ligands, with affinities ranging from the subnanomolar range (DES), to the high micromolar range (NET). They are easily identified as binders and clearly distinguishable from the nonbinders. In order to optimize the throughput, in a final set of experiments mixtures of compounds, consisting of one to three binders and seven nonbinders, were screened. Similar to a library screening methodology, all compounds were present in the

Figure 5. Mixture containing norethisterone (NET) and seven other compounds is analyzed. ESI(+) data is shown for, consecutively, the total ion current (TIC) of an SPE-LC-MS analysis of the entire mixture, an extracted ion chromatogram for the mass of NET in the same experiment, the TIC of the affinity assay, an extracted ion chromatogram for NET of the affinity assay. Finally, a mass spectrum of the measured peak in the affinity assay is shown, corresponding with the structure of NET.

Figure 6. Results of incubations of eight-compound mixtures, normalized to the response of a standard injection in a SPE-LC-MS system. The second series (dark color) represents the nonspecific binding to the magnetic beads.

mixture in the same concentration (1 µM) as in the single incubations. This is essential for the successful application of the method. If mixtures of compounds have different concentrations, the protein-ligand ratio will vary per compound, and the normalization leading to the final result cannot be performed. An

example of the data generated when measuring such a mixture is shown in Figure 5. No control experiment is used, so no corrections are made for nonspecific binding. In a real screening situation only a very small number of compounds will show affinity for the target. All mixtures containing a binder are then reanalyzed Analytical Chemistry, Vol. 81, No. 11, June 1, 2009

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in single-compound incubations. These additional experiments are performed in order to prevent false negatives (more than one hit in one mixture) or false positives (extremely high nonspecific binding). The results (see Figure 6) are normalized against the maximum possible SPE-LC-MS signal and clearly demonstrate that the binders are easily distinguishable from the nonbinding compounds. The first difference with the results shown in Figure 4 is the significantly higher signal for the nonbinders, caused by the fact that these measurements are not corrected for nonspecific binding. However, in order to prove the concept, the nonspecific binding has been measured by repeating the experiments without the presence of ERR. The determined nonspecific binding is presented in Figure 6, normalized to the same scale. However, this data only represents nonspecific binding to the magnetic beads. The difference between the binding and nonspecific binding of the nonbinders is most likely caused by the nonspecific binding to the protein that is not included in this data. The second difference is an increase in the standard deviation, which is most likely caused by the increased complexity of the sample. However, the distinction between binders and nonbinders remains very clear. In order to mimic a situation in which false positives or negatives might occur, mixtures were screened consisting of three binders with seven nonbinders. Only a mixture containing E2 and CMS combined with either NET or EQ resulted in a double hit (E2 and CMS). In all other mixtures, the weaker binders were displaced by the strongest one, producing only one hit. These results support the concept that every mixture producing a hit should be split up in separate incubations for every constituent

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of the mixture. If these experiments are not performed, false negatives will occur. The results represent a screening experiment that is able to screen 40 compounds in 45 min, providing simultaneously information on the bioaffinity and chemical characteristics of active compounds. Conclusion. The methodology described in this paper employs magnetic nanoparticles in a novel screening methodology that combines the advantages of in-solution incubation with the efficiency of online analysis using SPE-LC-MS analysis. A main benefit of the method, compared with previously reported methodologies, is that also binders with a very weak affinity can be determined. However, methodologies employing covalently immobilized proteins can be used to rank the compounds in order of affinity. Magnetic bead dynamic protein-affinity selection only provides a yes or no answer, determining whether a compound is a binder or a nonbinder. A further advantage is the high throughput of the method because of the two parallel used SPE modules allowing the simultaneous screening and analysis of 1300 compounds per day. The optimized system has been operational for over 150 runs per C18 cartridge, without any changes in reproducibility or system deterioration. Furthermore, this methodology can offer possibilities in the study of membrane receptors, such as GPCRs, because of the relative ease of separating the bound and unbound fraction using the magnetic trapping device. Received for review January 12, 2009. Accepted April 17, 2009. AC9000755