Anal. Chem. 2005, 77, 1157-1162
Technical Notes
Surface Plasmon Resonance/Mass Spectrometry Interface Jens Grote,† Nico Dankbar,‡ Erk Gedig,‡ and Simone Koenig*,†
Integrated Functional Genomics, Interdisciplinary Center for Clinical Research, Medical Faculty, University of Muenster, Roentgenstrasse 21, 48149 Muenster, Germany, and XanTec Bioanalytics GmbH, Muenster, Germany
A strategy for combining surface plasmon resonance (SPR) biomolecular interaction analysis, and matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. Both techniques are highly complementary but need separate optimization to improve their individual specificity and sensitivity. Sensor surfaces that are optimal for kinetic analysis are not well suited for MALDI-MS and vice versa. In addition, the transfer of analyte from SPR to MS is crucial and often accompanied by sample loss. To address both of these points, a bifunctional SPR fluid cell was constructed where optimized surfaces can be used for binding studies and MS simultaneously with regard to the special need of each technique. The setup guarantees that the SPR and the loading experiment for MS are performed at identical conditions. A removable pin carries the affinity-surfacebound analyte to the mass spectrometer so that handling is minimized, avoiding analyte elution. Functionalized transfer pins can also be used independently of SPR for microaffinity capture-MS. In recent years, surface plasmon resonance (SPR) has become a standard technique for quantitative and kinetic analysis of biomolecular interactions. SPR uses light of defined wavelength, polarity, and angle of incidence on a gold-coated sensor chip to measure changes in refractive index on the opposite side of the gold film. The sensor chip surface is usually coated with a thin hydrogel matrix bearing covalently immobilized receptor molecules such as antibodies for specific analyte capture. Complex formation with ligands leads to a change in the refractive index, which can be monitored in real time. The signal increases proportional with the amount of bound molecules, allowing the determination of association and dissociation kinetics by varying the analyte concentration.1 Labeling of biomolecules is not required in SPR, and complex analytes such as serum can be analyzed directly at picogram sensitivities. SPR offers a number of advantages with respect to analytical speed and throughput as * To whom correspondence should be addressed. Phone: xx-(0)251-8357164. Fax: xx-(0)251-8357255. E-mail:
[email protected]. † University of Muenster. ‡ XanTec Bioanalytics GmbH. (1) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid. Interface Sci. 1991, 143, 513-526. 10.1021/ac049033d CCC: $30.25 Published on Web 01/13/2005
© 2005 American Chemical Society
well as sensitivity and flexibility compared to other methods such as protein affinity chromatography2 and two-hybrid experiments.3 Therefore, much effort is now focused on the adaptation of highly sensitive techniques with massive parallelization potential such as DNA microarray technology in order to use them in protein assay and affinity studies.4,5 Notwithstanding its clear advantages over conventional techniques, SPR suffers from the inherent drawback that no structural information about the captured analyte is generated. In this respect, matrix-assisted laser/desorption ionization (MALDI)-MS seems to be the perfect complement. Not only does it offer all the advantages related to the determination of molecular masses of biomolecules as is presented in a wealth of scientific literature but it is also tolerant to functionalized hydrogel surfaces, which should allow the direct analysis of bound species on the sensor chip surface.6,7 Therefore, both techniques have been applied in parallel, but there have also been several efforts to integrate SPR and MS synergistically.6,8-17 For this combination, the analyte is either eluted from the SPR chip and respotted onto the MS target,15,18,19 or the SPR chip (mostly known from Biacore) is taken (2) Formosa, T.; Alberts, B. M. Cold Spring Harbor Symp. Quant. Biol. 1984, 49, 363-370. (3) Bartel, P. S.; Fields, S. Methods. Enzymol. 1995, 254, 241-263. (4) Abbott, A. Nature 1999, 402, 715-720. (5) Gedig, E. In Analyzing Gene Expression; Lorkowski, S., Cullen, P., Eds.; Wiley-VCH: New York, 2003; Vol. 2, pp 660-670. (6) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 43634368. (7) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1993, 7, 576580. (8) Nelson, R. W.; Krone, J. R.; Granzow, R.; Jansson, O.; Sjoelander, S. Pharmacia Biosensor Ab. PCT Int. Appl. WO 9709608, 1997. (9) Williams, C., Addona, T. A. TIBTECH 2000, 18, 45-48. (10) Lofas, S. Am. Biotechnol. Lab. 2003, 21, 16F-16G. (11) Nedelkov, D.; Nelson, R. W. Trends Biotechnol. 2003, 21, 301-305. (12) Seymour, C. Proteomics 2003, 3, 809-810. (13) Zhukov, A.; Suckau, D.; Buijs, J. Am. Biotechnol. Lab. 2002, 20, 10, 12. (14) Zhukov, A.; Buijs, J.; Suckau, D. Biochemist 2002, 24, 21-23. (15) Gilligan, J. J.; Schuck, P.; Yergey, A. L. Anal. Chem. 2002, 74, 20412047. (16) Nedelkov, D.; Nelson, R. W. J. Mol. Recognit. 2003, 16, 15-19. (17) Zhukov, A.; Schu ¨ renberg, M.; Jansson, O ¨ .; Areskoug, D.; Buijs, J. J. Biomol. Tech. 2004, 15, 112-119. (18) Soenksen, C. P.; Nordhoff, E.; Jansson, O.; Malmqvist, M.; Roepstorff, P. Anal. Chem. 1998, 70, 2731-2736. (19) Nelson, R. W.; Jarvik, J. W.; Taillon, B. E.; Tubbs, K. A. Anal. Chem. 1999, 71, 2858-2865.
Analytical Chemistry, Vol. 77, No. 4, February 15, 2005 1157
Figure 1. Photograph and schematic of an IBIS fluid cell where openings for transfer pins were added.
Figure 2. Modified TofSpec targets for the analysis of (a) SPR chips (left Biacore, right IBIS) and (b) transfer pins. (c) Pins coated in a stainless steel pin holder (top) or immersed in reaction solution (bottom).
from its holder and refitted into a custom-made MS target.20 While the first method keeps the biosensor chip reusable, but is hampered by time- and sample-consuming transfer steps,21 the second is more direct, but involves currently the destruction of the sensor chip and some manufacturing. Even more important is the partial incompatibility of the surface requirements of both techniques: SPR-based kinetic analysis of biomolecular interaction requires low receptor densities in order to avoid potential diffusion limitations.22 Furthermore, computer simulations and experimental data have shown that the at present commonly employed ∼100-nm-thick carboxymethyldextran (CMD) hydrogel layers limit the diffusion of the analyte and can cause rebinding and steric effects on analyte binding, which lead to discrepancies between SPR-measured kinetic parameters and solution data.23-26 This is especially the case at conditions where mass transport effects have to be considered.27 As it could be demonstrated that employing a self-assembled monolayer (SAM) instead of a hydrogel matrix reduces these effects,28 it would only be consequent to use very thin hydrogel layers with a thickness below 20 nm and immobilization capacities of CMD2000 as opposed to CMD100, but also leveling off at ∼CMD2000. CMDs can vary in length, shape, and density, and all of those features influence the SPR and MS results, although there is no detailed study available, yet. Commercial SPR chips often rely on CMD500 (Biacore CM530). The compatibility of polysaccharide hydrogels with MS has been shown before also by other authors.21,29,31 They do not cause problems concerning background ions, and they can easily be functionalized with conventional covalent coupling chemistry.32 N’-Ethyl-N’-[(dimethylamino)propyl]carbodiimide)/N-hydroxysuccinimide (EDC/NHS) coupling, as was used in this note, is frequently employed to bind proteins or peptides to the carboxylated surface. It is not surprising that a higher surface capacity would benefit MS output since more analyte molecules are available for detection, and although an MS signal can be obtained from common sensor chips, we have come to value the added sensitivity, quality, and flexibility that is achievable with the redesigned SPR/MS interface and optimized surfaces described in this note.33,34 (29) (30) (31) (32)
Koenig, S., Grote, J., Gedig, E. Int. Biotechnol. Lab. 2002, 20, 10. Bergstro ¨m, J. PCT patent WO 90/05303, 1990. Nelson, R. W.; Krone, J. R. J. Mol. Recognit. 1999, 12, 77-93. Johnsson, B.; Lofas, S.; Lindquist, G. Anal. Biochem. 1991, 198, 268-277.
Figure 3. MALDI-MS on CMD2000 pin of BSA bound to immobilized mIgG R BSA (top) and control without immobilized antibody (bottom).
EXPERIMENTAL SECTION Experiments were performed using an IBIS instrument (IBIS Technologies BV, Hengelo, The Netherlands) for SPR and TofSpec-2E (Micromass, Manchester, U.K.) for MS. Special targets were manufactured at the workshop of the Institute of Experimental Biomechanics (University of Muenster). Unless otherwise noted, all chemicals were purchased from Sigma and solvents were of HPLC-grade purity. Screws (DIN 7991, M2, A2, length 8 mm; Hummer & Riess GmbH, Nu¨rnberg, Germany) Figure 2c) were shortened to the height of a TofSpec-2E target plate (Figure 2a,b), galvanized with gold (jeweller Teufel, Muenster, Germany), and CMD custom-coated by XanTec. For the experiment shown in Figure 3, a CMD2000 pin was derivatized with mouse polyclonal anti-bovine serum albumin (antiBSA) IgG. To that end, possible contaminants were eluted with 2 M sodium chloride containing 0.01 M sodium hydroxide. Activation was carried out with NHS (0.5 M in 0.5 M 2-(N-morpholino)ethanesulfonic acid, pH 6) containing 0.5 M EDC. After immobilization of anti-BSA (0.01 mg/mL in 5 mM sodium acetate (NaAc), pH 5), excess NHS esters were quenched with 1 M ethanolamine hydrochloride at pH 8.5. The pin was equilibrated with phosphatebuffered saline (PBS; 20 mM sodium phosphate, 150 mM NaCl, pH 7.2) and incubated with BSA (MW 66 430) solution (0.02 mg/ mL in PBS) for 20 min. The pin was washed with water, and 300 nL of matrix (50 mM sinapinic acid in water/acetonitrile (ACN) 60/40 v/v containing 0.1% trifluoroacetic acid (TFA, Merck, Darmstadt, Germany)) was applied to the pin. The pin was kept in a humid atmosphere for ∼10 min. Additional matrix solvent was added if necessary to avoid crystallization before protein diffusion out of the hydrogel layer was expected to be sufficiently complete. After this slow-drying process the pin was inserted into the modified MALDI target plate (Figure 2b). For control, a second pin was treated in the same way except for antibody immobilization. (33) Grote, J.; Dankbar, N.; Gedig, E.; Koenig, S. [i]lab workshop: Molecules as Modulators: Systems Biology Challenges Chemistry, Eberbach Monastery, January 29-31, 2004. (34) Koenig, S., Grote, J., Dankbar, N., Gedig, E. Patent application to the University of Muenster, 7/1/2003, release 10/24/2003.
In Figure 4a-d, a CMD500 biosensor chip and a CMD2000 transfer pin were used in the bifunctional fluid cell (Figure 1). An additional CMD2000 transfer pin was prepared for a binding experiment in an Eppendorf cap (Figure 4e). Rabbit polyclonal antibody against human myeloid-related protein 14 (MRP14; 0.1 mg/mL in 5 mM NaAc, pH 4.5) was immobilized as described above. Pins and chip were equilibrated with Tris-buffered saline (TBS; 50 mM Tris, 150 mM sodium chloride, pH 7,6), and 0.063 mg/mL His-tagged MRP14 (MW 14 780) in TBS was added. All steps were monitored by SPR. The fluid cell was cleaned from TBS with two injections of distilled water, and the batch pin with distilled water in an Eppendorf cap. Sinapinic acid matrix was applied for MALDI measurement. MRP14 and anti-MRP14 were a kind gift from C. Kerkhoff (Institute of Experimental Dermatology, University of Muenster). In Figure 5, a CMD6 biosensor chip and a CMD2000 transfer pin were used in the bifunctional SPR fluid cell. They were immobilized with rabbit polyclonal antibody against human brain fatty acid binding protein (anti-hB-FABP; 0.05 mg/mL in 5 mM NaAc, pH 4.5) and then exposed to 0.035 mg/mL hB-FABP after equilibration with PBS. All steps were monitored by SPR and sinapinic acid was used for MS. hB-FABP (recombinant from Escherichia coli, MW 14 889) and anti-hB-FABP were a kind gift from T. Hanhoff (Institute of Biochemistry, University of Muenster). In Figure 6, a CMD2000 transfer pin was used. A 0.5-µL aliquot of 100 µg/mL cytochrome c (MW 12 361) in 5 mM NaAc at pH 6 was applied, keeping the pin in a humid environment. After washing with water to remove residual protein not taken up by interaction with the hydrogel, sinapinic acid matrix was added for MW determination. The pin was then removed from the MALDI target and washed with sinapinic acid-free matrix solvent. A 0.5-µL aliquot of 100 ng/µL trypsin (Roche Diagnostics, Mannheim, Germany) in 50 mM NH4HCO3 was added. The pin was kept at 37°C in a humid environment overnight. A 0.5-µL aliquot of matrix (10 mg/mL R-cyano-4-hydoxycinnamic acid in 50/50 v/v ACN/H2O containing 0.1% TFA) was added, and the peptide map was measured in reflectron mode. The database Analytical Chemistry, Vol. 77, No. 4, February 15, 2005
1159
Figure 4. SPR/MS of MRP14 using bifunctional fluid cell (CMD500 chip, CMD2000 transfer pin). (a) Immobilization of antibody (8.3 ng/mm2). (b) Binding of antigen (1 ng/mm2; 70 fmol/mm2). MALDI-MS on (c) sensor chip. (d) Transfer pin. (e) Pin coated in batch experiment.
search was performed with Mascot (Matrix Science, London, U.K.) in the NCBI database. TECHNICAL SETUP AND RESULTS Some SPR instruments such as those from Biacore use a flowthrough system controlling the sample homogeneity, that is, the concentration, above the affinity surface by flow rate adjustments. A loop limits the available volume of applied solutions. Although sample solutions could be reused, they have to be recovered and reinjected, which takes up time and disturbs the experiment. In contrast, IBIS instruments avoid concentration gradients above the sensor chip over extended periods of time using an automatic dispenser, which performs slight in/out pumping of the sample in the measuring chamber (principle of jet-to-wall). In this way, the sample is used very efficiently and the kinetic measurement is not affected.35,36 This particular geometry of the IBIS cuvette allowed us to insert two removable bioactive pins for MS (Figure 1). The pins are located close to the sensor chip surface, and they are exposed to the same experimental conditions as the chip itself. They can be coated with surfaces different from those used for the SPR experiment, and in this way, it is possible to individually optimize the surface properties for SPR and MS. To measure functionalized chips or pins with MS, the probe target had to be modified to hold the respective analyte carrier. For direct measurement from Biacore or IBIS chips, it was sufficient to fit the chips into the stainless steel plate (Figure 2a). The chips are then kept in place by clear tape. Conductivity has (35) deMol, N. J.; Plomp, E.; Fischer, M. J.; Ruijtenbeek, R. Anal. Biochem. 2000, 279, 61-70. (36) Ward, L. D.; Winzor, D. J. W. Anal. Biochem. 2000, 285, 179-193.
1160 Analytical Chemistry, Vol. 77, No. 4, February 15, 2005
to be ensured between the metal target and the hydrogel/goldcoated sensor chips applying silver conductive paste (Conrad). The transfer pins were manufactured from commercial screws. Accordingly, the MALDI target was modified to provide screw threads where they could be mounted (Figure 2b). It is important that only the top of the pin is coated with hydrogel (Figure 2c, top). In cases where the CMD covers the whole pin, analyte and matrix spread down over the entire screw thread and are partially lost for analysis (Figure 2c, bottom). Therefore, the pins were inserted into a special holder, which was built from gold-coated stainless steel. Alternatively and more simple, the pins can be screwed into a reaction vial. The SPR sensor disk used in the IBIS instrument provides six measuring points across the disk, while there are four points on a Biacore chip. However, the chips are otherwise fully coated, and those points are only separated by physical barriers when the chips are docked in the SPR instrument. Difficulties occur at preparation of these chips for MS, because some cross-contamination and analyte dilution among the measuring points is unavoidable when matrix is applied. Due to the high surface energy of hydrogels, solvents tend to spread out over the whole surface taking locally applied analyte from the SPR measuring points with it. Several measures have been discussed to improve on this.20 For the transfer pins, the problem does not arise, because one pin represents one particular experiment. We also noted a dependence of the MS signal on the time the matrix was allowed to interact with the hydrogel. The application of the high-organic matrix solution lowers the pH on the surface and breaks the affinity bond. As described before,31 the protein is desorbed from the gel to the surface and can be cocrystallized
Figure 5. SPR/MS of hb-FABP using bifunctional fluid cell (CMD6 chip, CMD2000 transfer pin). (a) Immobilization of antibody (7 ng/mm2). (b) Binding of antigen (0.3 ng/mm2; 20 fmol/mm2). MALDI-MS on (c) transfer pin used in (a) and (b). (d) Sensor chip of comparative experiment.
Figure 6. MAC-MS for cytochrome c uptake into CMD2000. Bottom left: Linear MS for MW determination. Middle: Reflectron-measured peptide map after tryptic digestion. Top right: Mascot search identifies several cytochrome c homologues.
with matrix, which we confirmed with SPR experiments (not shown). Conclusively, diffusion effects need to be considered, and we now allow incubation in a humid environment for ∼10 min, depending on the hydrogel. To test the bifunctional cuvette and the transfer pins, several experimental combinations have been evaluated. First, an affinity experiment was performed using the standard protein BSA and its immobilized monoclonal antibody to demonstrate the off-line binding procedure and the MS measurement of noncovalently bound BSA on a CMD2000 transfer pin (Figure 3, top). Impor-
tantly, it could be shown in the control experiment without antibody that nonspecific binding due to electrostatic or hydrophobic interactions of the protein with the hydrogel can be kept at an absolute minimum, adjusting buffer conditions with respect to the isoelectric point of the proteins (Figure 3, bottom). In a second experiment, a more interesting pair of binding partners was chosen to demonstrate the SPR response and the MS signal from an CMD500 chip and a CMD2000 transfer pin using the bifunctional IBIS cuvette (Figure 4a-d). For comparison, an additional pin was reacted in the identical way in an Eppendorf Analytical Chemistry, Vol. 77, No. 4, February 15, 2005
1161
vial and the MS signal is shown in Figure 4e. Interestingly, the MS response on chip (Figure 4c) and pin (Figure 4d) is comparable, notwithstanding their different surface coating, while the batch experiments demonstrates the expected improvement in signal intensity (Figure 4e). This was also observed for the interaction of FABP with its immobilized antibody (CMD6 chip, Figure 5d; CMD2000 pin, Figure 5c). Therefore, it appears that there are processes during the binding experiment in the cuvette that are not fully understood yet, and the result suggests that inserted pins with CMDs of lower capacity may give little MS signal and not be suitable for use in the bifunctional cuvette at all. Investigations to clarify those issues are underway. Finally, in preparation for proteomic approaches, where the identification of proteins is sought via their enzymatic digest as was already shown with other commercial chips,37,38 we have also developed a method to address this issue. It was possible to obtain MS results for both the intact cytochrome c and its peptide map in sequence on one sample from a CMD2000 pin (Figure 6). The database search confidently found cytochrome c homologues with these data. It is understood that binding experiments would be harder to follow with the described protocol, because the immobilized antibody will interfere with peptide mapping. However, subtractive approaches can be used to a certain extend eliminating peptides resulting from the known antibody. (37) Zhukov, A. S. M.; Jansson, O ¨ .; Areskoug, D.; Buijs, J. J. Biomol. Tech. 2004, 15, 112-119. (38) Kussmann, M. N., E.; Rahbeck-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601.
1162
Analytical Chemistry, Vol. 77, No. 4, February 15, 2005
CONCLUSION A bifunctional SPR fluid cell for SPR/MS has been introduced above. It represents a convenient way of combining both techniques, thus solving the dilemma of different surface requirements of optical sensors and MALDI-MS. Sample handling is simplified, and probes can be optimized separately. With the proposed cuvette design, the SPR experiment is carried out in the regular manner with the added functionality of preparing the MS probe. Not only is it possible to generate kinetic and mass data in one combined experiment, but the results of the experiments also influence each other. Complex protein profiles detected with MS might indicate unspecific binding and lead to further optimization of the SPR experiment. On the other hand, a lack of signal in SPR can explain unsuccessful MS measurements. The use of removable transfer pins adds flexibility to MAC-MS in that various surfaces can be used at low cost compared to commercial sensor chips. It further allows easy access to bioaffinity MS experiments when no SPR instrument is available. Moreover, we find the transfer pins very useful for experiments involving CMDs modified in such a way that surfaces present chromatographic properties (C18 or ion exchangers) for profiling experiments. ACKNOWLEDGMENT The work was supported by the Interdisciplinary Center for Clinical Research Mu¨nster. This work is part of the Ph.D. theses of J.G. and N.D. Received for review July 1, 2004. Accepted November 12, 2004. AC049033D
