Anal. Chem. 1997, 69, 4363-4368
Surface Plasmon Resonance Biomolecular Interaction Analysis Mass Spectrometry. 1. Chip-Based Analysis Randall W. Nelson*,† and Jennifer R. Krone†
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604 Osten Jansson
Biacore AB, Uppsala, Sweden
The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in concert with surface plasmon resonance-based biomolecular interaction analysis (SPR-BIA) is reported. A chip-based biosensor unit was used to simultaneously monitor biomolecular interactions taking place on four different regions of the sensor chip (flow cells). Species retained during SPR-BIA were then identified by performing MALDI-TOF directly from within the area of the flow cells. Analyses were performed on an antibody/antigen/antibody system with detection limits in the low-femtomole range. The combined assay demonstrates the use of SPRBIA to evaluate the relative stability of sequential solutionphase interactions, as well as, upon MALDI-TOF analysis, the ability to unambiguously confirm the presence of species retained during the interaction analysis. Critical to the detailed understanding of biomolecular processes (e.g., structure/function relationship, biomolecular recognition events, and receptor/ligand interaction) are analytical techniques capable of rapid, sensitive, and accurate analyses. Two such techniques, surface plasmon resonance-based biomolecular interaction analysis (SPR-BIA)1 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF),2 have over the past decade evolved to the point of routine application in the biological sciences. The first of these techniques, SPR-BIA, is used primarily in the characterization of noncovalent interactions between surface-immobilized ligands and solution-borne analytes. Analyses can be performed as a function of analyte availability (by varying analyte concentration) in order to evaluate association and dissociation kinetics, from which the overall affinity constant(s) of the interaction may be derived.3 SPRBIA also finds application in the mapping of epitopes involved in antigen/antibody interaction,4 monitoring the actions of DNA polymerases and reverse transcriptases,5 and the screening of † Present address: Intrinsic Bioprobes Inc., 2009 E. 5th St., Ste. 11, Tempe, AZ 85281. (1) Lo¨fås, S.; Malmqvist, M.; Ro ¨nnberg, I.; Stenberg, E.; Liedberg, B.; Lundstro¨m, I. Sens. Actuators 1991, B5, 79-84. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Myszka, D. G. Curr. Opin. Biotechnol. 1997, 8, 50-57. (4) Malmqvist, M. Methods 1996, 9, 525-532. (5) Buckle, M.; Williams, R. M.; Buc, H; Negroni, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 889-894.
S0003-2700(97)00538-6 CCC: $14.00
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small molecules as potential therapeutics.6 The second technique, MALDI-TOF, has found much use in the accurate weighing of virtually all classes of polypeptides and, to a lesser degree, nucleic acids and carbohydrates. This ability has made possible the routine application of MALDI-TOF in the verification or identification of protein sequenceseither by direct molecular weight determination or through methods such as mass mapping7 and protein ladder sequencing.8 Other applications of MALDI-TOF extend into the investigation of higher order protein structure, for example, protein/ligand interactions9 or conformational differences in allosteric proteins.10 Although SPR-BIA and MALDI-TOF have each found appropriate (and independent) applications in bioanalytical chemistry, when brought together, the combination of the two affords an extremely powerful approach to biomolecular characterization. The novelty of such biomolecular interaction analysis mass spectrometry (BIA/MS) stems from the fact that the component techniques operate on mutually exclusive detection principles and address different facets of biomolecular characterization: SPRBIA is a nondestructive, optical-based biosensor technology used in interaction kinetic/affinity evaluations; MALDI-TOF is a destructive, vapor-phase ion technology used in structural analysis. Thus, performance of MALDI-TOF on analytes retained during SPR-BIA yields an analysis capable of determining the kinetic and affinity parameters of the interaction, as well as unambiguously identifying the (bound) binding partners. Accordingly, we previously investigated the coupling of SPR-BIA with MALDI-TOF mass spectrometry.11 The exploratory studies were centered on a wellcharacterized antibody/antigen system addressing the peptidebased toxin, myotoxin a. An approach was taken in which retained analytes were mass spectrometrically analyzed directly from the sensor surface used for SPR-BIA. A chip-based biosensor capable of simultaneously supporting multiple (up to four) SPR analyses on a single sensor chip was used in the studies. In brief, the (6) Taremi, S. S.; Prosise, W.; Windsor, W. T.; Rajan, N.; O-Donnell, R. A.; Le, H.V. Biochemistry 1996, 35, 2322-2331. (7) Nelson, R. W.; Dogruel, D.; Krone, J. R.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 1380-1385. (8) Chait, B. T.; Wang, R.; Beavis, R. C., Kent, S. B. H. Science 1993, 262, 89-92. (9) Cohen, S. L.; Ferre-D’Amare, A. R.; Burley, S. K.; Chait, B. T. Protein Sci. 1995, 4, 1088-1094. (10) Lewis, J. K.; Nelson, R. W. In preparation. (11) Krone, J. R.; Nelson, R. W.; Dogruel, D.; Williams, P.; Granzow, R. Anal. Biochem. 1997, 244, 124-132.
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studies demonstrated the ability to view the retention of compounds from a natural biological carrier (snake whole venom) in real-time, followed by the unambiguous determination of the retentate at a subpicomole level (20 fmol). Also demonstrated was the ability to individually target the spatially resolved flow cells present on the biosensor chip. Given here are further uses of chip-based BIA/MS in the investigation of a multiple component affinity interaction system. An affinity system of polyclonal antihuman myoglobin IgG/human myoglobin/monoclonal anti-human myoglobin was investigated. Central points of investigation were the ability to detect multiple retained analytes (both serially during SPR-BIA and simultaneously during MALDI-TOF), the ability to detect analytes over a moderate dynamic range (both in amount and molecular weight), and the optimization of experimental parameters used in sample preparation and data acquisition. EXPERIMENTAL SECTION Chip-based SPR-BIA analyses were performed on a rabbit polyclonal anti-human IgG/human myoglobin/monoclonal antihuman myoglobin system using a Pharmacia Biosensor (now Biacore AB) BIAcore 2000 (Uppsala, Sweden). Stabilizing agents were washed from a CM5 (carboxylated dextran) sensor chip by rinsing the entire surface of the chip with five successive 200 µL aliquots of distilled water. The chip was then immediately introduced into the biosensor. All four flow cells of the sensor chip were activated through exposure to N-hydroxysuccinimide [0.1 M prepared in 20 mM HEPES, 0.005% surfactant P20, 150 mM NaCl, 3 mM EDTA, pH 7.4 (HBS) containing 0.1 M Ν′-ethylN′-[(dimethylamino)propyl]carbodiimide], derivatized by exposure to rabbit polyclonal anti-human myoglobin IgG (0.05 mg/mL in 5 mM sodium maleate buffer, pH 6.0) and then blocked by exposure to 1 M ethanolamine hydrochloride (in HBS adjusted to pH 8.5). The flow cells were monitored simultaneously using SPR during all steps of the derivatization process. Individual flow cells were exposed to human myoglobin (400 ng/mL, prepared in HBS with 40 mg/mL human serum albumin, HSA) for times ranging from 0 to 15 min, followed by exposure to monoclonal anti-human IgG (0.001 mg/mL, prepared in HBS) for 3 min. The sensor chip was then removed from the biosensor and immediately (before allowing sufficient time to dry) rinsed with two successive 200 µL aliquots of distilled water. The chip was stored at ambient conditions until preparation for mass spectrometry. The chip was prepared for mass spectrometry by applying 100-200 nL of sinapinic acid matrix (∼50 mM dissolved in 1:2 acetonitrile/1.5% trifluoroacetic acid) to each of the four flow cells using a thin-gauge wire. The matrix was allowed to air-dry before chips were inserted into a MALDI-TOF mass spectrometer. The mass spectrometer used in these studies was home-built, equipped with a nitrogen laser, and of simple, linear design (flight distance ∼1.5 m) (further details of the mass spectrometer can be found in ref. 13). Samples inserted into the instrument resided on a two-dimensional (and rotary) translation stage. Different areas of the sensor chips were targeted for analysis by either translation or rotation of the chip (under a stationary laser spot) or, alternatively, by movement of the laser spot (while the chip remained stationary). MALDI-TOF mass spectrometry was performed by targeting each flow cell with the laser individually while (12) Jansson, O.; Krone, J. R.; Nelson, R. W. In preparation. (13) Nelson, R. W.; Krone, J. R.; Dogruel, D.; Tubbs, K. A.; Granzow, R.; Jansson, O. Techniques in Protein Chemistry VIII; Academic Press: San Diego, CA, 1997; pp 493-504.
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viewing the operation using a video camera. Time-of-flight spectra were acquired in the positive-ion mode (using a 500 Ms/s transient recorder) with the instrument operating in a continuous-extraction mode (at 30 kV acceleration potential). All spectra shown were the signal average of multiple (∼50) laser desorption/ionization events and were calibrated using an equation generated by equating the flight times and mass-to-charge (m/z) values of the singly and doubly charged signals of horse heart cytochrome c (MW ) 12 360.7: the calibration sample was prepared on a discrete area of the sensor chip). RESULTS Figure 1 shows a general overview of the chip-based BIA/MS technique. Microfluidics capable of precisely delivering nanoliter volumes of solution are used to route derivatization agents, or analyte, through individual flow cells (either in parallel or serially) present on the surface of the sensor chip. SPR is used to simultaneously monitor all flow cells throughout each step in the ligand immobilization process, as well as in the evaluation of interactions between the surface-immobilized ligand and solutionborne analytes. Data derived from SPR-BIA can then be used in the evaluation of ligand surface density and analyte retrieval from solution as a function of time, the real-time data affording information on the association and dissociation parameters of the interaction. Upon removal of the sensor chip from the biosensor, an appropriate matrix is applied within the confines of each flow cell and species retained during BIA analyzed directly from the surface of the chip using MALDI-TOF mass spectrometry. Flow cells are targeted for analysis individually with the mass spectral data yielding qualitative information on the retained analytes. The chip-based biosensor used during these studies was capable of supporting the simultaneous analysis of four individual flow cells. Figure 2 shows sensorgrams resulting from the serial derivatization (activating, derivatization, and blocking chemicals flowed from one flow cell through the next) of the four flow cells present on the SPR sensor chip. The three plateaus represent the activation of the sensor surface, the immobilization of the polyclonal anti-human myoglobin, and the blocking of remaining active sites. The loading capacity of each flow cell (maximum possible amount of analyte that can be bound) can be estimated from the change in resonance response observed throughout the derivatization process. A change in resonance response of ∆RU ∼ 12 500 is observed for all flow cells indicating the covalent immobilization of ∼85 fmol of antibody IgG. Considering an antibody valence of 2, a maximum loading capacity of ∼170 fmol is estimated for each flow cell. Figure 3 shows sensorgrams resulting from the parallel incubation of the flow cell surfaces with human myoglobin in the HSA carrier (for various amounts of time), followed by incubation with monoclonal anti-human myoglobin IgG. Flow cells 1-3 were incubated with myoglobin and antibody, while flow cell 4 was reserved as a control to check for leakage between the flow cells (the flow cell was not exposed to either myoglobin or antimyoglobin). Changes in resonance response of ∆RU ) 1900, 1800, and 180 indicate ∼110, 105, and 10 fmol of myoglobin retained in flow cells 1, 2, and 3, respectively. The sensorgram for flow cell 4 shows no net change in response. The flow cells were next exposed to monoclonal anti-human myoglobin IgG. Overall response changes of ∆RU ) 11 780, 11 100, and 3800, indicate 78, 74, and 25 fmol of secondary antibody IgG retained
Figure 1. General scheme of chip-based SPR-BIA/MS. Microfluidics capable of nanoliter delivery of solution are used to route solution through flow cells either serially or in parallel (each flow cell has the dimension of 0.5 mm × 2.0 mm). SPR is used to monitor biospecific interactions as a function of time, from which the association and dissociation parameters of the interaction may be determined. MALDI-TOF analysis of the flow cells directly from the surface of the sensor chip is then used to evaluate analytes retained during BIA.
Figure 2. Sensorgrams of polyclonal anti-human myoglobin immobilization using an amine-coupling procedure (sensorgrams are reported in terms of resonance units (RU) with 1000 RU ) 1 ng of material retained within the confines of the flow cell). All four flow cells were derivatized with polyclonal anti-human myoglobin IgG to levels of ∼12 500 RU (∼12.5 ng; ∼85 fmol of antibody/flow cell). Activation, immobilization, and blocking steps are as indicated.
in flow cells 1, 2, and 3, respectively. Again, no change in response is observed for flow cell 4. Figure 4 shows MALDI-TOF mass spectra resulting from the targeting of the individual flow cells. Spectra obtained from flow cells 1 and 2 exhibit strong ion signals for myoglobin (m/z ) 17 200 Da) and the anti-myoglobin IgG (m/z ) 144 500 Da). The myoglobin and anti-myoglobin signals are also observed in the spectrum obtained from flow cell 3, albeit with less intensity and a lower signal-to-noise ratio. The blank spectrum obtained from flow cell 4 indicates no discernable seepage between flow cells
(no signals consistent with myoglobin) or reversal of the antibody coupling procedure (no signals consistent with the antibody). No additional artifacts (e.g., due to retention of nontargeted species) are observed in any spectra. DISCUSSION Sample Preparation/Data Acquisition. The data shown in Figures 2-4 are representative of sensorgram sets and ∼60 mass spectra (∼5 spectra/flow cell, per sensor chip), obtained from replicate experiments utilizing three different sensor chips. In Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
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Figure 3. Sensorgrams showing the retention of human myoglobin followed by the retention of secondary (monoclonal) anti-myoglobin IgG. Myoglobin was retained at levels ranging between 110 and 10 fmol, whereas monoclonal anti-myoglobin IgG was retained at levels ranging between 78 and 25 fmol. Dissociation rates of 1 × 10-4-5 × 10-4 s-1 were estimated from the data.
Figure 4. MALDI-TOF mass spectra of analytes retained during the SPR-BIA analysis of the myoglobin/anti-myoglobin system. The spectra were generated directly from the surface of the sensor chip by targeting each of the individual flow cells. Ion signals consistent with the retention of human myoglobin (m/z ) 17 200 Da) and antihuman myoglobin IgG (m/z ) 144 500 Da) were observed in spectra obtained from flow cells 1-3 (see text for amounts). Ion signals were not observed from the blank flow cell.
general, a high experimental reproducibility was experienced throughout the replicate experiments provided that requisite sample preparation and analytical procedures were followed. First and foremost, it was found necessary to remove stabilizing agents from the surface of the sensor chip in order to readily achieve ion signal during MALDI. The sensor chips are coated with a relatively large amount of stabilizing agent that was found to interfere with the MALDI process. Removal of the stabilizing agents prior to BIA (by successive water rinses) made possible the repetitive and reproducible generation of ion signal from the surface of the sensor chip during MALDI. The second rinsing proceduresthe water rinse following the BIA (after the chip was undocked from the biosensor)swas not as critical for generating ion signal; however, it was necessary to minimize mass spectral contributions due to surfactants present in the running buffer. The (commercially available) running buffer normally used with the biosensor units contains a significant amount of poly(ethylene glycol)-based surfactant. Without the final rinse, residual buffer 4366 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
components left on the chip after BIA were observed in the MALDI-TOF mass spectra. A second issue of sample preparation regards matrix application. Matrix was applied to the surfaces of the individual flow cells using a thin wire. This procedure was found preferable to matrix application using a micropipetter because surface adhesive forces (toward the sensor chip) were sufficient enough to draw an excess amount of matrix solution from the micropipetter (compromising the spatial resolution of the flow cells). Overall, matrix application using a thin wire was sufficient in the routine preparation of samples on the sensor chips. However, other, more sophisticated (and expensive) approaches to controlled matrix application have been explored, with the methods showing the most promise being matrix delivery using either a piezoelectrically driven dispenser, or a micro syringe. In either case, defined regions inside the dimensions of the flow cells could be addressed discretely with different spots of matrix (i.e., up to six discrete matrix spots could be placed within the confines of an individual flow cell), thereby allowing MALDI analysis of retained analyte using a variety of matrices. A full report is forthcoming.12 Finally, because of the small amounts of sample present within the flow cells (and the comparably small amounts of applied matrix), ion signal was found to be fleeting, lasting on any one targeted spot from 20 to 100 laser shots. As such, and considering the possibility of ablating sample from the sensor chip when not actually acquiring data, the laser was not rastered over sample surface while at a high intensity. A general procedure of targeting a sample spot, increasing laser irradiance while acquiring signal, and then lowering laser irradiance prior to moving to a new spot was followed. Data Evaluation. (1) Estimate of Valence. The amount of myoglobin retained in flow cells 1-3 corresponds to, at most, an occupancy of 65% (110 fmol/170 fmol) of primary antibody valence. The less than unity retention of myoglobin is not unusual because of unavoidable variations in antibody orientation experienced during immobilization using the amine-coupling procedure. The amount of secondary antibody retained in flow cells 1 and 2 corresponds to ∼70% of the amount of myoglobin present in the flow cells. The amount of antibody retained in flow cell 3,
however, corresponds to roughly 250% of the amount of myoglobin present in the flow cell. Obviously, some form of nontargeted retention is responsible for the abnormally high reading (such nontargeted interaction having the most influence during the analysis of small quantities of myoglobin). Assuming that nonspecific binding contributes universally to the response observed from flow cells exposed to the antibody, a simple linear fit of the data can be used to estimate the relative contribution due to background interactions. Considering data obtained from flow cells 1-3, the binding efficiency of the secondary antibody is estimated to be ∼50%, with a background reading (due to nontargeted secondary antibody) of ∼3000 RU (∼20 fmol of IgG). Background interactions of this nature, with approximately the same response and effect, were encountered in the replicate experiments, suggesting the possibility of secondary antibody interacting with an element of the sensor surface (including the primary antibody). An alternate explanation might be the retention of a low molecular weight fragment of the myoglobin, which possesses the epitope targeted by the secondary antibody. The retention of such small fragments has been observed in previous BIA/MS studies of a similar antigen/antibody system,13 with the origin of the species being a breakdown product of the analyte. The mass spectra shown in Figure 4, however, do not indicate the retention of any such low molecular species, suggesting interaction of the secondary antibody with a component of the sensor surface. (2) Complex Avidity. The SPR-BIA data shown in Figure 3 does not support the rigorous determination of the association rates involved in the formation of the antibody/antigen complexes [because of the high levels of analyte bound, the presence of a second biological carrier (HSA), and because analyses at variable analyte concentration were not performed]. Therefore, the determination of the overall affinity constants of the interactions is not possible. It is possible, however, to estimate the dissociation rates of the complexes once formed (because of the zero-order nature of the dissociation). As gauged using the evaluation software supplied with the biosensor, the off-rates of the two interactions (primary antibody/antigen and antigen/secondary antibody) fall in the same order of magnitude: dissociation rate ∼1 × 10-4-5 × 10-4 s-1. In general, a measure of dissociation rates is of some value (even without accompanying association rate data) because the rates are an indication of avidity of the complexes, which in turn governs the availability of analyte (after SPR-BIA) for MALDI-TOF analysis. That is, analyte as part of a low-avidity (rapid dissociation rate) complex can be readily washed from the sensor chip and not be available for MALDI-TOF analysis. Alternatively, analyte as part of a (relatively) high-avidity complex is retained during BIA (and rinses) and available for MALDI-TOF analysis (as demonstrated in the analyses reported here). In a different role, measurement of the avidity of a complex is important when correlating the observance of noncovalently bound species in the vapor phase (during MALDI-TOF analysis) with true biospecific interactions. It is generally thought that noncovalently bound complexes are not of sufficient strength to sustain throughout the sample preparation and laser desorption/ionization steps of MALDI. It is possible, however, to generate noncovalent complexes during MALDI-TOF analysis given the appropriate circumstances (high quantities of analyte, high laser irradiances, appropriate matrix conditions). As of yet, very few of the these
Figure 5. Enlargement of the MALDI-TOF mass spectrum obtained from flow cell 1. Signals consistent with myoglobin (m/z ) 17 200 Da), anti-myoglobin IgG (m/z ) 144 500 Da), and the antigen/antibody complex (m/z ) 161 700 Da) were observed. Myoglobin and antimyoglobin IgG were retained within the flow cell (during SPR-BIA) at levels of 110 and 78 fmol, respectively.
complexes are known to be of any biological relevance. Possible exceptions are functional proteins comprised of multiple subunits of the same or a limited number of polypeptide species, for example, the membrane protein porin14 or the immune complement C1q.15 An interesting detail of the studies reported here was the observation of antigen/antibody complex in the vapor phase. Figure 5 shows an expansion of Figure 4 (flow cell 1). Clearly, ion signal is observed for the myoglobin/anti-myoglobin complex at m/z ∼ 161 600 Da. Due to the limited nature of the present studies, the exact nature of the antigen/antibody complex (i.e., the fortuitous vapor-phase interaction or a sustained biomolecular interaction) remains to be discerned. A relatively low dissociation constant, between the myoglobin and monoclonal IgG (5 × 10-4 s-1), suggests the possibility of true biomolecular interactions sustained in the vapor phase. Without proper controls, however, the generation of fortuitous vapor-phase clusters can be as strongly argued (especially when considering that the interaction between the myoglobin and the anchored primary antibody is just as strong). Regardless, SPR-BIA/MS seems ideally suited for the rapid screening of sustained vaporphase complexes as a function of solution-phase affinity/avidity (determination of solution-phase affinity vs observation of complex in the vapor phase) and further studies are certainly warranted. CONCLUSION The use of SPR-BIA/MS to analyze sequential biomolecular recognition events has been demonstrated. Polyclonal anti-human myoglobin IgG was covalently immobilized to a sensor surface and used to specifically address human myoglobin (as retained from solution). A secondary (monoclonal) antibody IgG was then introduced for binding to the retained myoglobin. SPR-BIA analysis yielded quantitative information on the sequential recognition events, indicating a depletion of primary antibody valence (upon immobilization), and the nonspecific retention of secondary antibody. Further, SPR-BIA was used to estimate the relative (14) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (15) Nelson, R. W.; Krone, J. R.; Dogruel, D.; Tubbs, K. A. New Methods for the Study of Molecular Aggregates; Kluwer Academic Press: Norwell, MA, in press.
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avidity between the myoglobin and either of the antibodies. MALDI-TOF mass spectrometry was used to confirm the retention of both binding partners (myoglobin and monoclonal anti-myoglobin IgG) and the apparent lack of other species responsible for the background retention of the secondary antibody (suggesting the background retention due to secondary antibody interaction with an element of the sensor surface). Ion signals consistent with the myoglobin/monoclonal anti-myoglobin IgG were also observed in the mass spectra, indicating the possibility of sustained biospecific vapor-phase interaction throughout the MALDI process. Investigations covered a moderate dynamic range (∼1 decade) with respect to both analyte molecular weight
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and quantity, and limits of detection in the low (10-25) femtomole range were experienced. ACKNOWLEDGMENT The financial and technical support of Biacore AB has been graciously appreciated. Data presented in part at the 6th Biasymposium, Washington, D.C., October 8-10, 1996. Received for review May 27, 1997. Accepted August 25, 1997.X AC970538W X
Abstract published in Advance ACS Abstracts, October 1, 1997.
