Anal. Chem. 1998, 70, 2731-2736
Combining MALDI Mass Spectrometry and Biomolecular Interaction Analysis Using a Biomolecular Interaction Analysis Instrument Carsten P. So 1 nksen,† Eckhard Nordhoff,† O 2 sten Jansson,‡ Magnus Malmqvist,‡ and ,† Peter Roepstorff*
Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense, Denmark, and Biacore AB, Rapsgartan 7, S-754 50 Uppsala, Sweden
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with biomolecular interaction analysis (BIA) in a Biacore instrument. A method has been developed for the recovery of the affinity-bound molecules from the sensor chip in a few microliters ready for mass spectrometric analysis. The procedure is illustrated with two molecular systems which exemplify antibody-antigen and DNA-protein interactions. In both cases, femtomole quantities of the affinitybound proteins were eluted and subsequently detected by MALDI-MS. Whereas the Biacore analysis yields the surface concentration of protein bound to the sensor chip, identity of the bound compounds is revealed in the second step by accurate molecular mass determination. Combining the information of the two analyses allows calculation of the total surface molar concentration of affinitybound molecules. Biomolecular interaction analysis (BIA) is an affinity-based biosensor technology optimized for monitoring interactions between biomolecules in real-time.1 The detection relies on the optical phenomenon of surface plasmon resonance (SPR), which can be used to detect changes in the refractive index close to the sensor chip surface. These changes are displayed as response units (RU) versus time in a sensorgram and are in linear correlation to changes in surface concentration for molecules with approximately the same refractive index, e.g., as for proteins. For proteins, a specific response correlation factor of close to 1000 RU per ng of protein/mm2 was found for surface concentrations ranging from 2 to 50 ng/mm2.2 However, the specific correlation factor for nucleic acids has not been determined in Biacore instruments. In a typical experiment, a specific interactant (ligand) is first immobilized on the carboxymethyl-dextran/gold surface of the sensor chip. In the next step, analyte solution is passed over the sensor chip, and binding of analyte molecules to the ligand is †
Odense University. Biacore AB. (1) Jo ¨nsson, U.; Fa¨gestam, L.; Ivarsson, B.; Johnsson, B.; Karlsson, R.; Lundh, K.; Lo ¨fås, S.; Persson, B.; Roos, H.; Ro¨nnberg, I.; Sjo¨lander, S.; Stenberg, E.; Ståhlberg, R.; Urbaniczky, S.; O ¨ stlin, H.; Malmqvist, M. BioTechniques 1991, 11, 620-627. (2) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513-526.
monitored. When an interaction cycle is finished, the active surface of the sensor chip can be regenerated by standard procedures such as employed in affinity chromatography. Applications of BIA analysis include the study of proteinprotein and protein-nucleic acid interactions as well as the binding of signaling substances and drugs to proteins or nucleic acids.3,4 In such experiments, quantitative information of kinetic and affinity constants can be obtained. Examples for the use of BIA are elucidation of the chaperone mechanism, investigation of the gene expression mechanism for a number of DNA-binding proteins, epitope mapping, and the kinetic analysis of antigenantibody interactions.3,5 According to system specifications, myoglobin can be detected in concentrations down to 10 pM under favorable conditions in a sandwich assay. Mass spectrometry is one of the most sensitive and specific techniques for the identification and characterization of biomolecules, especially for peptides and proteins.6 Two techniques have proven efficient for the analysis of large biomolecules, i.e., matrixassisted laser desorption/ionization mass spectrometry (MALDIMS) and electrospray ionization mass spectrometry (ESI-MS). MALDI-TOF-MS was exclusively used in this study, due to its high sensitivity and relative tolerance for contaminants such as salts, detergents, and buffer components. The routinely achieved sensitivity for analysis of peptides and proteins by MALDI-MS is typically in the low- to mid-femtomole range. The mass accuracy is highly dependent on the molecular mass. For peptides, mass accuracy in the 10-50 ppm range can be obtained.7 For proteins up to 20 kDa, 50-100 ppm is obtainable if sample heterogeneity does not degrade the signal resolution.6,7 However, above 30 kDa, the mass accuracy frequently drops to 0.1-0.2%. For molecules with molecular masses in the range 200-50 000 Da, the quantities that can be bound to the BIA chip are the same order of magnitude as those typically needed for mass spectrometric analysis, indicating that the coupling of the Biacore with MALDI-MS or ESI-MS is possible. However, it has to be considered that the sensitivity of BIA analysis increases with increasing molecular mass of the analyte whereas, in general, the
‡
S0003-2700(98)00045-6 CCC: $15.00 Published on Web 05/21/1998
© 1998 American Chemical Society
(3) Szabo, A. S. L.; Granzow, R. Curr. Opin. Struct. Biol. 1995, 5, 699-705. (4) Malmqvist, M.; Karlsson, R. Curr. Opin. Chem. Biol. 1997, 3, 378-383. (5) Johne, B. G. M.; Hansen, K. J. Immunol. Methods 1993, 160, 191-198. (6) Roepstorff, P. Curr. Opin. Biotechnol. 1997, 8, 6-13. (7) Jensen, O. N.; Podtelejnikov, A.; Mann, M. Rapid Commun. Mass Spectrom. 1996, 10, 1371-1378.
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sensitivity of both MALDI- and ESI-MS decreases with increasing mass. One strategy for combining mass spectrometry and BIA is to use the sensor chips as MALDI-MS target as recently demonstrated by Krone et al.8 and Nelson et al.9 In this approach, the analyte is bound to the ligand present on the sensor chip and washed in the Biacore instrument. Subsequently, the sensor chip is taken out, matrix solution is applied to the active surface, the chip is loaded into the MALDI mass spectrometer, and the bound molecules are analyzed. A similar approach has been used to perform MALDI mass spectrometric analysis of the molecules bound to the external fiber-optic sensor probe of the Biacore Probe instrument.10 In the approach reported here, the bound analyte is instead eluted from the chip inside the BIA instrument and then analyzed by MALDI-MS. The interactions studied include binding of myoglobin to an immobilized monoclonal IgG directed against human myoglobin and capturing of the DNA-binding protein ParR by a biotinylated double-stranded (ds) DNA probe bound to the sensor chip via biotin-streptavidin interaction. Detection of myoglobin (17 kDa) is important as a clinical trial for the diagnosis of acute myocardial infarction.11 In Escherichia coli cells, protein ParR (13 kDa) binds cooperatively to 11 nucleotide direct repeats of the parA promotor partition site parC of plasmid R1. If the direct repeats are occupied by ParR, its own expression and the expression of the protein parM are suppressed. Both proteins take part in the partitioning of the plasmid R1 upon cell division.12,13 EXPERIMENTAL SECTION Materials. Certified Biacore running buffer (HBS) containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% of the surfactant polyoxyethylenesorbitan (P20, Biacore A/B), sensor chips CM5 and SA5, and the Biacore amine coupling kit containing N-hydroxysuccinimide, N-ethyl-N′-(3-diethylaminopropyl)carbodiimide, and ethanolamine hydrochloride, 0.5% SDS solution (v/v), 50 mM glycine buffer adjusted to pH 9.5 with NaOH and monoclonal anti-human-myoglobin IgG were obtained from Biacore AB (Uppsala Sweden). The control double-stranded DNA probe containing no specific ParR binding sites with the sequences 5′-biotin-C6-d(GCG CAG GAG TCA GTG GGC GTT GCG CCA CGA TCT CTC TCC A)-3′ 3′-d(CGC GTC CTC AGT CAC CCG CAA CGC GGT GCT AGA GAG AGG TG)-5′
and the ParR binding double-stranded DNA probe containing three direct binding repeats (indicated by boldface letters) with the sequences13 5′-biotin-C6-d(TTCCGC AAACAAAACCC AAAAACAACCC ATACCCAACCC TCGTGC)-3′ 3′-d(AAGGCG TTTGTTTTGGG TTTTTGTTGGG TATGGGTTGGG AGAACG)-5′
were custom synthesized by DNA Technology ApS (Aarhus, (8) Krone, J. R.; Nelson, R. W.; Dogruel, D.; Williams, P.; Granzow, R. Anal. Biochem. 1997, 244, 124-132. (9) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 4363-4368. (10) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 4369-4374. (11) Ishii, J.; Wang, J. H.; Naruse, H.; Taga, S.; Kinoshita, M.; Kurokawa, H.; Iwase, M.; Kondo, T.; Nomura, M.; Nagamura, Y.; Watanabe, Y.; Hishida, H.; Tanaka, T.; Kawamura, K. Clin. Chem. 1997, 43, 1372-1378. (12) Breuner, A.; Jensen, R. B.; Dam, M.; Pedersen, S.; Gerdes, K. Mol. Microbiol. 1996, 20, 581-592.
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Denmark). The used ds DNA probes were prepared following standard laboratory procedures.14 Poly [d(I-C)] was purchased from Boehringer Mannheim. Eppendorf Eurotips (No. 0030 063.619) were obtained from Eppendorf Nethelm-Hinz. Cyano-stabilized human myoglobin was obtained from DAKO A/S. The MALDI matrix used was R-cyano-4-hydroxycinnamic acid (HCCA) purchased from Sigma. The following solutions were used: HCCA solution I, 20 µg/µL in acetonitrile/0.1% trifluoroacetic acid (TFA) (70:30, v/v); HCCA solution II, 20 µg/µL in acetone/water (99:1, v/v). Methods. BIA Analysis of Myoglobin. All analyses were performed on a Biacore X instrument (Biacore A/B Uppsala, Sweden) using HBS buffer as running buffer at 5 µL/min in a continuous flow with flow cells 1 and 2 in series. Anti-myoglobin antibody was covalently immobilized in both flow cells on a CM5 sensor chip using the amine coupling method15 and monoclonal antibody dissolved in 35 µL of 10 mM sodium acetate, pH 4.7, to a concentration of 43 µM. Unspecific bound antibody was then washed off by injection of 10 µL of 2% formic acid, pH 2. Human myoglobin was specifically captured from a 40-µL sample with a human myoglobin concentration of 0.1 µM and a physiological serum concentration of 60 µM human serum albumin in HBS buffer. Human serum albumin was included as an internal control for the detection of nonspecific protein binding. A volume of ∼2 µL of this analyte solution was also analyzed by MALDI-MS prior to BIA analysis. BIA Analysis of the DNA-Binding Protein ParR. All analyses were performed on a Biacore X instrument. In this case, buffer A (25 mM Hepes, pH 8.0, 1 mM EDTA, and 50 mM NaCl) was used as running buffer at 5 µL/min in a continuous flow. Flow cells 1 and 2 were run in series except for the binding of the DNA probes, where the flow cells were addressed in separate cycles. The biotinylated ds DNA probe containing no specific binding sites for ParR (control) was captured in flow cell 1 by the immobilized streptavidin of a SA sensor chip. The ds DNA probe containing three specific binding repeats (sample) was bound in flow cell 2 on the same sensor chip. In each case, 100 ng/mL DNA dissolved in 40 µL of buffer A modified to 1 M NaCl was injected and passed over the sensor chip. Afterward, 20 µL of buffer A modified to 120 mM NaCl with ∼0.5 µM ParR 0.3 ng/µL poly [d(I-C)], and 1 µM each of insulin (porcine), myoglobin (horse) and β-lactoglobulin (variants A and B, bovine milk) was injected. The proteins insulin, myoglobin, and β-lactoglobulin were included as internal control for the detection of nonspecifically bound proteins. A volume of ∼2 µL of this analyte solution was also analyzed by MALDI mass spectrometry prior loading onto the Biacore. BIA System Cleaning. Before affinity-bound analytes were eluted from the sensor chip, a cleaning procedure of the Biacore X flow system was performed. The cleaning procedure includes the following steps: (1a) The sensor chip with the affinity-bound analyte is removed from the Biacore instrument. (1b) A blank chip is inserted. (1c) The system is washed with HBS buffer (13) Dam, M.; Gerdes, K. J. Mol. Biol. 1994, 236, 1289-1298. (14) Mandrup, S.; Højrup, P.; Kristiansen, K.; Knudsen, J. Biochem. J. 1991, 276, 817-823. (15) Johnsson, B.; Lo¨fås, S.; Lindquist, G.; Edstro¨m, Å.; Mu ¨ ller Hillgren, R.-M.; Hansson, A. J. Mol. Recognit. 1995, 8, 125-131.
extraction. The default calibration of the instrument was used for calibration. Typically 100-150 single-shot spectra were averaged. Protein samples were prepared for MALDI-MS using our previously described sandwich preparation method.16 This involves first preparation of a thin layer of matrix by adding 0.7 µL of HCCA (II) in acetone on the target. After solvent evaporation, 0.4 µL of 5% TFA was added, followed by the eluate from BIA (∼3 µL) and finally 0.4 µL of matrix solution of 20 mg/mL HCCA (I). After ∼1 min, when crystallization of the matrix was observed, a washing procedure was performed by adding 100 µL of 0.1% TFA solution to the sample droplet. A few seconds later, the remaining liquid droplet was carefully removed with a 100-µL pipet tip without removing the matrix crystals and a second wash was performed with another 100 µL of 0.1% TFA solution. The remaining solvent was then allowed to evaporate, and the sample analyzed by MALDI-MS.
Figure 1. (a) Principle of the sandwich elution, developed for subsequent mass spectrometric analysis. Typically 3 µL of elution solvent is passed over the chip, separated from the system buffer by two air bubbles. (b) Typical sensorgram of an protein elution for subsequent mass spectrometric analysis.
(“Prime” procedure). (1d) The system is washed with SDS and glycine buffer (BIA “Desorb” procedure). (1e) The injection block is removed, sonicated for 5 min in 50% ethanol, and remounted. (1f) The system is checked for cleanness by performing an elution procedure followed by mass spectrometric analysis of the eluate (see below). If the system is not clean, steps 1d-f are repeated. If no contaminants are observed, then steps 1g and 1h are performed. (1g) The blank chip is replaced by the chip with the bound analyte. (1h) A “Prime” procedure is performed with the respective running buffer. Elution of the Bound Analyte. (2a) A 100-µL pipet tip is prepared containing 20 µL of water, 5 µL of air, 3 µL of elution solvent, and 10 µL of air as shown in Figure 1a. The lower ∼25 µL is injected into the sample loop. (2b) The first ∼20 µL of the content from the sample loop is passed over the sensor chip. (2c) When 16 µL has passed the sensor chip (indicated on the screen) a 100-µL pipet tip is placed in the “Flow cell out” port and the eluate is collected until the second air bubble moves into the pipet tip. At this point, the pipet tip is removed and the content analyzed by MALDI-MS. The elution solvent in step 2a was 2% formic acid for the antimyoglobin sensor chip and buffer A modified to 1 M NaCl for the sensor chip carrying ds DNA. The amount of material eluted from the chip was calculated from the change in baseline before and after elution. A typical sensorgram of the elution process is shown in Figure 1b. Mass Spectrometric Analysis. MALDI-MS measurements were performed on a Voyager Elite mass spectrometer (Perseptive Biosystems) in positive ion linear mode using delayed ion
RESULTS Development of the Washing and the Elution Procedure. A series of initial experiments performed without rinsing the system showed a considerable carry-over from proteins bound nonspecifically to the BIA flow system. Repeated performance of the Biacore standard washing procedures, “wash”, “flush”, “rinse”, and “prime” (1-3 min), was not sufficient to remove the nonspecifically bound proteins. Consequently, a harsher washing procedure was developed comprising application of a standard “desorb” (20 min) procedure to clean the flow system combined with subsequent cleaning of the injection block. To prevent loss of the specifically bound protein, the sensor chip with the bound protein was dismounted during the desorb procedure. In the elution step, two requirements were found to be essential. First, it was necessary to be able to collect the appropriate fraction of the eluate. Second, dilution of the eluted material should be minimized. Both were achieved by sandwiching the elution solvent (typically 3 µL) between two air bubbles. The air bubbles minimized mixing of solvents and were easily detected in the sensorgram on the screen as well as at the outlet port. Based on the above-mentioned experience, the elution procedure described in the Experimental Section was developed and used in the following experiments. Detection of Human Myoglobin. The analyte solution containing human myoglobin and a 600-fold molar excess of human serum albumin was analyzed by MALDI-MS prior to BIA analysis (Figure 2a). The major peak series correspond to human serum albumin (HSA) molecular ions carrying from one to eight positive charges. Human myoglobin is not detected due to the high molar excess of HSA in the analyte solution. Upon ligand immobilization, 14.6 kRU of anti-myoglobin antibody was covalently bound in flow cell 1 and 15.0 kRU in flow cell 2. The difference in response units before and after sample transfer is 2.2 kRU in each flow cell (Figure 3). According to the change of the baseline level in the sensorgram, 2.0 kRU of protein was removed from the sensor chip upon elution corresponding to a total of 4 ng of protein (2 ng/flow cell)2. The difference of 0.2 kRU between the amount of protein originally bound and the (16) Kussmann, M.; Nordhoff, E.; Nielsen, H. R.; Haebel, S.; Larsen, M. R.; Jakobsen, L.; Gobom, J.; Mirgorodskay, K.; Kristensen, A. K.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 483-493.
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Figure 2. (a) MALDI mass spectrum obtained from 1 µL of the analyte solution containing 120 fmol of human myoglobin (17 052 Da) and 600 pmol of human serum albumin (hsa, 66.4 kDa). Myoglobin was not detected due to the large excess of human serum albumin. (b) MALDI mass spectrum of the sample eluted from the antimyoglobin antibody sensor chip. Only human myoglobin (17 052 Da) was detected. The displayed section shows the signals for singly, doubly, and triply charged molecular ions.
Figure 3. Protein-binding sensorgram from flow cells 1 and 2, both containing immobilized anti-myoglobin antibody. A volume of 40 µL of sample solution containing 4.8 pmol of human myoglobin and 24 nmol of human serum albumin were passed over the sensor chip in series. The change in baseline after the injection corresponds to 2.2 kRU of protein bound in each flow cell.
eluted amount indicates sample loss during the washing procedure in step 1h. In the mass spectrum of the eluate (Figure 3b), singly, doubly, and triply charged myoglobin ions are detected but no molecular ions for human serum albumin. This result strongly indicates that myoglobin was effectively bound to the antimyoglobin sensor chip and that this contained no nonspecific bound human serum albumin. In addition, no serum albumin was eluted from places other than the sensor chip; i.e., the sample loop and the inlet and outlet channels demonstrated the efficiency of the system cleaning procedure. The resolution of the myoglobin peaks is ∼120 fwhm, which is lower than normally observed 2734 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
for proteins in this mass range. This is most likely due to adduct ion formation with salts or the surfactant component P20 of the used system buffer (HBS buffer). In these studies, the standard system buffer was used in order to maintain the well-established standard conditions for the detection of human myoglobin by BIA analysis.17 Use of alternative buffers including omission or replacement of the surfactant P20 is currently under investigation. A potential alternative to P20 is n-octyl glucopyranoside, which has proven beneficial rather than disturbing for the detection of peptides and proteins by MALDI-MS.18,19 The immobilized antibody was tested for loss of activity caused by sample elution with 2% formic acid. After 100 elution runs, each performed with 24 µL of 2% formic acid, a loss in binding capacity of 25% was observed. Furthermore, a series of experiments were performed in order to test whether the air bubbles injected during elution affect the subsequent BIA analysis or reuse of the affinity sensor chip. A series of elutions with and without air bubbles were compared. No significant differences were observed. Thus, the binding capacity of the antibody is only marginally affected by the used elution procedure. The mass spectrometric analysis detected exclusively myoglobin in the eluate. Therefore, it can be assumed that the 2.0 kRU of protein bound per flow cell all represent myoglobin. The molar ratio between monoclonal antibody and myoglobin is calculated to 1.3. This is below the theoretical maximal ratio of 2, indicating that part of the antibody binding sites may not be available for binding. Detection of the DNA-Binding Protein ParR. Based on the immobilization sensorgram (not shown), 3.4 kRU biotinylated ds DNA was bound to streptavidin in flow cell 2 and ∼2.9 kRU in flow cell 1 (control). Prior to injection of the analyte sample into the Biacore instrument, a 2-µL aliquot was analyzed by MALDIMS (Figure 4a). Strong signals for singly and multiply charged molecular ions of myoglobin and insulin are observed. In addition, less abundant, molecular ions of β-lactoglobulin (variants A and B) and ParR are also detected. As typically observed for analysis of protein mixtures by MALDI-TOF-MS, the signal intensities for the different compounds do not reflect the quantitative composition of the analyte solution. Figure 5 shows the sensorgram of the interaction experiment, where 5.9 kRU of protein was bound in flow cell 2 containing the specific ParR binding whereas only 0.4 kRU was bound in flow cell 1 with the control DNA. Binding of a small amount ParR to the control DNA was expected since most, if not all, DNA-binding proteins with a specific recognition sequence motif also exhibit a considerable nonsequence-specific affinity for DNA due to electrostatic interactions with the deoxyribose phosphate backbone of the ds DNA. In the initial experiments, nonspecific binding was much more abundant. Therefore, poly [d(I-C)] was included in the sample solution as competitor for nonspecific binding.20 A series of Biacore experiments without subsequent mass spectrometric analysis were performed with varying concentrations of ParR, NaCl, and the competitor poly[d(I-C)] to optimize the ratio (17) Getting Started BIAcore 2000, June 1995 ed.; Biacore AB. (18) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (19) Vorm, O.; Chait, B. T.; Roepstorff, P. 41st ASMS Conference on Mass Spectrometry and Allied Topics, May 31-June 4, San Francisco, CA, 1993; p 621a. (20) Larouche, K.; Bergeron, M. J.; Leclerc, S.; Guerin, S. L. Biotechniques 1996, 20, 439-444.
signal broadening for any of the detected proteins (Figure 4a). The most likely reason for the observed degradation of signal resolution is the combination of the low signal intensity due to the small amount of eluted ParR and the high concentration of NaCl (1 M) in the elution solvent. Other sample preparation techniques such as the dried-droplet or thin-layer methods16 were also applied but did not yield any protein signals at all. Since it was suspected that the high NaCl content in the elution buffer was responsible for the peak broadening, another series of experiments were performed in which the DNA-binding protein was eluted with water/acetonitrile/triflouroacetic acid (88:10:2, v/v/v). This resulted in significantly improved peak shape but also in considerable loss of binding capacity of the sensor chip in subsequent experiments. Most likely, dissociation and degradation of the immobilized ds DNA due to the low pH of the elution solvent is responsible for the latter observation. We are currently investigating alternative elution procedures that might result in improved quality of spectra without degradation of the sensor chip.
Figure 4. (a) MALDI mass spectrum obtained from 1 µL of analyte solution containing ∼0.5 pmol of ParR (parR, 13 325 Da) and 1 pmol of each insulin (ins, 5772 Da), myoglobin (myo, 16 951 Da), and β-lactoglobulin (lac, variant A 18 363 Da; variant B 18 277 Da). (b) MALDI mass spectrum of the protein fraction eluted from both flow cells of the used ds DNA sensor chip.
Figure 5. Protein-binding sensorgram from flow cells 1 and 2 containing immobilized ds DNA. In flow cell 1, each ds DNA probe contains three direct binding repeats for the protein ParR (ParR specific) whereas in flow cell 2 no binding repeats are present (control). A volume of 20 µL of sample solution containing 0.5 pmol of ParR and 1 pmol each of insulin, myoglobin, and β-lactoglobulin was passed over the sensor chip
between specific and nonspecific binding. A sample solution with ∼0.5 µM ParR, 120 mM NaCl, and 0.3 ng/µL poly[d(I-C)] was found to give good results. After the Biacore flow system was cleaned, the bound proteins were eluted in series from the ParR specific and nonspecific ds DNA and analyzed by MALDI-MS. From the sensorgram of the elution process, the amount of eluted protein was 3.3 and 0.05 kRU for the target (flow cell 2) and the control DNA (flow cell 1), respectively. Only molecular ions from ParR were detected in the mass spectrum (Figure 4b). The signal resolution is rather low and considerable peak tailing toward higher masses is observed. In comparison, the spectrum obtained from the analyte solution prior to injection does not display such
DISCUSSION We have developed a procedure that allows elution and mass spectrometric analysis of the proteins bound to the sensor chip in a Biacore X instrument. Mass spectrometric analysis of the eluate is a very efficient test for the specificity of the binding to the sensor chip provided that the remaining part of the integral fluidic system does not contain nonspecifically bound compounds. Initial experiments demonstrated considerable nonspecific binding to the surfaces of the flow system as well as of the injection block. This does not affect the BIA measurement but brings about difficulties for the subsequent mass spectrometric analysis. Therefore, a procedure for cleaning of the Biacore flow system was developed. In addition, an elution technique was developed which allows elution of the bound molecules in a minimal volume, thereby ensuring optimal sample concentration for the mass spectrometric analysis. The method has been applied to myoglobin bound to immobilized anti-myoglobin antibody and to the DNA-binding protein ParR bound to ds DNA immobilized onto the sensor chip via streptavidin/biotin binding. In both cases, a single elution run yielded sufficient sample amounts for mass spectrometric analysis. Because the method can be used to test the specificity of the sensor chip, it also allows absolute quantification based on the sensorgrams and the mass spectrum of the eluate provided that only one component is bound. The fact that neither insulin nor myoglobin and β-lactoglobulin, added as internal control for nonspecific binding, were detected in the eluate demonstrates selective binding of ParR onto the sensor chip surface. In addition, the result demonstrates that the used cleaning procedure is effective. The sensorgrams show that 56 and 13% of the total protein bound to the ParR specific DNA and the control DNA, respectively, were eluted. The differences between the amounts bound to the sensor chip and the amounts eluted indicate losses in the washing step (1h, “Prime”) performed after remounting the sensor chip. This step is needed to equilibrate the flow systems including the sensor with an appropriate buffer prior to elution and to remove air from the flow system. A series of experiments confirmed this observation (data not shown). Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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Inclusion of mass spectrometric analysis further expands the use of the Biacore instrument for screening for binding partners to any given immobilized compound. In the case of proteins, the identification may be performed by mass spectrometric peptide mapping of the eluted proteins using the same procedure as applied for the identification of proteins in gels.21 In cases where only a few protein species are still bound, these may be identified by their molecular mass or by direct peptide mapping. If many proteins are bound, a number of eluates might be pooled and the proteins separated by gel electrophoresis followed by identification of the proteins by in-gel proteolytic digestion and subsequent mass spectrometric peptide mapping. The developed elution procedure also allows the use of ESIMS and ESI-MS/MS provided that an appropriate desalting step is included.22 This might be the procedure of choice for identifying small ligands, e.g., drugs. The method described here is more flexible in terms of choice of analytical procedure than the method reported by Krone et al.8 and Nelson et al.,9 which uses the Biacore sensor chip as MALDI-MS target. When this latter approach is used, only a small part of the bound molecules is accessible for MALDI mass spectrometric analysis. Elution of the bound (21) Patterson, S. D. Anal. Biochem. 1994, 221, 1-15. (22) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8.
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molecules should allow recovering of the total amount and consequently result in a higher sensitivity. The potential of using different mass spectrometric techniques (e.g., electrospray), and reusing the sensor probe, has also been discussed by Nelson et al.10 Finally, elution of the bound molecules allows testing of the specificity of a prepared sensor chip prior to its subsequent use for a series of BIA analysis, because the sensor chip as demonstrated is not degraded by the elution procedure. ACKNOWLEDGMENT Marie Godtfredsen and Kenn Gerdes are acknowledged for the supply of the protein ParR. PerSeptive Biosystems and Biacore AB are acknowledged for the loan of a Voyager Elite MS and a Biacore X instrument, respectively. Financial support by the Commission of the European communities (TMR; ERBFMBICT950446) and the Danish biotechnology program is greatly appreciated.
Received for review January 12, 1998. Accepted March 25, 1998. AC9800457
