Anal. Chem. 1998, 70, 498-503
On-Probe Immunoaffinity Extraction by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Xiaoli Liang and David M. Lubman*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 David T. Rossi, Gerald D. Nordblom, and Charles M. Barksdale
Department of Pharmacokinetics/Drug Metabolism, Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105-2430
A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses. Immunoassay is a highly specific separation/detection technique often used for biological samples. Coupling this technique with mass spectrometric analysis is very attractive by virtue of the high sensitivity and unambiguous and simultaneous detection of the analytes of interest in a single assay. In this combination, the affinity method serves as a purification and preconcentration step. In initial work Hutchens and Yip,1 used a surface-enhanced affinity capture (SEAC) method using agarose beads chemically derivatized with single-stranded DNA to capture a human glycoprotein, lactoferrin, from preterm infant urine. The beads were then removed, washed to remove impurities, and placed on a MALDI-MS probe tip where analysis was performed using standard MALDI procedures. Tomer et al. used MALDI MS to directly analyze affinity-bound analytes where this method was characterized by minimal sample manipulation and high sensitivity with low-picomole to high-femtomole levels of analytes being readily observed.2 Immunoprecipitation has also been combined (1) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1993, 7, 576580. (2) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-2613.
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with mass spectrometry as illustrated by Wang et al.3 and Nelson et al.4 In their work, antibodies, which were covalently immobilized to a solid support, were incubated in the antigencontaining solution. The resulting antigen-antibody complex was washed repetitively, and the antigen was eluted directly onto a probe tip for MALDI MS analysis. A typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. Also, the combination of fast atom bombardment MS5 or electrospray MS6 and immunoaffinity chromatography was performed using liquid chromatography (LC) as the interface. LC was used to remove the nonvolatile elution buffer, concentrate the eluted sample, and separate multiple antigens. An alternative method to perform affinity extraction is to immobilize antibodies directly on the MALDI probe tip, thus eliminating the need to elute the antigen from the medium. This procedure is more efficient in terms of the low volume of sample required and because the additional separation needed for immunoprecipitation is no longer required. Two major requirements for the immobilization in immunoaffinity extraction must be fulfilled: (a) a stable linkage between the insoluble surface and the antibody and (b) retention of specific binding characteristics of the immobilized antibody. Brockman and Orlando covalently immobilized antibodies onto the gold surfaces via their primary amino groups.7,8 The new immobilization scheme they proposed8 has been demonstrated to possess approximately 500 times more analyte binding sites than the original immobilization procedure.7 However, the immobilization procedure is time-consuming (7 days for one probe) and limited binding sites are available because of random immobilization via amino groups. A nitrocellulose (NC) film prepared by dissolving a NC membrane in acetone has been used previously as a substrate (3) Wang, R.; Sweeney, D.; Gandy, S. E.; Sisodia, S. S. J. Biol. Chem. 1996, 271, 31894-31902. (4) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (5) Davoli, E.; Fanelli, R.; Bagnati, R. Anal. Chem. 1993, 65, 2679-2685. (6) Rule, G. S.; Mordehai, A. V.; Henion, J. Anal. Chem. 1994, 66, 230-235. (7) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (8) Brockman, A. H.; Orlando, R. Rapid Commun. Mass Spectrom. 1996, 10, 1688-1692. S0003-2700(97)00885-8 CCC: $15.00
© 1998 American Chemical Society Published on Web 02/01/1998
Figure 1. Amino acid sequence of SNX-111.
for MALDI MS.9,10 Protein or DNA samples remain on the NC film due to the strong interaction with the film, while salts and other contaminants can be selectively and effectively removed by washing with water or dilute acid. The mechanism of the interactions between proteins and NC is complex and not well resolved. Hydrophobic interactions are believed to dominate under normal conditions, but electrostatic interactions may be involved as well.11 In this work, a simple and effective method has been proposed for combination of immunoaffinity extraction with MALDI MS. It is based on the strong interaction of the IgG with the NC film. In this method, a NC film is coated on the stainless steel probe tip and purified polyclonal IgG antibodies raised against a 25-amino acid peptide are then assembled on the NC film. The whole immobilization procedure can be accomplished within minutes and still meet the two requirements discussed above. This allows the corresponding antigen to be selectively extracted and concentrated on the probe tip from a complex plasma sample prior to MALDI MS analysis. The resulting probe is used in analysis of SNX-111 and its [Met12]-sulfoxide metabolite. This method could provide improved results compared to the more traditional radiolabeled approach because of the built-in extraction selectivity provided by the immunoaffinity extraction and because of the speed, sensitivity, and structural information provided by mass spectrometric detection. SNX-111, a potential therapeutic drug, is a 25-amino acid peptide (2637 molecular weight) containing three internal disulfide bonds, manufactured by solid-phase synthesis, as shown in Figure 1. It is identical in chemical structure to Conotoxin MVIIA obtained from the venom of Conus magus, a fish-hunting cone snail.12 Analysis of therapeutic peptides is difficult because very low doses are usually required for an efficacious therapy, while multiple metabolites may be present in the plasma solution. Direct extraction of the target antigen using the immobilized IgG antibody probe not only overcomes the suppression effect but also concentrates the analyte onto the probe. Another advantage of mass spectrometric detection is the ability to unambiguously identify the captured antigen on the basis of the characteristic mass-to-charge ratio. Therefore, it can be used to simultaneously determine multiple antigens present in solution. This is especially important in analysis of therapeutic drugs and metabolites in plasma solution. EXPERIMENTAL SECTION Materials. SNX-111 acetate (a ω-conotoxin MVIIA) was manufactured by Adria-SP, Inc. (Albuquerque, NM). Nitrocel(9) Liu, Y.-H.; Bai, J.; Liang, X.; Venta, P. J.; Lubman, D. M. Anal. Chem. 1995, 67, 3482-3490. (10) Liang, X.; Zheng, K.; Qian, M. G.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1996, 10, 1219-1226. (11) Gershoni, J. M.; Palade, G. E. Anal. Biochem. 1983, 131, 1. (12) Olivera, B. M.; Cruz, L. J.; de Santos, V.; LeCheminant, G. W.; Griffin, D.; Zeikus, R.; McIntosh, J. M.; Galyean, R.; Varga, J.; Gray, W. R.; Rivier, J. Biochemistry 1987, 26, 2086-2090.
lulose (Protran BA 83, pore size 0.2 µm) was obtained from Schleicher & Schuell (Keene, NH). R-Cyano-4-hydroxycinnamic acid (R-CHCA) and trifluoroacetic acid (TFA) were obtained from Sigma (St. Louis, MO). 2,5-Dihydroxybenzoic acid (DHB), HPLCgrade acetone, and acetonitrile were from Aldrich (Milwaukee, WI). All the chemicals were used without further purification. Rat plasma was produced by heparinization of the blood from male Wistar rats (Charles River Corp., Portage, MI). Antibody Preparation. Preparation of SNX-111-Bovine Serum Albumin. Bovine serum albumin (BSA), was dissolved at 20 mg/ mL in 14 mL of distilled water in a 50-mL polypropylene flask. SNX-111, at 10 mg/mL in 7 mL of water, was added (2:1 ratio, v/v), and the mixture was slowly stirred at room temperature. The conjugation was initiated by the dropwise addition of 1-ethyl3-[(3-dimethylamino)propyl]carbodiimide, at 50 mg/mL in 700 µL of water. The reaction was gently stirred overnight at 4 °C. The mixture was transferred to an Amicon Centricon-10 filter apparatus and spun at 3500 rpm at 4 °C for 20 min in a Beckman TJ6 centrifuge. The resulting retentate (20 mL) was mixed with 50 mL of 0.01 M ammonium bicarbonate buffer, pH 8.0. Aliquots (1 mL; equivalent to 1 mg of SNX-111 + 4 mg of BSA) were placed in 5-mL screw cap vials, lyophilized to dryness, and stored at 4 °C until used. Spectral analysis of the resulting conjugate indicated 4.5 mol of SNX-111/mol of BSA. Immunization of Rabbits. Antibody was raised in rabbits at Josman Laboratories (Napa, CA). Four rabbits were immunized with 1.25 mg of SNX-111-BSA immunogen in a Freund’s complete adjuvant and normal saline (1:1, v/v) emulsion with a biweekly schedule of injections over a period of 3 months until adequate titers and sensitivities were obtained. The serum from the best responding rabbits (NXJ 149, NXJ 150) was harvested and subsequently pooled to provide SNX-111 antiserum used in the RIA and for MALDI MS investigations.13 Rabbit Anti-SNX-111 Immunoglobulin G Isolation. Pooled polyclonal antiserum (15 mL) from the rabbits was loaded on a protein-Sepharose-4B column (2 × 6 cm, glass) obtained from BioRad (Hercules, CA) and the IgG eluted with 30 mL of glycine buffer (0.1 M, pH 3.0). The eluted IgG was then dialyzed against sodium bicarbonate buffer (0.1 M, pH 8.8) and aliquoted for longterm storage at -20 °C.14 Before use in the MALDI MS procedures, an IgG aliquot was thawed and dialyzed against 4 L of ammonium bicarbonate buffer, pH 8.0. Probe Preparation. Nitrocellulose solution (3 µL; 10 mg/ mL in acetone with 1% TFA) was applied onto the stainless steel probe tip and air-dried to form a thin film. A 3-µL aliquot of 14 µg/mL purified IgG solution was placed directly onto the nitrocellulose thin film. The probe tip was dried completely and then rinsed with deionized water to wash away the unbound antibody. Antigen Extraction and Sample Preparation for MALDI MS. SNX-111-containing plasma solutions were prepared by the combination of 495 µL of plasma solution and 5 µL of a stock dilution of pure SNX-111 solution in water. This resulted in a fixed analytical volume in which the concentration of plasma proteins was constant and the concentration of SNX-111 variable; thus, the (13) Barksdale, C. M.; Nordblom, G. D.; Miljanich, G. P.; Tarczy-Hornoch, K.; Kristipati, R.; Kugler, A. R. J. Clin. Ligand Assay 1996, 19, 229-233. (14) Kagel, J. R.; Rossi, D. T.; Nordblom, G. D.; Dudeck, R. C.; Barksdale, C. M.; Kuo, B.-S.; Wright, D. S. J. Pharm. Biomed. Anal. 1995, 13, 12051213.
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Figure 2. MALDI MS spectra of 2 pmol/µL SNX-111 in plasma solution (a) with and (b) without the NC film.
detection limit of SNX-111 could be estimated. The probe coated with immobilized antibody was immersed into a water or plasma solution containing SNX-111. Approximately 20 min was allowed for antibody-antigen binding. The probe was then rinsed with ample water to eliminate any unbound components in the solution. Matrix solution (3 µL), containing saturated R-CHCA in 35:65 (v/ v) acetonitrile/water with 1% TFA, was added on the probe and dried. The probe was then inserted into the mass spectrometer for analysis. In the direct analysis, the analyte solution and matrix were premixed before being placed on the probe tip. Alternatively, the analyte solution was added onto the nitrocellulose film and allowed to dry. The probe was rinsed with water to wash away the unbound components. The matrix was subsequently applied on top of the sample layer. MALDI-MS. The time-of-flight mass spectrometer (TOFMS) used in these experiments was a modified Wiley-McLaren design (R. M. Jordan Co., Grass Valley, CA). All the spectra were obtained using 355-nm radiation from a DCR-11 Nd:YAG laser system (Spectraphysics, Mountain View, CA). The detector was a triple microchannel plate (MCP) detector with a CuBe conversion dynode for postacceleration capability. The postacceleration stage can operate up to -15 kV to enhance the detection efficiency of heavy species, but at the expense of the resolution. Pulsed delayed extraction was applied in these experiments to improve the resolution to a typical value of 500, ultimately limited by the postacceleration stage.15 A digital delay generator (Model DG535, Stanford Research Systems, Sunnyvale, CA) was used to initiate an in-house-designed high-voltage pulser which was connected (15) Zhu, Y.; He, L.; Srinivasan, J. R.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1997, 11, 987-992.
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Figure 3. MALDI MS spectra of 0.2 fmol/µL SNX-111 in water solution extracted by IgG, which is immobilized on (a) NC film and (b) stainless steel probe tip.
to the second acceleration plate. In this system, the ions formed by MALDI are produced in a field-free region and then extracted by a high-voltage pulse after a specified delay time. Data were recorded using a LeCroy 9350M digital oscilloscope and processed by a Gateway P5-133 computer. All the spectra were obtained as 100-shot averages. The MALDI mass spectra initially obtained were calibrated by repeating the experiment using internal standards such as gramicidin or bovine insulin added to the sample and matrix on the probe tip. RESULTS AND DISCUSSION Suppression effects are typically encountered in the analysis of complex biological mixtures by MALDI MS, which may result from the presence of buffers or other components in a mixture.10 This effect can result in reduced intensity for a component of interest in a complex mixture, limiting the general applicability of the direct analysis approach. The use of a nitrocellulose film as a substrate has been shown to greatly facilitate the analysis of complex biological solutions. The suppression effect can be reduced significantly because of the more uniform sites formed by the NC substrate compared with the stainless steel probe tip. In Figure 2 are shown the MALDI MS spectra of the SNX-111 in the presence of the plasma with and without the NC film, respectively. No peaks could be observed without the NC film. The use of a NC film can alleviate the suppression effect as shown in Figure 2a, where a peak of SNX-111 was obtained. However, the S/N ratio was still not sufficient for our experiments, probably due to the interference of other plasma components present in the solution.
Figure 4. MALDI MS spectra of 0.2 pmol/µL SNX-111 in cytochrome c digest solution (a) with and (b) without extraction by immobilized IgG probe.
Mass spectrometry has been used increasingly in pharmaceutical applications due to its high sensitivity and ability to provide structural information. In applications such as analysis of in vivo metabolism of therapeutic drugs from biological samples, sample pretreatment techniques are often used.16 Affinity chromatography is probably the most highly specific technique currently available.17 Direct coupling of antibodies and mass spectrometry has been performed by immobilizing the binding molecules directly onto the MALDI probe surface.7,8 However, the timeconsuming immobilization procedure can limit the use of this method for real applications. A simple and efficient approach has been developed in this paper to immobilize the antibody onto the probe via a nitrocellulose film. Comparison experiments have been performed to demonstrate the excellent retention of the antibody on the nitrocellulose film rather than the stainless steel surface. Figure 3 shows the spectra obtained on the nitrocellulose film and on the stainless steel probe, respectively. In these experiments, all the conditions remained the same except the initial surface. Almost no signal could be resolved on the stainless steel probe, while a sharp peak of SNX-111 was clearly observed on the nitrocellulose film, demonstrating the improved retention of the antibody on the film even after extensive washing steps. Direct extraction of the target antigen using the immobilized probe not only overcomes the suppression effect but also concentrates the analyte of interest onto the probe. In Figure 4 are shown the MALDI MS spectra of SNX-111 extracted from a mixture of cytochrome c digest products and the mixture itself. (16) Krishnan, T. R.; Ibraham, I. J. J. Pharm. Biomed. Anal. 1994, 12, 287-294. (17) de Frutos, M.; Regnier, F. E. Anal. Chem. 1993, 65, 17A-25A.
Figure 5. MALDI MS spectra of different concentrations of SNX111 in plasma solution extracted by IgG-immobilized probe.
Clearly, only SNX-111 was extracted and concentrated on the probe tip while other components were washed off, demonstrating the excellent selectivity of the IgG. Figure 5 shows the MALDI MS spectra of different concentrations of SNX-111 extracted by probe-immobilized IgG from plasma solution. The IgG was adsorbed onto the nitrocellulose film directly. A concentration as low as 2 fmol/µL SNX-111 can be detected. This detection limit is only slightly less than that obtained in water solution (∼2 fmol/µL). In addition, there are peaks other than SNX-111 detected in the spectrum which might be derived from nonspecific interaction of plasma proteins with the antibody or the NC film. This can be explained in terms of the high viscosity of the plasma solution and the competition of limited binding sites by other plasma proteins. The control spectrum (data not shown) also shows a similar pattern of background signals. Although the signals due to the plasma proteins are present, they did not interfere with the unambiguous determination of SNX-111, indicated by the signal at m/z ) 2638 Da. A progressive decrease in the ion signal of the analyte was observed relative to the signals of the nonspecifically retained proteins. SNX-111 (2 fmol/µL) present in the 500-µL samples corresponds to a maximum coverage of 1 pmol of sample on the probe if the efficiency of every step involved is 100%. This limit is comparable to the detection limit of the mass spectrometer itself. Although the exact amount of antibody captured onto the probe tip via immobilization has not been determined at this moment, the sensitivity obtained was sufficient for analysis of human clinical therapeutic samples. Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
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Figure 6. MALDI MS spectrum of SNX-111 and its metabolite in water solution coextracted by immobilized IgG probe. The calculated m/z are 2638 and 2654 for SNX-111 and its metabolite, respectively. The measured masses as shown are m/z 2638.0 and 2654.3 for SNX111 and its metabolite.
Another advantage of mass spectral detection is the ability to unambiguously identify the captured antigen on the basis of the characteristic mass-to-charge ratio. The method can thus be used to simultaneously determine multiple antigens present in the solution. This is especially important in analysis of SNX-111 and its metabolite in plasma. Analysis of therapeutic peptides is difficult because very low doses are usually required for a therapeutic effect, and the analysis becomes more difficult when multiple antigens exist in the plasma caused by metabolism. A sample fortified with the authentic metabolite was used to demonstrate this feasibility. Water solution (500 µL) containing 4 fmol/µL SNX-111 and 2 fmol/µL of its metabolite was used for immersion. As illustrated in Figure 6, SNX-111 and its metabolite were easily resolved in the MALDI mass spectrum, although both were captured together on the probe. A mass difference of 16 between SNX-111 and its metabolite has been obtained from the spectrum, which indicates the peptide has been oxidized to produce SNX-111-[Met12]-sulfoxide. In real applications, the volume of the available sample is usually under 0.5 mL, making extraction by immersion into the solution difficult. In addition, immersion into the solution would contaminate the whole solution, making further analysis questionable and impractical. In this case, 3 µL of sample was applied onto the top of the antibody layer directly instead of immersing the probe tip into the solution. In Figure 7 are shown the mass spectra of SNX-111 extracted by this method. Although the amount of sample is limited by the volume applied, the sensitivity is still sufficient for real dose analysis. Figure 8 shows the mass spectrum of sample collected from a dosed rat. The concentration of SNX-111 is estimated to be at 112 ng/mL which corresponds to about 40 fmol/µL. The peak of SNX-111 is clearly observed. However, the metabolite of SNX-111 cannot be detected because 502 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
Figure 7. MALDI MS spectra of SNX-111 extracted by applying the plasma solutions on the probe directly.
Figure 8. MALDI MS spectrum of sample collected from a dosed rat.
of the limited amount present. The metabolite was estimated to be only 5-10% SNX-111 by comparison of the peak area from an LC chromatogram. In this case, only 6-12 fmol of metabolite was contained in the sample. CONCLUSIONS The combination of immunoaffinity extraction with MALDI on one probe tip eliminates the need to elute the antigen from a
separate medium and offers a number of advantages. It combines the high specificity of immunoaffinity extraction with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. The whole process can be completed within 1 h. This method has been used successfully to elucidate the presence of a therapeutic peptide at relevant doses.
ceutical Research Division of Warner-Lambert Co. X.L. acknowledges a Sokol Fellowship for financial support.
Received for review August 14, 1997. Accepted November 6, 1997.X AC9708856
ACKNOWLEDGMENT This work received support from the National Institutes of Health under Grant R01GM49500 and from Parke-Davis Pharma-
X
Abstract published in Advance ACS Abstracts, December 15, 1997.
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