A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass

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Anal. Chem. 2001, 73, 4012-4019

A General Strategy for Epitope Mapping by Direct MALDI-TOF Mass Spectrometry Using Secondary Antibodies and Cross-Linking Jochen F. Peter and Kenneth B. Tomer*

National Institute of Environmental Health Sciences, National Institute of Health, P.O. Box 12233, MD F0-03, Research Triangle Park, North Carolina 27709

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra. Epitopes, or antigenic determinants, are defined as the immunologically active region of an immunogen, that bind to antigenspecific membrane receptors on lymphocytes or to secreted antibodies.1 Antibodies are a major tool in any diagnostic serum test. In the development of modern diagnostic test systems, the knowledge of the epitope recognized by each antibody used is imperative. Knowledge of the epitope recognized by an antibody is also important for understanding antibody-antigen interactions,2-4 in the investigation of the pathogenesis of autoimmune diseases and in the production of peptide vaccines.3-5 A number of methods for the elucidation of epitopes have been developed. The most precise method for the determination of * Corresponding author: (phone) (919) 541-1966; (fax) (919) 541-0220; (email) [email protected]. (1) Kuby, J. Immunology, 3rd ed.; W. H. Freeman and Co.: New York, 1997. (2) Laver, W. G.; Air, G. M.; Webster, R. G.; Smith-Gill, S. J. Cell 1990, 61, 553-556. (3) Barlow, D. J.; Edwards, M. S.; Thornton, J. M. Nature 1986, 322, 747748. (4) Berzosky, J. A. Science 1985, 219, 932-940. (5) Murray, K.; Shiau, A.-L. Biol. Chem. 1999, 380, 277-283 (6) Ogawara, K. Microbiol. Immunol. 1999, 43 (10), 915-923. (7) Schmidt, M. A. Biotechnol. Adv. 1989, 7, 187-213.

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protein-antibody interactions is performed by X-ray crystallographic analysis,8-11 but obtaining suitable crystals can be difficult. Currently, only 60 antigen-antibody structures are in the PDB database.12 NMR spectroscopy has also been used but may not be suitable for the rapid determination of unknown epitopes on a protein interacting with an antibody.13,14 PEPSCAN15 and phage-display libraries16,17 have been useful for rapid epitope determination. The PEPSCAN technique uses sets of overlapping peptides, which must cover the entire sequence of a protein if the epitope region is totally unknown. This makes the technique very expensive and dependent on the purity and reproducibility of the synthesized peptides. Epitope mapping with phage-display libraries involves random peptide sequences displayed on the surface of filamentous bacteriophages. Neither method, however, is suitable for the detection of conformational or posttranslationally modified epitopes. The approach we have been using to map unknown epitopes on an intact protein is based on limited proteolysis, which was first reported by Jemmerson and Paterson.18 This approach was combined with the use of immobilized antibodies and mass spectrometry by Suckau et al.,19 using plasma desorption mass spectrometry, and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)20 by this laboratory21-24 and by (8) Padlan, E. A.; Silverton, E. W.; Sheriff, S.; Cohen, G. H.; Smith-Gill, S. J.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1989, 86 (15), 5938-5942. (9) Tormo, J.; Blaas, D.; Parry, N. R.; Rowlands, D.; Stuart, D.; Fita, I. Embo J. 1994, 13 (10), 2247-2256. (10) Lescar, J.; Stouracova, R.; Riottot, M. M.; Chitarra, V.; Brynda, J.; Fabry, M.; Horejsi, M.; Sedlaced, J.; Bentley, G. A. J. Mol. Biol. 1997, 267, 12071222. (11) Bentley, G. A.; Boulot, G.; Riottot, M. M.; Plojak, R. J. Nature 1990, 348, 254-257. (12) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. H.; Weissig, H. Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 235-242 (Web address: www.rcsb.org). (13) Scherf, T.; Anglister, J. Biophys. J. 1993, 64, 754-761. (14) Anglister, J.; Scherf, T.; Zilber, B.; Levy, R.; Zvi, A.; Hiller, R.; Feigelson, D. FASEB J. 1993, 7, 1154-1162. (15) Geysen, H. M.; Rodda, S. J.; Mason, T. J.; Tribbick, G.; Schoofs, P. G. J. Immunol. Methods 1987, 102, 259-274. (16) Scott, J. K.; Smith, G. P. Science 1990, 249, 386-390. (17) Devlin, J. J.; Panganiban, L. C.; Devlin, P. E. Science 1990, 249, 404-406. (18) Jemmerson, R.; Paterson, Y. Science 1986, 232, 1001-1004. (19) Suckau, D.; Kohl, J.; Karwath, G.; Schneider, K.; Casaretto, M.; BitterSuermann, D.; Przybylski, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (24), 9848-9851. (20) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (21) Papac, D. I.; Hoyes,J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-2613. 10.1021/ac010258n Not subject to U.S. Copyright. Publ. 2001 Am. Chem. Soc.

Published on Web 07/19/2001

others.25-28 MALDI-MS has also been used with nonimmobilized antibodies29 and with antigens captured by antibodies during surface plasmon resonance experiments.30 Our approach is different from others in that we directly analyze the affinity-bound antigen-containing sequences by spotting aliquots of the affinity media onto the MALDI target, using the MALDI matrix to release the antigen from the affinity media. This reduces the amount of sample handling and, more importantly, by allowing sequential proteolytic steps, e.g., trypsin followed by carboxypeptidase, permits definiton of the fine epitope structure. Unfortunately, because many antibodies of interest are obtained as crude solutions, such as ascites or cell supernatants, impurities can interfere with the coupling of the antibody to the activated surface (cyanogen bromide- or tosyl-activated Sepharose beads). This can also lead to low binding capacity of the bound antibody because of the stereochemistry of the resulting bound antibody. The use of secondary antibodies that recognize the Fc region of the primary antibody could be an attractive modification of our procedures. Using the secondary antibody should result in a better orientation of the binding region (CDR region) of the primary antibody, thus improving accessibility of the primary antibody for the capture of the antigen. By using a secondary antibody, it should be possible to purify the primary antibody from a crude solution. Two potential drawbacks, however, are that some of the primary antibody could be lost during the subsequent washing steps and there could be increased chemical background during the proteolysis reactions and direct analysis of the affinity beads by MALDI-MS due to increased accessibility of the antibody to the enzyme as well as background due to ions arising from desorption of the antibody. To reduce loss of the primary antibody and to reduce the chemical background, we have incorporated an additional step in which the primary antibody is cross-linked to the secondary antibody.31 In this paper, we report the results of our study to examine the utility of using secondary antibodies to capture primary antibodies from crude solutions followed by cross-linking for epitope mapping. In this study, we have used a mouse anti-ACTH antibody (clone 58) to test our procedure. MATERIALS AND METHODS Reagents. Adrenocorticotropin (ACTH) was purchased from Bachem, California Inc. (Torrance, CA) and monoclonal IgG1 antiACTH (clone 58) from Biodesign International (Kennebunk, ME). (22) Parker, C. E. Papac, D. I.; Trojak, S. K.; Tomer K. B. J. Immunol. 1996, 157, 198-206. (23) Hochleitner, E. O.; Borchers C.; Parker C.; Bienstock, R.; Tomer, K. B. Protein Sci. 2000, 9 (3), 487-496. (24) Hochleitner, E. O.; Gorny, M. K.; Zolla-Pazner, S.; Tomer, K. B. J. Immunol. 2000, 164 (8), 4156-4161. (25) Zhao, Y.; Muir, T. M.; Kent, S. B. H.; Tischer, E.; Scardina, J. M.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4020-4024. (26) Macht, M.; Fiedler, W.; Kuerzinger, K.; Przybylski, M. Biochemistry 1996, 35, 15633-15639. (27) Yu, L. Gaskell, S. J.; Brookman, J. L. J. Am. Soc. Mass Spectrom. 1998, 9, 208-215. (28) Legros, V.; Jolivet-Reynaud, C.; Battail-Poirot, N.; Saint-Pierre, C.; Forest, E. Protein Sci. 2000, 9 (5), 1002-1010. (29) Kiselar, J.; Downard, K. M. Anal. Chem. 1999, 71, 1792-1801. (30) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 4369-4374. (31) Sisson, T. H.; Castor, C. W. J. Immunol. Methods 1990, 127, 215-220.

Polyclonal IgG goat anti-mouse Fc-specific (immunopurified) and mouse IgG (immunopurified) were supplied from Sigma (St. Louis, MO). Endoproteinases Lys-C and Glu-C, trypsin, and carboxypeptidase Y were purchased from Roche Diagnostics Corp. (Indianapolis, IN). Leucine aminopeptidase M was purchased from Sigma. The R-cyano-4-hydroxycinnamic acid was supplied by Aldrich Chemical Co. (Wilwaukee, WI). Acetonitrile, HPLC grade, was purchased from Fisher Scientific (Fair Lawn, NJ). Trifluoroacetic acid, bis(sulfosuccinimidyl) suberate (BS3), and the BCA protein assay kit were purchased from Pierce (Rockford, IL). Cyanogen bromide-activated Sepharose 4B beads were obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). NuPAGE 4-12% Bis-TRIS gels and 3-(N-morpholino)propanesulfonic acid (MES) buffer were obtained from Novex (San Diego, CA). Coomassie brilliant Blue G-250 was from Serva (Paramus, NJ). Compact reaction columns (CRC) were purchased from USB (Cleveland, OH). Microcon30 microconcentrators were obtained from Amicon, Inc. (Beverly, MA). Instrumentation. The HPLC system used was an HP1100 Series HPLC-system (Hewlett-Packard, Waldbronn, Germany). The analytical column used for the separations was a Poros R1/H 2.1 × 30 mm column (PerSeptive Biosystems, Framingham, MA). Mass spectrometric analyses were carried out on a Voyager-DESTR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser (λ ) 337 nm). The accelerating voltage used was 20 kV, and the spectra were measured in the positive mode with delayed extraction. The matrix R-cyano-4-hydroxycinnamic acid (R-cyano), (Aldrich) was recrystallized in hot methanol prior to use. For each sample, 1 µL of the immunocomplex bound to the CNBr-activated Sepharose beads was mixed on the target with 0.5 µL of saturated R-cyano in EtOH/water/HCOOH 45/45/10 (v/v/v), and mass spectra were collected by summing the spectra obtained from 100 laser shots. The mass accuracy of this instrument using internal calibration is typically better than 30 ppm. SDS-PAGE was performed with the Xcell II Mini-Cell (San Diego, CA). Procedures. Determination of the Binding Capacity of the Secondary Antibody, Goat Anti-Mouse Fc-Specific IgG Bound to CNBr-Activated Beads for the Primary Antibody, Mouse IgG. The CNBr-activated Sepharose beads were activated as recommended by the manufacturer but on a smaller scale.32 To determine the binding capacity of the beads for the secondary antibody, 200-µL aliquots of different goat anti-mouse Fc-specific IgG solutions (concentration: 2.40, 1.20, 0.60, 0.30, 0.15, and 0.00 mg/mL) in PBS/0.1 M NaHCO3, pH 8.4, were reacted with 10 µL of beads for 1 h at room temperature. The beads were washed alternately with 0.1 M NaOAc/0.5 M NaCl, pH 4.0 and 0.1 M TRIS, pH 8.0, and then 3 times with PBS. A 500-µL aliquot of a mouse IgG solution (concentration, 40 µg/mL) was added to the beads and reacted for 1 h at room temperature. For the assay, 10 µL of each IgG solution was mixed with 190 µL of the BCA solution in a microtiterplate well and reacted at 60 °C for 30 min. Absorption was measured at 562 nm with an MRX Microplate reader from (32) Parker, C. E.; Tomer, K. B. In Protein and Peptide Analysis in New Mass Spectrometric Applications; Chapman, J. R., Ed.; Methods in Molecular Biology 146; Humana Press: Totowa, NJ, 2000; pp 185-201.

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Dynex Technologies (Chantilly, VA). The concentration of each IgG solution was calculated from an IgG calibration curve. The amount of IgG bound to the beads was calculated by subtracting the values of the solutions before and after the binding experiment. The bound IgG amounts were then plotted semilogarithmically against the total IgG. Immobilization of Mouse Anti-ACTH IgG as the Primary Antibody on CNBr-Activated Sepharose Beads. Prior to addition of the antibody solution, the CNBr-activated Sepharose beads were prepared as recommended by the manufacturer. For each sample, 20 µL of the bead slurry was pipetted into compact reaction columns and washed three times with PBS/0.1 M NaHCO3, pH 8.2. The beads were reacted for 1 h at room temperature with 50 µL of mouse anti-ACTH IgG (c ) 200 µg/ mL), either in PBS/0.1 M NaHCO3, pH 8.2, or in PBS/0.1 M NaHCO3, pH 8.2 + 20% bovine serum albumin (BSA). The beads were washed alternately with 0.1 M NaOAc/0.5 M NaCl, pH 4.0, and 0.1 M TRIS, pH 8.0, and then 3 times with PBS, pH 7.2/0.1% Tween 20 and PBS, pH 7.2. Binding of the Antigen ACTH to Mouse anti-ACTH IgGCoated CNBr Beads. The beads prepared above were reacted with 100 µL of ACTH solution (concentration, 10 µg/mL) in PBS, pH 7.2 for 1 h at room temperature. The immunocomplex, which was later used as a control, was then incubated with PBS, pH 7.2. The CRCs were centrifuged, and the eluates were collected for later HPLC analyses. The beads were then washed three times and stored in PBS, pH 7.2, prior to the proteolysis experiments. Immobilization of Goat Anti-Mouse Fc-Specific IgG as the Secondary Antibody on CNBr-Activated Sepharose Beads. A 20-µL aliquot of the bead slurry was used for each sample and was prepared as described above. Coating was performed by the addition of 100 µL of purified goat anti-mouse Fc-specific IgG (c ) 400 µg/mL) in PBS, pH 7.2, and 0.1 M NaHCO3/0.15 M NaCl, pH 8.2 (binding buffer), to the beads and incubating for 1 h at room temperature. The antibody solution was removed, and 200 µL of 0.1 M TRIS, pH 8.0, was added to the beads for 1 h at room temperature, to block unreacted binding positions. The beads were then washed alternately three times with 0.1 M NaOAc/0.5 M NaCl, pH 4.0, and 0.1 M TRIS, pH 8.0, and then with PBS, pH 7.2. Binding of the Primary Antibody Mouse Anti-ACTH IgG to Goat Anti-Mouse Fc-Specific IgG-Coated CNBr Beads. The coated beads were then reacted for 1 h at room temperature with 50 µL of mouse anti-ACTH IgG (c ) 200 µg/mL), either in PBS, pH 7.2, or in PBS, pH 7.2/20% BSA. After removing the solutions, the beads were washed three times with PBS/0.1% Tween 20 and three times with PBS, pH 7.2. Cross-linking was performed with 20 mM BS3 in PBS, pH 7.2, for 30 min in the dark. Excess crosslinker was quenched with 0.1 M TRIS-HCl, pH 8.0, at room temperature for 15 min. Binding of ACTH to Mouse Anti-ACTH IgG Immobilized on Goat Anti-Mouse Fc-Specific IgG-Coated CNBr Beads. The antibody beads prepared above were washed several washing times with PBS, pH 7.2. A 100-µL solution of ACTH (10 µg/mL) in PBS was added and allowed to react for 1 h at room temperature. The immunocomplex, which was later used as a control, was then incubated with PBS, pH 7.2. The CRCs were centrifuged, and the eluates were collected for further HPLC 4014

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determinations. The beads were then washed three times and stored in PBS prior to the proteolysis experiments. Determination of the Amount of ACTH Bound to the Immunocomplex. The concentration of each ACTH solution (before and after incubation) was determined on an HP1100 Series HPLC system. The ACTH solutions (100 µL each) were treated with 10 µL of acetonitrile and mixed vigorously. A 100-µL aliquot of each solution was analyzed on a Poros R1/H 2.1 × 30 mm column using a linear gradient of 0-80% acetonitrile in water containing 0.085% TFA (v/v) over 10 min, at a flow rate of 1 mL/ min, with detection at 210 nm. The amount of ACTH in each injection was calculated from a calibration curve, and the ACTH bound to the immunocomplex was calculated by subtracting the amount after immunosorption from the amount prior to immunosorption. Enzymatic Proteolysis of ACTH (Epitope Excision). The immunocomplexes were treated successively with several enzymes in two different experiments: (a) Lys-C, Glu-C, trypsin, carboxypeptidase Y, and aminopeptidase M and (b) Glu-C, LysC, trypsin, carboxypeptidase Y, and aminopeptidase M. Buffer conditions for each protease were according to the manufacturers’ recommendations. Specifically, digestion with Lys-C was performed in 50 mM TRIS-HCl buffer, 1 mM EDTA, pH 8.5, digestion with Glu-C was done in 25 mM NH4HCO3 buffer, pH 7.8, and the tryptic digestion was done in 50 mM TRIS-HCl/1 mM CaCl2. Digestion with carboxypeptidase Y was performed in 50 mM NH4OAc buffer, pH 4.5, and digestion with leucine aminopeptidase was done in 100 mM sodium phosphate, pH 7.0. The digestion temperatures were 35 °C for Lys-C, trypsin, carboxypeptidase Y, and leucine aminopeptidase, and 25 °C for Glu-C. The enzyme solution (50 µL) was added to the beads in a 20:1 substrate-to-enzyme ratio for Lys-C, Glu-C, trypsin, and leucine aminopeptidase M. For carboxypeptidase Y, the ratio was 3:1. The beads were incubated overnight for all enzyme reactions. After each proteolysis, the beads were washed three times with PBS to remove unbound products, and the affinity-bound peptides were characterized by MALDI-TOF mass spectrometry. Derivatization of Mouse IgG with Bis(sulfosuccinimidyl) Suberate in Solution. Mouse IgG (50 µg) was dissolved in PBS, pH 7.2, at a concentration of 1 mg/mL. A 50-µL solution of 40 mM bis(sulfosuccinimidyl) suberate in PBS, pH 7.2, was added to the IgG solution, and the reaction was performed for 30 min at room temperature in the dark. BS3 is a bifunctional aminoreactive cross-linker that reacts under neutral pH conditions only with the -amino group of lysine residues. Excess reactive cross-linker was quenched by the addition of 100 µL of 0.1 M TRIS, pH 8.0, and incubation for 30 min at room temperature. The IgG solution was dialyzed against 20 mM TRIS/1 mM EDTA, pH 8.5, using a microconcentrator with a 30-kDa cutoff membrane and then concentrated by centrifugation, using a concentration unit with a 30-kDa membrane (Microcon30). Following this concentration step, 20 mM TRIS/1 mM EDTA was added to give a final IgG concentration of 1 mg/mL. Digestion of Derivatized Mouse IgG with Endoproteinase Lys-C. Next, 25 µL of endoproteinase Lys-C (concentration, 100 ng/µL in 50 mM HEPES/10 mM EDTA, pH 8.0/5% raffinose (w/ v)) was added to the cross-linked and non-cross-linked IgG solutions (50 µL each with c ) 1 mg/mL). The digestion was

Scheme 1. Comparison of Direct and Indirect Immobilization of Antibodies

performed at 35 °C, and 15-µL aliquots were removed after 0, 2, 4, 8, 24, and 48 h and stored at -80 °C. Gel Electrophoresis. SDS-PAGE was performed with NuPAGE 4-12% Bis-TRIS gels and MES buffer. Approximately 3.3 µg of IgG/lane was loaded on the gel, and electrophoresis was run at 200 V for 30 min with the protein bands being visualized with Coomassie brilliant Blue G-250. RESULTS AND DISCUSSION The series of experiments described here are designed to test our hypotheses that the use of a secondary antibody to capture the primary antibody will improve sensitivity and that, when combined with cross-linking to immobilize both antibodies, chemical background in the MALDI mass spectra will be reduced. In these experiments, comparisons are made between (a) the efficiencies of direct versus indirect binding of the primary antibody to the immobilizing media and (b) the efficiencies of capture of an antigen by a directly coupled primary antibody versus an indirectly coupled primary antibody (Scheme 1). We then compare (c) the chemical background resulting from proteolysis of directly coupled primary antibody versus an

indirectly coupled primary antibody. Finally, we test our hypothesis with an epitope-mapping study using either (c) directly coupled antibodies or (d) antibodies that are indirectly coupled and cross-linked. (A) Determination of the Binding Capacity of the Immobilized Goat Anti-Mouse Fc-Specific IgG for Mouse IgG. The amount of mouse IgG bound to the goat anti-mouse Fcspecific IgG-coated beads was determined by incubating the activated beads with different amounts of the goat anti-mouse Fcspecific secondary antibody followed by reaction with a constant amount of mouse IgG. We observed that 4 µg of Fc-specific secondary antibody bound to 1 µL of bead solution was sufficient to bind 1 µg of mouse IgG (data not shown). The following experiments were therefore carried out using a 4:1 ratio of goat anti-mouse Fc-specific IgG secondary antibody/mouse IgG primary antibody. (B) Comparison of the ACTH Capture Efficiencies of the Mouse Anti-ACTH Antibody Bound Directly to the Sepharose Beads and Bound to the Secondary Antibody, and from Pure and Crude Antibody-Containing Solutions. Many antibodies, Analytical Chemistry, Vol. 73, No. 16, August 15, 2001

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Figure 1. Theoretical binding capacities of the four different immunocomplexes. Samples: (1) direct method with pure antibody solution; (2) direct method with crude antibody solution; (3) indirect method with pure antibody solution; (4) indirect method with crude antibody solution.

especially human antibodies, are expressed in culture media, which requires the addition of 10-20% fetal calf serum. To mimic such crude antibody solutions, we added 20% bovine serum to the pure mouse anti-ACTH Ab solution. Four different situations were investigated: For the direct method, mouse anti-ACTH antibody from either a pure or crude antibody solution was immobilized directly on the beads. For the method using a secondary antibody, the pure or crude mouse anti-ACTH antibody solution was bound after reacting the beads with goat anti-mouse Fc-specific IgG. The two antibodies were then cross-linked covalently with BS3. In the final step, ACTH was bound to the immunocomplex. The amount of bound ACTH was determined using HPLC, and our results showed that direct coupling of the pure antibody solution led to a binding capacity of 13.5 ng of ACTH/µL of bead solution (2.97 pmol of ACTH/µL of bead solution) (Figure 1). Using the crude antibody solution, only 7.5 ng (1.67 pmol) of ACTH was bound to 1 µL of the bead solution. Using the secondary antibody, we were able to bind 21 ng of ACTH/µL of beads (4.67 pmol/µL), using either pure or crude antibody solutions. This amount of ACTH corresponds to a theoretical binding capacity of 68%, indicating that the second binding region is partially occupied. This also shows that the cross-linking procedure did not adversely affect the mouse anti-ACTH Ab binding sites. (C) Effects of Cross-Linking with BS3 on resistance of the antibodies to Proteolysis. The effect of cross-linking the primary/secondary antibody complex with BS3 was investigated by MALDI-TOF-MS. After cross-linking, the signals due to mouse anti-ACTH IgG in the mass spectrum decreased significantly (Figure 2, A1 and B1). This indicates that the mouse anti-ACTH Ab was covalently attached to the immobilized secondary antibody. This was also shown by SDS-PAGE (data not shown). Using a very high laser power, ions due to the cross-linked anti-ACTH Ab molecule could be observed in the MALDI spectrum and showed an increase in the molecular weight of ∼6000, compared to the non-cross-linked IgG. The cross-linking procedure also had a significant effect on proteolysis of the antibodies by Lys-C. In the absence of crosslinking, the signal abundances due to the intact IgG molecule significantly decreased after Lys-C digestion for 24 h, while the 4016 Analytical Chemistry, Vol. 73, No. 16, August 15, 2001

Figure 2. MALDI-TOF mass spectra of the immunocomplex goat anti-mouse Fc-specific IgG and mouse IgG: (A1) immunocomplex without cross-linker; (A2) immunocomplex without cross-linker after digestion with endoproteinase Lys-C for 24 h; (B1) cross-linked immunocomplex; (B2) cross-linked immunocomplex after digestion with endoproteinase Lys-C for 24 h.

signal abundance in the lower mass range, indicating the presence of IgG digestion products, increased significantly (Figure 2, A2). After cross-linking, only minor differences in the IgG ion abundances were seen after digestion, indicating that the Ab was more stable to Lys-C proteolysis (Figure 2, B2). The effect of crosslinked IgG on proteolysis with Lys-C was also investigated by SDS-PAGE with similar results (Figure 3). (D) MALDI-TOF-MS of ACTH Bound to the Immobilized Immuncomplexes. The mass spectra of ACTH bound to the different immunocomplexes were acquired. For both immunocomplexes, singly and doubly protonated ions were observed. (Figure 4). Because the bead-matrix mixture forms a nonhomogeneous surface, a low mass accuracy (∼0.1%) was observed using external calibration. Recalibrating spectra internally, after acquisition using known ions, resulted in a mass accuracy of 0.01% or better. A better signal-to-noise ratio was observed using the beads with the cross-linked primary and secondary antibodies, compared to the direct method. This indicated a higher analyte binding capacity using the secondary antibody and was in accordance to the previous binding experiments. Using the secondary antibody method, however, two additional ions were observed at masses 156 and 312 Da above that of ACTH (Figure 4B). We have determined that this is a result of migration of hydrolyzed cross-linker from the attached antibodies to the tyrosine residues of ACTH (data not shown). Epitope Excision Experiments. ACTH bound to the different immunocomplexes was digested successively with the enzymes Lys-C, Glu-C, trypsin, carboxypeptidase Y, and aminopeptidase M. The possible cleavage sites of the endoproteinases Lys-C, GluC, and trypsin are illustrated in Figure 5. After each proteolysis, the beads were washed three times with PBS to remove unbound products, and the affinity-bound peptides were characterized by MALDI-TOF mass spectrometry. After digestion with Lys-C for 24 h at a protein/enzyme ratio of 20:1, ions arising from cleavages on the C-terminal sides of

Figure 3. Comparison of the rates of proteolysis of IgG and IgG cross-linked with BS3, as determined by SDS-PAGE.

Figure 5. Amino acid sequence of human ACTH showing the possible sites of cleavage by the endoproteinases Lys-C, Glu-C, and trypsin.

Figure 4. MALDI-TOF mass spectra of ACTH bound by the direct method (A) and the indirect method (B).

Lys15, Lys16, and Lys21 were observed for ACTH immobilized by either the direct or indirect method (Figure 6A and B, respectively). The ions corresponded to intact ACTH [(M + H)+, obs 4542.12 Da, calc 4542.13 Da], aa1-21 [(M + H)+, obs 2574.98 Da, calc 2575.05 Da], aa1-16 [(M + H)+, obs 1937.93 Da, calc 1938.26], and aa1-15 [(M + H)+, obs 1810.08 Da, calc 1810.08 Da]. No signals were observed for aa16-39, aa17-39, or aa2239. Using the direct method, many nonspecific ions were observed, in addition to ions arising from ACTH residues (Figure 6B). The spectrum obtained from the proteolysis of the indirect complex showed a lower background and an improved signal-to-noise ratio compared to the direct complex, and only signals specific to ACTH were observed. An additional ion was observed at a mass 156 Da above the ion due to aa1-21, as seen previously for the ACTH

molecular ion, and is assigned as arising from the migration of free cross-linker. The remaining affinity-bound ACTH peptides from the Lys C digest were then treated consecutively with Glu-C and trypsin. Only one ion specific to ACTH, aa1-21 (m/z ) 2574.54 Da, calc 2575.05 Da), was observed from the direct complex (Figure 7A). The ion of the highest relative abundance observed in the spectrum of the indirect complex (Figure 7B) was also due to aa1-21. An adduct ion was also observed 156 Da higher in mass in the spectrum of the indirect complex. Additionally, an ion of m/z ) 1212.11 Da (calc 1212.44 Da) was observed which corresponds to aa6-15 (HFRWGKPVGK) (Figure 7B). No ions arising from cleavage between Arg8 and Trp9 were observed after digestion with trypsin, indicating protection of the Arg8-Trp9 bond by the antibody. Further digestions of the immunocomplexes with aminopeptidase M and carboxypeptidase Y with analysis by MALDI produced no smaller peptide fragments (data not shown), indicating that the epitope recognized by the antibody had the amino acid sequence HFRWGKPVGK. Enzymatic digestion of ACTH bound to mouse anti-ACTH antibody from the crude solution, immobilized by the direct method, resulted in a very high background with a very low signalto-noise ratio, making the identification of ions specific to ACTH very difficult. To confirm these results, we cleaved ACTH bound to the immobilized antibodies using consecutive digestion with the Analytical Chemistry, Vol. 73, No. 16, August 15, 2001

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Figure 6. MALDI-TOF mass spectra of the direct immunocomplex (A) and indirect immunocomplex (B) after digestion with Lys-C for 24 h.

endoproteinases Glu-C, Lys-C, and trypsin. Digestion with Glu-C for 24 h at a protein/enzyme ratio of 20:1 led to ions arising from cleavages on the C-terminal side of Glu5, Glu30, and Glu33 and produced four peptides: aa6-30 [(M + H)+, obs 2952.63 Da, calc 2952.39 Da], aa6-33 [(M + H)+, obs 3239.67 Da, calc 3239.66 Da], aa1-30 [(M + H)+, obs 3550.96 Da, calc 3550.03 Da], and aa1-33 [(M + H)+ obs 3837.39 Da, calc 3837.31 Da)]. No ions were seen in the spectrum due to cleavage of the bond between Glu28 and Gln29. Protection of the bond between Glu and Gln from cleavage is in accordance with investigations on insulin by Sørensen et al.,33 who also observed no cleavage by Glu-C between these two residues. The remaining affinity-bound ACTH peptides were then treated with Lys-C, and again four peptides, aa6-15 [(M + H)+ ) 1212.44 Da], aa6-16 [(M + H)+ ) 1340.64 Da], aa6-21 [(M + H)+ ) 1977.16 Da], and aa1-21 [(M + H)+ ) 2575.56 Da], were observed in the spectrum (Table 1). Further digestions of the immunocomplexes with trypsin, aminopeptidase M, and carboxypeptidase Y again showed no additional peptide fragments in the MALDI spectra smaller than residue 6-15. These results, therefore, confirmed that the epitope recognized by the antibody has the amino acid sequence HFRWGKPVGK. CONCLUSIONS The method described in this study allows the identification of epitopes by limited proteolysis combined with mass spectrometry without previous purification of the antibody of interest by using an Fc-specific capture antibody and cross-linking. Normally, epitope mapping combined with mass spectrometry works best with purified antibodies, but purification, especially of low amounts of antibody, involves several steps, is time-consuming, and may lead to loss of sample. During the elution step, which is normally (33) Sørensen, S. B.; Sørensen, T. L.; Breddam, K. FEBS J. 1991, 294 (3), 195197.

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Figure 7. MALDI-TOF mass spectra of the direct complex (A) and indirect complex (B) after consecutive digestions with the endoproteinases Lys-C, Glu-C, and trypsin. Table 1. Peptides Observed by MALDI-TOF of the Immobilized Antigen-Antibody Complexes following Enzymatic Cleavage with Glu-C and Glu-C plus Lys-C

enzyme (1) Glu-C

(1) Glu-C + (2) Lys-C

(M + H)+ ions observed calculated 2952.63 3239.67 3550.96 3837.39 1212.44 1340.64 1977.16 2575.56

2952.39 3239.66 3550.03 3837.31 1212.42 1340.61 1977.41 2575.05

amino acid sequence 6-30 6-33 1-30 1-33 6-15 6-16 6-21 1-21

accomplished under harsh, e.g., acidic conditions, a high loss of protein or a loss in binding capacity of the antibody may occur. Using our approach, the antibody only encounters neutral buffer conditions, because no elution step is required. The antibody of interest is immunopurified with Fc-specific IgG-coated beads and then the two antibodies are attached covalently with a cross-linker. This leaves the binding region fully accessible to the antigen. An additional advantage is that the secondary antibody functions as a spacer, reducing steric hindrance and ensuring optimal orientation of the second antibody. This results in a better binding capacity than does the direct binding of the antibody to the activated surface. Covalent cross-linking of the two antibodies

prevents the loss of antibody during the extensive washing steps required for proteolytic footprinting and has no effect on the binding capacity of the antibody. When the crude antibody solution was directly bound to the Sepharose beads, a very high abundance of nonspecific ions was seen in the mass spectra during epitope excision, because nonspecific proteins had also been covalently bound to the surface. Use of the capture antibody with subsequent cross-linking resulted in a dramatic reduction of background in the mass spectra during the epitope excision experiments and permits, therefore, easier identification of the ions in the mass spectra that correspond to specific antigenic residues. Because cross-linking occurs via the

lysine residues, the antibodies are more resistant to digestion with endoproteinase Lys-C in the epitope excision experiments. The method presented here, which combines improved immunopurification with cross-linking, should prove useful for epitope identification via proteolysis and mass spectrometry. ACKNOWLEDGMENT The authors thank Dr. Carol E. Parker for assistance in the preparation of the manuscript. Received for review March 1, 2001. Accepted June 5, 2001. AC010258N

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