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  Section: Cell Biology Methods » Antibodies » Production and Purification of Antibodies
 
 
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Epitope Mapping by Mass Spectrometry

 
     
 
Epitope Mapping by Mass Spectrometry


I. INTRODUCTION
Antigenic determinants, or epitopes, are the specific chemical structures within an antigen molecule that are recognized by either B-cell or T-cell antibodies. Knowledge of the structure of the epitope recognized by an antibody is important for the development of vaccines, for understanding antibody-antigen interactions, for validating some diagnostic tests, and in the investigation of the pathogenesis of autoimmmune diseases. There are a number of approaches for determination of epitopes currently in use, including peptide reactivity, nuclear magnetic resonance (NMR), crystallography, mutation analysis, and phage display and limited proteolysis (Jemmerson and Paterson, 1986). There are various limitations to each of these techniques. For example, NMR can be limited by relatively high sample levels needed and the antibody-antigen mass. Crystallography requires high analyte levels and crystal formation. Mutation analysis can be misleading when a mutation distant from the epitope induces a change in the conformation of the epitope. Peptide scanning and phage displays can be used to determine linear epitopes but are less applicable to conformational epitopes or epitopes containing posttranslational modifications. Limited proteolysis involves the comparison of proteolytic products from the antigen with those arising from the antigen-antibody complex. Enzymatic cleavage sites that are part of the epitope will be protected from cleavage in the presence of the antibody but not in the free antigen. This approach requires a detection system that can provide structural information about the proteolytic fragments.

The high sensitivity and specificity provided by high-accuracy mass measurement have made mass spectrometry the detection method of choice for use with limited proteolysis experiments (Suckau et al., 1990; Zhao et al, 1996; Macht et al., 1996; Yu et al., 1998; Legros et al., 2000). Our approach involves the use of antibodies immobilized on Sepharose beads (Papac et al., 1994; Parker et al., 1996; Jeyarajah et al., 1998). After binding the antigen, the complex is treated sequentially with one or more proteolytic enzymes. The nonspecifically bound peptides are washed off the beads, and then an aliquot of the beads is placed on a matrixassisted laser desorption/ionization mass spectrometry (MALDI/MS) target. The MALDI matrix is then applied to the beads, releasing the affinity-bound peptides, which are then analyzed. By using an endopeptidase such as trypsin or Lys-C followed by an exopeptidase such as aminopeptidase or carboxypeptidase, the fine structure of the epitope can be determined.

We have modified our procedure to incorporate the use of a secondary Fc-specific antibody to capture the primary antibody from crude biological preparations (Fig. 1) (Peter and Tomer, 2001; Parker et al., 2001). The two antibodies are then cross-linked. Incorporating this step has the advantage of significantly reducing the chemical background in MALDI spectra and increasing the antibody-binding capacity. Few ions are observed in the mass spectrum that can be attributed to proteolysis of the antibodies. This is due to a combination of factors: antibodies in general appear to be resistant to proteolysis, possibly due to resistance to denaturation and to extensive glycosylation shielding proteolytic sites; proteolytic cleavages do not always give rise to free peptides because of the presence of disulfide bonds; peptides originating from proteolysis at sites that do not disrupt antigen-antibody binding are washed off prior to analysis; and, when the primary and secondary antibodies are cross-linked, many potential proteolysis sites for the most commonly used enzymes are blocked. The capabilities of this approach are illustrated by mapping the epitope on adrenocorticotropin recognized by a mouse anti- ACTH IgG antibody (Peter and Tomer, 2001).


FIGURE 1 Scheme of proteolytic footprinting incorporating capture antibodies with cross-linking. [Adapted with permission from Peter and Tomer (2001). Copyright 2001 American Chemical Society.]
FIGURE 1 Scheme of proteolytic footprinting incorporating capture antibodies with cross-linking.
[Adapted with permission from Peter and Tomer (2001). Copyright 2001 American Chemical Society.]


II. MATERIALS AND INSTRUMENTATION
Adrenocorticotropin is from Bachem, California (Cat. No. Hl160). Monoclonal IgG1 anti-ACTH (clone 58) is from Biodesign International (Cat. No. E54008M). Polyclonal IgG goat antimouse Fc specific is from Sigma (Cat. No. M-4280), as are Trizma-HCl, (Cat. No. T-6666), Trizma-base (Cat. No. T-6791), Tween 20 (Cat. No. P-8942, 10% in water), NaH2PO4- H2O (Cat. No. S-9638), leucine aminopeptidase M (Cat. No. L-0632), and EDTA (Cat. No. E-5513). Endoproteinases are obtained from Roche Diagnostics (Lys-C Cat. No. 1 047 825; Glu-C Cat. No. 1 047 817; trypsin Cat. No. 1 418 025; and carboxypeptidase Y Cat. No. 1 111 914). Formic acid, 96% (Cat. No. 25,136-4), (α-cyano- 4-hydroxycinnamic acid (Cat. No. 47,687-0), and NH4HCO3 (Cat. No. 28,509-9) are from Aldrich Chemical Co. Bis(sulfosuccinimidyl) suberate, BS3 (Cat. No. 21580), is from Pierce. Cyanogen bromide-activated Sepharose 4B beads are from Amersham Biosciences (Cat. No.17-0820-01). Compact reaction columns, CRC (Cat. No. 13928), and 35µM compact column filters (Cat. No. 13912) are from USB. HCl, 1M (Cat. No. 920-1), and NaOH, 1M (Cat. No. 930-65), are from Sigma Diagnostics. CaCl2·2H2O (Cat. No. 4160), Na2HPO4·3H2O (Cat. No. 7914), NaHCO3 (Cat. No. 7412), NH4OAc (Cat. No. 3272), and NaOAc-7H2O (Cat. No. 7364) are from Mallinckrodt. NaCl is from Baker (Cat. No. 3624-05), and ethyl alcohol is from Pharmco Products. Becton Dickinson Falcon tubes, 15 ml, No. 352059, are from Fisher Scientific (Cat. No. 14-959-11B). The rotating incubator is a Lab Line hybridization incubator or similar. Deionized water is obtained from a Hydro Picopure 2 system (Hydro Systems, Research Triangle Park, NC).

MALDI mass spectra are obtained on a Voyager DESTR mass spectrometer (Applied Biosystems). Similar results should be obtained from similar instrumentation.


III. PROCEDURES
A. Immobilization of Secondary Antibody to Cyanogen Bromide-Activated Sepharose Columns
Solutions
  1. Phosphate buffer, pH 7.2: Prepare 100ml 0.1M Na2HPO4 by dissolving 2.68 g Na2HPO4·7H2O in water and 100ml 0.1M NaH2PO4 by dissolving 1.38g NaH2PO4-H2O in water. Use 100ml of the 0.1M Na2HPO4 solution and titrate to pH 7.2 with the 0.1M NaH2PO4 solution (approximately 37ml necessary).
  2. Phosphate-buffered Saline (PBS), pH 7.2: To make 100ml of PBS, pH 7.2, containing 0.1M phosphate buffer and 150mM NaCl, dissolve 0.88 g NaCl in previously prepared 100ml 0.1M phosphate buffer.
  3. 1 mM HCl: To make 1 liter of 1 mM HCl, dilute 1 ml 1M HCl to 1000 ml.
  4. Coupling buffer: To prepare 50ml of coupling buffer, add 0.42g sodium bicarbonate to sufficient deionized water to make 50 ml of solution. The pH will be between 8.2 and 8.35 and does not need further adjustment.
  5. 0.1M NaHCO3/O.15M NaCl: To make 50ml of solution, add 0.42g sodium bicarbonate and 0.44g NaCl to sufficient deionized water to make 50ml of solution. Again, the pH does not need to be adjusted any further.
  6. Antibody solution: Mix 20µl of goat anti-mouse Fc-specific IgG (supplied as a 2.0-mg/ml solution in 10 mM phosphate buffered saline, pH 7.4, containing 15mM sodium azide) with 80µl 0.1M NaHCO3/ 0.15 M NaCl.
  7. 0.1M Tris, pH 8.0: To make 50ml of solution, add 0.79g Trizma-HCl to approximately 45ml deionized water and adjust pH with 1M NaOH. Add sufficient deionized water to yield 50ml final volume.
  8. 0.1 M NaOAc/0.5 M NaCl, pH 4.0: To prepare 50 ml of solution, add 0.68 g NaOAc·3H2O and 1.46 g NaCl to approximately 45ml deionized water and adjust pH with HCl. Add sufficient deionized water to make 50 ml final volume.


Steps
  1. Place ca. 0.2 g of dry CNBr-Sepharose beads into a Falcon tube.
  2. Add 10ml of 1 mM HCl. Mix gently and equilibrate for 15 min.
  3. Place ca. 20µl of the wet beads in a CRC and then drain the column.
  4. Wash the column six times with 0.8 ml 1 mM HCl and then six times with 0.4ml coupling buffer (0.1M NaHCO3).
  5. Add 100µl of antibody solution with a final concentration of 400µg/ml to the beads and incubate for 1 h at room temperature.
  6. Remove the antibody solution and then, to block unreacted binding positions, add 200µl of 0.1M Tris, pH 8.0, and react for 2h at room temperature with slow rotation.
  7. Wash alternately three times with 0.1M NaOAc/ 0.5M NaCl, pH 4.0, 0.1M Tris, pH 8.0, and PBS, pH 7.2.
  8. Check immobilization of the secondary antibody by MALDI/MS using a 1-µl aliquot. Ions attributable to the antibody should be observed in low abundance only.


B. Binding of Primary Antibody to Immobilized Secondary Antibody
Solutions
  1. PBS/0.1% Tween 20: To make 5ml of solution, add 50 µl of 10% Tween 20 in water to 5 ml of previously prepared PBS, pH 7.2.
  2. 10mM BS3 in PBS, pH 7.2: For a 1-ml solution, dissolve 5.72mg BS3 in 1 ml previously prepared PBS, pH 7.2.
  3. 0.1M Tris, pH 8.0: To make 50ml of solution, add 0.79g Trizma-HCl to approximately 45ml deionized water and adjust pH with NaOH. Add sufficient deionized water to yield 50ml final volume.
  4. Anti-ACTH IgG solution: Prepare initial solution by dissolving 200 µg anti-ACTH IgG in 1 ml previously prepared PBS, pH 7.2.


Steps
  1. Add 50µl of mouse anti-ACTH IgG (200pg/ml in PBS, pH 7.2) to the beads from step 7 and react for 1 h at room temperature.
  2. Drain solution and wash the beads three times with 0.5ml PBS/0.1% Tween 20 and three times with 0.5 ml PBS, pH 7.2.
  3. Check that the primary antibody is bound to the immobilized secondary antibody by obtaining a MALDI/MS from a 1-µl aliquot. Ions arising from the antibody should be observed (Fig. 2A). If no ions are detected the procedure has not worked.
  4. To cross-link the primary antibody to the secondary antibody, add a 10-µl aliquot of a 10mM solution of BS3 in PBS, pH 7.2, to the beads. Incubate the mixture at RT in the dark with rotation for 45 min.
  5. Drain and then quench the cross-linker with 400µl 0.1M Tris, pH 8.0, for 15min at room temperature.
  6. Wash the beads twice with 100µl of 0.1M Tris, pH 8.0.
  7. Wash three times with 400 µl PBS, pH 7.2.
  8. Set aside one-fourth of the beads for use as a control. Note: All further experiments should be performed on the control except that no ACTH should be bound to the immunocomplex. This will provide information about chemical background for subsequent MALDI analyses.
  9. Check that the primary antibody has been crosslinked to the secondary antibody by MALDI/MS using a 1-µl aliquot. Ions attributable to the antibody should be of low abundance. (Fig. 2B, cf. Fig. 2A).


FIGURE 2. (A) Direct MALDI/MS of mouse anti-ACTH antibody from immunocomplex with Sepharose-bound antimouse Fcspecific IgG without cross-linking. (B) Direct MALDI/MS of mouse anti-ACTH antibody from immunocomplex with Sepharose-bound antimouse Fc-specific IgG with cross-linking. (C) Direct MALDI/MS of ACTH bound to cross-linked immunocomplex. [Adapted with permission from Peter and Tomer (2001). Copyright 2001 American Chemical Society.]
FIGURE 2. (A) Direct MALDI/MS of mouse anti-ACTH antibody from immunocomplex with Sepharose-bound antimouse Fcspecific IgG without cross-linking. (B) Direct MALDI/MS of mouse anti-ACTH antibody from immunocomplex with Sepharose-bound antimouse Fc-specific IgG with cross-linking. (C) Direct MALDI/MS of ACTH bound to cross-linked immunocomplex. [Adapted with permission from Peter and Tomer (2001).
Copyright 2001 American Chemical Society.]

C. Binding of Antigen (ACTH) to Secondary Antibody

Solutions
  1. Phosphate buffer, pH 7.2: Prepare 100ml 0.1M Na2HPO4 by dissolving 2.68g Na2HPO4·7H2O in water and 100 ml 0.1M NaH2PO4 by dissolving 1.38g NaH2PO4·H2O in water. Use 100ml of the 0.1M Na2HPO4 solution and titrate to pH 7.2 with the 0.1M NaH2PO4 solution (approximately 37ml necessary).
  2. PBS: To make 100ml of PBS, pH 7.2, containing 0.1M phosphate buffer and 150mM NaCl, dissolve 0.88 g NaCl in 100 ml previously prepared 0.1M phosphate buffer.
  3. 10 µg/ml ACTH in PBS: Dissolve 10 µg ACTH in 1 ml PBS from solution 2.


Steps
  1. Drain the beads.
  2. Add 100-µl aliquot of a 10-µg/ml solution of ACTH in PBS to the remainder of the beads from step 7. Allow reaction at room temperature for 1 h with slow rotation.
  3. Incubate the control aliquot from step 8 in a CRC with 100 µl PBS, pH 7.2, for 1 h at room temperature with rotation.
  4. Drain the two CRCs and wash the beads three times with 400µl PBS and store in sufficient PBS to keep the beads moist.
  5. Remove a 1-µl aliquot of the beads for MALDI/MS analysis (Fig. 2C).


D. Proteolytic Footprinting (Epitope Excision)
Successive enzymatic digestions can be performed. In this example, the immunocomplex is treated successively with Lys-C, Glu-C, trypsin, carboxypeptidase Y, and aminopeptidase M. Prepare all proteinase-containing solutions just prior to use to limit autolysis.

Solutions
  1. Lys-C solution: To prepare lµg/µl Lys-C stock solution, dissolve 50µg endoproteinase Lys-C in 50µl deionized water. To make 50ml stock buffer solution, dissolve 0.30 g Trizma-base and 18.6mg EDTA disodium salt dihydrate in approximately 45 ml deionized water and adjust pH to 8.5 with HCl. Add sufficient deionized water to yield 50ml final volume. To 90 µl of the buffer stock, add 10 µl of the Lys-C stock solution. Take 10µl of this 0.1-µg/µl Lys-C solution and dilute it further with 990 µl of the buffer stock.
  2. Phosphate buffer, pH 7.2: Prepare 100ml 0.1M NaRHPO4 by dissolving 2.68 g Na2HPO4·7H2O in water and 100ml 0.1M NaH2PO4 by dissolving 1.38g NaH2PO4·H2O in water. Use 100ml of the 0.1M Na2HPO4 solution and titrate to pH 7.2 with the 0.1M NaH2PO4 solution (approximately 37 ml necessary).
  3. PBS, pH 7.2: To make 100ml of PBS, pH 7.2, containing 0.1M phosphate buffer and 150mM NaCl, dissolve 0.88g NaCl in 100ml 0.1M phosphate buffer.
  4. Glu-C solution: To prepare lµg/µl Glu-C stock solution, dissolve 50 µg Glu-C in 50 µl deionized water. To make 50ml stock buffer solution, dissolve 98.8 mg ammonium bicarbonate in 45 ml deionized water and adjust pH to 7.8 with NaOH. Add sufficient deionized water to yield 50 ml final volume. To 90 µl of the buffer stock, add 10 µl of the Glu-C stock solution. Take 10 µl of this 0.1-µg/µl Glu-C solution and dilute it further with 990 µl of the buffer stock.
  5. Trypsin solution: To prepare 1 µg/µl trypsin stock solution, dissolve 25µg trypsin in 25µl deionized water. To make 50ml stock buffer solution, add 0.30g Trizma-base and 5.55 mg CaCl2 to approximately 45 ml deionized water and adjust pH to 8.5 with HCl. Add sufficient deionized water to yield 50ml final volume. To 90µl of the buffer stock, add 10µl of the trypsin stock solution. Take 10µl of this 0.1-µg/µl trypsin solution and dilute it further with 990µl of the buffer stock.
  6. Carboxypeptidase Y solution: To prepare 0.4µg/µl carboxypeptidase Y stock solution, dissolve 20µg carboxypeptidase Y in 50µl deionized water. Buffer stock solution, 50ml, is prepared by adding 193mg ammonium acetate to 45ml deionized water. Adjust the pH to 4.5 with acetic acid and add sufficient deionized water to yield 50ml final volume. Add 10 µl carboxypeptidase Y to 90 µl of the stock solution. Take 10µl of this 0.04-µg/µl carboxypeptidase Y solution and dilute it further with 90µl of the buffer stock.
  7. Phosphate buffer, pH 7.0: Prepare 100 ml 0.1M Na2HPO4 by dissolving 2.68 g Na2HPO4·7H2O in water and 100 ml 0.1M NaH2PO4 by dissolving 1.38 g NaH2PO4·H2O in water. Use 100ml of the 0.1M Na2HPO4 solution and titrate to pH 7.0 with the 0.1M NaH2PO4 solution (approximately 58 ml necessary).
  8. Leucine aminopeptidase solution: To prepare 1 µg/µl aminopeptidase M stock solution, dissolve 50µg aminopeptidase M in 50µl deionized water. Dilute 10µl of the aminopeptidase stock solution with 90µl phosphate buffer, pH 7.0. Take 10µl of this 0.1 µg/µl aminopeptidase M solution and dilute it further with 990 µl of the buffer stock.


Steps
1. Lys-C:
  1. Prepare a solution of Lys-C (1 ng/µl) in a solution of 50 mM Tris and 1 mM EDTA, pH 8.5. Add the enzyme solution (50µl) (approximately 20:1 substrate-to-enzyme ratio) to the immunocomplex in the CRC.
  2. Incubate the beads overnight at 37°C with slow rotation.
  3. Remove the enzyme solution and wash the beads three times with 0.4ml PBS, pH 7.2.
  4. Add sufficient buffer solution to keep beads moist.
  5. Remove a 1-µl aliquot for MALDI/MS analysis (Fig. 3A).


2. Glu-C:
  1. Prepare a solution of Glu-C (1ng/µl) in a solution of 25mM NH4HCO3, pH 7.8. Add the enzyme solution (50µl) (approximately 20:1 substrate-to-enzyme ratio) to the immunocomplex in the CRC.
  2. Incubate the beads overnight at 37°C with slow rotation.
  3. Remove the enzyme solution and wash the beads three times with 0.4 ml PBS, pH 7.2.
  4. Add sufficient buffer solution to keep beads moist.
  5. Remove a 1-µl aliquot for MALDI/MS analysis (Fig. 3B).


3. Trypsin:
  1. Prepare a solution of trypsin (1ng/µl) in a solution of 50mM Tris, l mM CaCl2, pH 8.5. Add the enzyme solution (50µl) (approximately 20:1 substrate-to-enzyme ratio) to the immunocomplex in the CRC.
  2. Incubate the beads overnight at 37°C with slow rotation.
  3. Remove the enzyme solution and wash the beads three times with 0.4 ml PBS, pH 7.2.
  4. Add sufficient buffer solution to keep beads moist.
  5. Remove a 1-µl aliquot for MALDI/MS analysis (Fig. 3C).


4. Carboxypeptidase Y:
  1. Prepare a solution of carboxypeptidase Y (4ng/µl) in a 50mM NH4OAc, pH 4.5, solution. Add the enzyme solution to the immunocomplex in the CRC at a 3:1 substrate-to-enzyme ratio (83 µl).
  2. Incubate the beads overnight at 37°C with slow rotation.
  3. Remove the enzyme solution and wash the beads three times with 0.4 ml PBS, pH 7.2.
  4. Add sufficient buffer solution to keep beads moist.
  5. Remove a 1-µl aliquot for MALDI/MS analysis (Fig. 3D).


5. Leucine aminopeptidase:
  1. Prepare a solution of leucine aminopeptidase (1 ng/µl) in a 100mM sodium phosphate, pH 7.0, solution. Add the enzyme solution to the immunocomplex in the CRC at a 20:1 substrateto- enzyme ratio.
  2. Incubate the beads overnight at 37°C with slow rotation.
  3. Removed the enzyme solution and wash the beads three times with 0.4 ml PBS, pH 7.2.
  4. Add sufficient buffer solution to keep beads moist.
  5. Remove a 1-µl aliquot for MALDI/MS analysis. In this experiment, no additional cleavages are observed.


E. Preparation of Samples for MALDI/MS Analysis
  1. Mix approximately 1 µl of the affinity beads on the MALDI target with 0.5-1.0µl of saturated α-cyano- 4-hydroxycinnamic acid in ethanol/water/formic acid (45/45/10 v/v/v).
  2. Let the target air dry.
  3. Obtain spectra as indicated by the instrument's user guide.


FIGURE 3. (A) Direct MALDI/MS of ACTH bound to the crosslinked immunocomplex after digestion with Lys-C. (B) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C. (C) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C and then trypsin. (D) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C, trypsin, and carboxypeptidase Y. Peak marked with an asterisk is background as observed in the control. [Adapted with permission from Peter and Tomer (2001). Copyright 2001 American Chemical Society.]
FIGURE 3. (A) Direct MALDI/MS of ACTH bound to the crosslinked immunocomplex after digestion with Lys-C. (B) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C. (C) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C and then trypsin. (D) Direct MALDI/MS of ACTH bound to the cross-linked immunocomplex after digestion with Lys-C followed by Glu-C, trypsin, and carboxypeptidase Y. Peak marked with an asterisk is background as observed in the control. [Adapted with permission from Peter and Tomer (2001).
Copyright 2001 American Chemical Society.]


IV. COMMENTS

This procedure works well for linear epitopes and many continuous conformational epitopes. Problems can be encountered for conformational epitopes that are conformationally constrained by the presence of disulfide bonds when the disulfide bond is lost during proteolysis. For discontinuous epitopes, a combination of limited proteolysis and chemical modification of surface accessible amino acids may provide the required structural information (Hochleitner et al., 2000).

The aforementioned order of proteolytic enzymes used was used to provide initially large proteolytic peptides, followed by enzymes that would cleave within the sequence of the smallest peptide observed (Fig. 4).

FIGURE 4. Proteolytic peptides observed for the various digestion steps involved in obtaining spectra shown in Fig. 3.
FIGURE 4. Proteolytic peptides observed for the various digestion steps involved in obtaining spectra shown in
Fig. 3.


V. PITFALLS
Do not use dithiothreitol because it can denature the antibody as well as the antigen.

Do not use too high a concentration of Ca2+ as this can interfere with the MALDI/MS analysis.

Although we have been able to perform four consecutive proteolysis experiments on one aliquot of antigen-antibody beads, the levels of background ions increase significantly while the abundance of analyte ions decreases.

References
Hochleitner, E. O., Borchers, C., Parker, C., Bienstock, R. J., and Tomer, K. B. (2000). Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis. Protein Sci. 9, 487-496.

Jemmerson, R., and Paterson, Y. (1986). Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes. Science 232, 1001-1004.

Jeyarajah, S., Parker, C. E., Sumner, M. T., and Tomer, K. B. (1998). MALDI/MS mapping of HIV-gp120 epitopes recognized by a limited polyclonal antibody. J. Am. Soc. Mass Spectrom. 9,157-165.

Legros, V., Jolivet-Reynaud, C., Battail-Poirot, N., Saint-Pierre, C., and Forest, E. (2000). Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping. Protein Sci. 9, 1002-1010.

Macht, M., Fiedler, W., Kuerzinger, K., and Przybylski, M. (1996). Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T. Biochemistry 35, 15633-15639.

Papac, D. I., Hoyes, J., and Tomer, K. B. (1994). Epitope mapping of the gastrin releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption mass spectrometry. Protein Sci 3, 1488-1492.

Parker, C. E., Deterding, L. J., Hager-Braun, C., Binley, J. M., Sch/ilke, N., Katinger, H., Moore, J. P., and Tomer, K. B. (2001). Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5. J. Virol 75, 10906-10911.

Parker, C. E., Papac, D. I., Trojak, S. K., and Tomer, K. B. (1996). Epitope mapping by mass spectrometry: Determination of an epitope on HIV-1IHB p26 recognized by a monoclonal antibody. J. Immunol. 15(1), 198-206.

Peter, J. E, and Tomer K. B. (2001). A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and crosslinking. Anal. Chem. 73, 4012-4019.

Suckau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter- Suermann, D., and Przybylski, M. (1990). Molecular epitope identification by limited proteolysis of an immobilized antigenantibody complex and mass-spectrometric peptide-mapping. Proc. Natl. Acad. Sci. USA 87, 9848-9851.

Yu, L., Gaskell, S. J., and Brookman, J. L. (1998). Epitope mapping of monoclonal antibodies by mass spectrometry: Identification of protein antigens in complex biological systems. J. Am. Soc. Mass Spectrom. 9, 208-215.

Zhao, Y., Muir, T. M., Kent, S. B. H., Tischer, E., Scardina, J. M., and Chait, B. T. (1996). Mapping protein-protein interactions by affinity- directed mass spectrometry. Proc. Natl. Acad. Sci. USA 93, 4020-4024.
 
     
 
 
     
     
 
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