Microscale Epitope Mapping by Affinity Capillary Electrophoresis

Figure 4 Epitope mapping of β-endorphin by ACE−UV: (A) tryptic digest, 2.9 pmol/μL, 5 s pressure injection; (B) tryptic digest, 5 s injection foll...
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Anal. Chem. 1997, 69, 3008-3014

Microscale Epitope Mapping by Affinity Capillary Electrophoresis-Mass Spectrometry Yelena V. Lyubarskaya, Yuriy M. Dunayevskiy, Paul Vouros,* and Barry L. Karger*

Barnett Institute and Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115

Using β-endorphin as a model system, a new microscale solution-based approach for linear epitope mapping based on affinity capillary electrophoresis-mass spectrometry (ACE-MS) is demonstrated. Tryptic peptides are separated in a neutral coated capillary and monitored by ultraviolet absorbance and electrospray mass spectrometry. Then, following injection of the peptide digest mixture, anti-β-endorphin antibody is injected. The peptide, which binds to the antibody, is captured and disappears from its migration time. Following this subtractionscreening procedure, the binding of the individually synthesized or isolated immunoreactive peptide is examined by the ACE-MS procedure to confirm that the epitope resides on the peptide. A series of truncated peptides can then be made and the precise epitope determined by ACE-MS. The method utilizes low femtomole amounts of antibody and peptide digest per run and is rapid and easily automatable. Epitope mapping, or determination of the antigen binding site to the antibody, is a useful tool for deciphering structure-function relationships of biological molecules. Several different approaches have been applied to study interactions of mono- and polyclonal antibodies with continuous (linear) or discontinuous (discrete or nonlinear) epitopes.1 Traditional methods for linear epitope mapping involve limited proteolysis of an antigen followed by immunoprecipitation or immunoblotting, with further isolation and molecular weight and/or sequence determination of the immunoreactive peptides.2,3 These procedures require tedious and timeconsuming isolation and sequencing of the peptides. Recently, mapping of antigenic determinants of proteins has been facilitated by rapid automated synthesis of selected peptides4-6 and combinatorial peptide libraries.7-9 Synthetic peptide mixtures are typically screened for the epitope by ELISA, for example by a competitive approach in which free and bound peptides compete (1) Morris, G. E. Methods in Molecular Biology; Epitope Mapping Protocols; Humana Press Inc.: Totowa, NJ, 1996; Vol. 66. (2) Pratt, L. H.; Cordonnier, M.-M.; Lagarias, J. C. Arch. Biochem. Biophys. 1988, 267, 723-731. (3) Shieh, H.-M.; Bass, R. T.; Wang, B. S.; Corbett, M. J.; Buckwalter, B. L. J. Endocrinol. 1995, 145, 169-174. (4) Bo¨ttger, V.; Stasiak, P. C.; Harrison, D. L.; Mellirick, D. M.; Lane B. Eur. J. Biochem. 1995, 231, 475-485. (5) Elsayed, S.; Stavseng, L. Int. Arch. Allergy Immunol. 1994, 104, 65-71. (6) Ko ¨nig, B.; Gra¨tzel, M. Biochim. Biophys. Acta 1994, 1223, 261-266. (7) Adler, S.; Frank, R.; Lanzavecchia, A.; Weiss, S. FEBS Lett. 1994, 352, 167170. (8) Savoca, R.; Schwab, C.; Bosshard, H. R. J. Immunol. Methods 1991, 141, 245-252. (9) Kramer, A.; Vakalopoulou, E.; Schleuning, W.-D.; Schneider-Mergener, J. Mol. Immunol. 1995, 32, 459-465.

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for the antibody.8 Although the method is reliable, nonspecific reactivity of the bound peptides can be an issue, giving rise to false positives. Screening of combinatorial libraries has also been used in conjunction with affinity chromatography.10 However, immobilization of the antibody onto a chromatographic column can result in steric hindrance of the antibody binding sites as well as, again, lead to nonspecific interactions. Other strategies of epitope determination involve recombinant DNA technologies, such as expression of random antigen fragments with modifications introduced at the DNA level.8,9,11-15 While these methods are mostly used for “fine-tuning” or thorough investigation of more precise epitopes, influence of a single amino acid or a group of amino acids on binding affinity, etc., they are time consuming and require significant a priori knowledge about an epitope of interest. To overcome the requirements of tedious procedures of traditional isolation and sequencing of the immunoreactive peptides, mass spectrometric approaches have recently been introduced.16 For example, mass spectrometry has been used for determination of the MW of the peptides selected by immunoprecipitation that allowed for rapid mapping of linear epitopes (“epitope extraction”).17 Identification of an epitope by limited proteolysis of an immobilized antigen-antibody complex with further molecular weight determination of the antibody-bound fragments by mass spectrometry (“epitope excision”) has also been demonstrated.16,18 These methods utilize solid phase approaches with the sequencing step performed off-line by MS. In the present study, we propose a new microscale solutionbased approach for linear epitope mapping using affinity capillary electrophoresis (ACE) coupled on-line with electrospray ionization mass spectrometry (ESIMS). ACE has recently been developed for solution-based binding studies19 and immunoassays.20-23 ACEMS has been successfully applied for combinatorial libraries (10) Evans, D. M.; Williams, K. P.; McGuinness, B.; Tarr, G.; Regnier, F.; Afeyan, N.; Satish, J. Nature Biotechnol. 1996, 14, 504-507. (11) Ellgaard, L.; Holtet, T. L.; Moestrup, S. K.; Etzerodt, M.; Thogersen, H. C. J. Immunol. Methods 1995, 180, 53-61. (12) Du Plessis, D. H.; Wang, L.-F.; Jordaan, F. A.; Eaton, B. T. Virology 1994, 198, 346-349. (13) Jorieux, S.; Gaucher, C.; Pie´tu, G.; Che´rel, G.; Meyer, D.; Mazurier, C. Br. J. Haematol. 1994, 87, 113-118. (14) Kam-Morgan, L. N. W.; Smith-Gill, S. J.; Taylor, M. G.; Zhang, L.; Wilson, A. C.; Kirsch, J. F. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3958-3962. (15) Frorath, B.; Abney, C. C.; Scanarini, M. J. Biochem. 1992, 111, 633-640. (16) Przybylski, M. Adv. Mass Spectrom. 1995, 13, 257-283 (17) Zhao, Y.; Muir, T. W.; Kent, S. B. H.; Tischer, E.; Scardina, J. M.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4020-4024. (18) Suckau, D.; Ko ¨hl, J.; Karwath, G.; Schneider, K.; Casaretto, M.; BitterSuermann, D.; Przybylski, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 98489852. (19) Chu, Y.-H.; Avila, L. Z.; Gao, J.; Whitesides, G. M. Acc. Chem. Res. 1995, 28, 461-468. (20) Shimura, K.; Karger, B. L. Anal. Chem. 1994, 66, 9-15. S0003-2700(97)00094-2 CCC: $14.00

© 1997 American Chemical Society

Table 1. Proteolytic Fragments of β-Endorphin Tryptic Digest 1 2 3 4 5 6

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys Asn-Ala-Ile-Ile-Lys Asn-Ala-Tyr-Lys Lys-Gly-Glu Gly-Glu

screening24 as an efficient method for identification of ligands that interact with the target. The ACE-MS methodology used in this work allows for rapid assessment of a protein binding site, followed by determination of the precise linear epitope. Furthermore, the solution method overcomes the drawbacks of the traditional heterogeneous assays, e.g., nonspecific interactions with the solid support, the need of immobilization chemistry, and the consequent steric hindrance of the binding sites. The approach combines the resolving power and microscale characteristics of CE (i.e., low femtomole quantities of analytes per run) with the on-line determination of both molecular weight and structure by mass spectrometry. The ACE-MS methodology for epitope mapping can be applied either in conjunction with limited proteolysis of an antigen or with synthesis of peptides corresponding to the sequence of the antigen. While previously ACE-MS was used for moderate affinity ligands with fast off-rates,24 the present approach can be potentially useful for screening of mixtures of peptides and other molecules for high binding affinity ligands to important protein targets, especially when the availability of the biological molecules is limited. The proposed methodology is demonstrated using human β-endorphin and its monoclonal antibody as a simple model system. Human β-endorphin is a potent neuropeptide and a major peptide hormone of the central nervous system and pituitary gland.25,26 Five N-terminal amino acids of β-endorphin constitute the linear epitope of this peptide (Table 1), which has high binding affinity to the human β-endorphin monoclonal antibody.25 EXPERIMENTAL SECTION Equipment. CE-UV experiments were performed using a P/ACE 2100 capillary electrophoresis system (Beckman Instruments, Fullerton, CA) in both polarity modes. Separation capillaries with covalent poly(vinyl alcohol) (PVA) coating27 were prepared in-house to reduce electroosmotic flow (EOF) and prevent adsorption to the capillary walls. Such columns are available commercially (Beckman Instruments). The capillaries had an effective length of 20-30 cm (total length 27-37 cm), with an inner diameter of 50-75 µm. Electrophoresis was monitored at 214 nm, and the temperature of the capillary was maintained at 25 °C. The samples were injected by pressure of 0.5 psi. The MS experiments were performed on a Finnigan TSQ 700 triple-quadrupole mass spectrometer (Finnigan MAT, San Jose, (21) Schmalzing, D.; Nashabeh, W.; Yao, X.-W.; Mhatre, R.; Regnier, F. E.;Afeyan, N. B.; Fuchs, M. Anal. Chem. 1995, 67, 606-612. (22) Pritchett, T.; Evangelista, R. A.; Chen, F.-T. A. Bio/Technology 1995, 13, 1449-1450. (23) Tao, L.; Kennedy, R. T. Anal. Chem. 1996, 66, 3899-3906. (24) Chu, Y.-H.; Dunayevskiy, Y. M.; Kirby, D. P.; Vouros, P.; Karger, B. L. J. Am. Chem. Soc. 1996, 118, 7827-7835. (25) Gramsch, C.; Meo, T.; Riethmu ¨ ller, G.; Herz, A. J. Neurochem. 1983, 40, 1220-1226. (26) Snyder, S. H.; Innis, R. B. Annu. Rev. Biochem. 1979, 48, 755-582. (27) Goetzinger, W.; Karger, B. L. PCT Int. Appl. WO9623220, August 1996.

CA) interfaced with a Finnigan atmospheric pressure ionization (API) source operated in the positive and negative electrospray ionization modes. For CE-MS experiments, the instrument was scanned over the mass range m/z 200-1400 at a rate of 2 s/scan. The ESI voltage was held at +4.5 kV (or -5 kV in negative ESI), with the heated capillary inlet at 150 °C (200 °C in negative ion mode analysis). The experimental design for on-line CE-MS consisted of a 1000R high-voltage power supply (Spellman, Plainview, NY), operated in the constant voltage mode and connected to a platinum electrode in a vial containing the running buffer. The CE capillary, coated with PVA, was 360 µm o.d. 50 µm i.d. and 35-40 cm in total length. The running buffers were either (a) 50 mM glycine/ acetic acid, pH 3.1; (b) 50 mM -aminocaproic acid/acetic acid, pH 5.1; (c) 20 mM Tris/cacodylic acid, pH 6.2; (d) 20 mM TES/ Tris, pH 7.5; or (e) 20 mM Tris/acetate, pH 8.0. The liquid sheath was a 1:3 (v/v) mixture of the running buffer/methanol at a flow rate of 1.5-2.5 µL/min. The analytes were separated as both cations and anions and detected in the positive or negative ESI modes, respectively. The applied voltage was 20-25 kV and the current 5-10 µA. The analytes were injected hydrodynamically at a height of 10-15 cm for 5-25 s, with injection volumes of 2-30 nL. Chemicals. Monoclonal human β-endorphin antibody was purchased from Boehringer Mannheim (Indianapolis, IN). Prior to CE-UV/MS, the antibody sample was reconstituted in water, partially desalted to approximately 50 mM NaCl or less, and concentrated to about 0.6-0.8 mg/mL of the antibody using Centricon-30 molecular weight cutoff membranes (Amicon, Beverly, MA). Tryptic digests were performed in 1 min at 37 °C, using a 1:100 ratio of enzyme/peptide in 50 mM Tris/HCl buffer, pH 8.5, with 10 µg of β-endorphin used for digestion. Trypsin (sequencing grade), Tris, TES, β-endorphin, Tyr-Gly-Gly-Phe-Met, and Tyr-Gly-Gly-Phe peptides were purchased from Sigma Chemical Co. (St. Louis, MO). Glycine, -aminocaproic acid, and acetic acid were from Fluka Chemical Corp. (St. Louis, MO). RESULTS β-Endorphin Binding by CE. CE-UV binding experiments were initially performed with undigested β-endorphin and its monoclonal antibody. In general, these experiments could be important to test initially the immunoreactive activity of the intact antigen under the conditions of the experiment. Human β-endorphin, 8 µg/mL, and the antibody, approximately 0.5 mg/mL, were analyzed individually by CE-UV. The experiments were performed at pH 6.2, where both β-endorphin and the antibody were positively charged (pI of β-endorphin is above 9; pI of the antibody is roughly 6.5-7.0). Sufficient separation of the analytes was achieved (Figure 1A,B), and subsequent binding experiments were carried out at this pH (Figure 1C,D). Human β-endorphin and the antibody were mixed and incubated off-line prior to electrophoresis. When the mixture was analyzed by CE, the peak of β-endorphin was not observed at its original position in the electropherogram, since it was presumably tightly bound to the antibody (Figure 1D). The antibody/β-endorphin complex comigrated with the free antibody peak, with only a slight change in the original shape. The influence of β-endorphin on the electrophoretic mobility of the formed complex was negligible due to the minor effect on the mass and charge that β-endorphin had on the mobility of the antibody. Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

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Figure 2. CE-MS of β-endorphin tryptic digest. Concentration, 46 pmol/µL. CE-MS conditions: buffer, 50 mM glycine/acetic acid, pH 3.1; capillary, 50 µm i.d., 35 cm total length, 20 kV, 7 µA, 10 s hydrodynamic injection. TIE, total ion electropherogram.

Figure 1. Binding of β-endorphin to its monoclonal antibody by ACE-UV: (A) β-endorphin, 2.9 pmol/µL, 5 s pressure injection; (B) antibody (AB), 4.7 pmol/µL, 5 s injection; (C) 1:1 mixture of β-endorphin and AB, 5 s injection; (D) 1:2 mixture of β-endorphin and AB, 5 s injection. CE conditions: buffer, 20 mM TRIS/cacodylic acid, pH 6.2; PVA coated capillary, 75 µm i.d., 20/27 cm length; voltage, 25 kV; current, 11 µA.

To minimize the amount of the antibody consumed in each binding experiment, mixing and interaction of β-endorphin with its antibody was performed inside the capillary. The individual analytes were sequentially injected into the CE capillary (“plugplug”),28,29 where on-line mixing was performed, as the binding on-rate for β-endorphin to the monoclonal antibody is presumably fast, with the off-rate slow.10 Since, as already shown in Figure 1, β-endorphin had a higher electrophoretic mobility than the antibody at the CE conditions used, injection of the antibody sample was followed by the injection of β-endorphin in order for the latter to migrate electrophoretically, without dilution, through the plug of the antibody while interacting with it. The antibody plugs of different length were introduced into a capillary, followed by a constant 5 s injection plug of β-endorphin. The peak of β-endorphin disappeared from the electropherogram when a sufficient amount of the antibody (∼60 fmol) was added. These results were identical to those presented in Figure 1. Tryptic Digestion of β-endorphin. The proteolytic digestion of β-endorphin was performed using trypsin. Normally an investigated protein would be first subjected only to limited (28) Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217-224. (29) Avila, L. Z.; Chu, Y.-H.; Blossey, E. C.; Whitesides, G. M. J. Med. Chem. 1993, 36, 126-133.

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proteolysis in order to minimize cleavage through the binding region. In the case of our model system, however, total tryptic digestion was appropriate. The digestion conditions were optimized by changing the reaction time, temperature, and enzyme/ peptide ratio. The optimized reaction was performed for 1 min at 37 °C in 10-20 µL volume using 0.8 mg/mL β-endorphin solution with a trypsin/ β-endorphin ratio of 1:100. Figure 2 shows the CE-MS analysis of tryptic digest performed in glycine/acetic acid CE buffer, pH 3.1, where all the formed peptides could be detected in one run. Each peak was identified by selected ion monitoring. Nonspecific digestion fragments and traces of autoproteolysis of trypsin were not detected. Originally, the formation of five digestion fragments (1, 2, 3, 4, and 6) was predicted (Table 1), since trypsin was expected to cleave every Lys-X bond. However, formation of Lys-Gly-Glu consistently took place, while only very low levels of peptide 6, Gly-Glu, were detected. The reason that trypsin did not completely hydrolyze peptide 5 was that the digestion activity of the enzyme is partially impaired at sites where several adjacent basic amino acid residues are located in the vicinity of an acidic amino acid residue,30 as in β-endorphin: (28)Lys-Lys-Gly-Glu(31). Determination of the Immunoreactive Fragment in the Tryptic Digest. Once the success of the proteolysis was established, the β-endorphin tryptic digest was investigated for the presence of the immunoreactive fragments. To optimize the conditions for ACE experiments, the digest and the antibody were analyzed individually under binding buffer conditions at different pHs. The peptides and the antibody were analyzed and detected as cations at pH 5.1 and 6.2, and as anions at pH 7.5 and 8.0. At pH 6.2, only three peptides (2, 3, and 4) were observed, since the other peptides were neutral or negatively charged at this pH. Under this condition, the antibody peak was close to its pI (6.57.0) and migrated slower than the three peptides (data not shown). On the other hand, at pH 5.1, the antibody peak migrated faster than any of the peptides of the digest (Figure 3A), and five fragments of the digest (1-5), well separated from each other, were detected (Figure 3B). Peptide 6, Gly-Glu, was negatively charged and was not observed. Alternatively, when analyzed as (30) Burrell, M. M. Methods in Molecular Biology; Enzymes of Molecular Biology; Humana Press Inc.: Totowa, NJ, 1993; Vol. 16.

Figure 4. Epitope mapping of β-endorphin by ACE-UV: (A) tryptic digest, 2.9 pmol/µL, 5 s pressure injection; (B) tryptic digest, 5 s injection followed by 5 s injection of AB, 4.7 pmol/µL; (C) tryptic digest, 5 s injection followed by 10 s injection of AB. CE conditions: buffer, 50 mM -aminocaproic acid/acetic acid, pH 5.1; capillary, 75 µm i.d., 20 cm effective length, 27 cm total length, 20 kV, 19-22 µA.

Figure 3. CE-UV of β-endorphin monoclonal antibody and β-endorphin tryptic digest at pH 5.1 (A,B) and 7.5 (3C,D): (A) AB, 4.7 pmol/µL, 5 s injection; (B) tryptic digest, 2.9 pmol/µL, 5 s injection; (C) AB, 4.7 pmol/µL, 10 s injection; (D) tryptic digest, 5 s injection, 4.6 pmol/µL. CE conditions at pH 5.1: buffer, 50 mM -aminocaproic acid/acetic acid; capillary, 75 µm i.d., 20 cm effective length, 27 cm total length, 20 kV, 19-22 µA. CE conditions at pH 7.5: buffer, 20 mM TES/TRIS; capillary, 75 µm i.d. 20 cm effective length, 27 cm total length, 20 kV, 16 µA.

anions at pHs 7.5 and 8.0, only two peptides, 1 and 5, were negatively charged and thus detected, whereas fragment 6, GlyGlu, was not observed at the level of the digest concentration used. The antibody again migrated faster than the peptides (Figure 3C,D). The identity of the tryptic digest fragments at pH 5.1 and 8.0 was determined in parallel CE-MS experiments. Since for this model example all the essential peptides were detected in one run and all the peptides were separated from the antibody at pH 5.1, this pH was selected for the corresponding CE and CEMS binding studies. The binding experiments were performed using the “plug-plug” injection procedure in the capillary. The antibody and β-endorphin tryptic digest were sequentially pressure injected into the capillary, after which high voltage was applied and electrophoresis carried out. At pH 5.1, the antibody peak migrated faster than any peptide of the digest; therefore, the plug of the digested sample was injected first into a CE capillary, followed by injection of the antibody. Analogous to the experiments described above with β-endorphin, the sequential injection and applied voltage allowed mixing and interaction of the components inside the capillary during the electrophoretic run. With increasing amounts of the antibody injected (Figure 4B,C), a peak of the immunoreactive peptide containing the epitope sequence decreased and then disappeared completely from the electropherogram. The complex formed was presumed to comigrate with the antibody. The same experiments with the “plug-plug” injection procedure were performed by CE-MS. Figure 5A,B presents extracted ion electropherograms of the peptides without and with the antibody injected, respectively. Upon the injection of ∼70 fmol of the antibody, the immunoreactive peptide disappeared from the

electropherogram. The two peptides (3 and 4), partially obscured by the antibody peak in CE-UV experiments (Figure 4), were unambiguously identified in the CE-MS run. Neither the antibody nor the antibody/immunoreactive peptide complex was detected in the mass range examined. Although in the model example shown, one set of experimental conditions was sufficient for identification of the binding fragment, in a more general case other acidic or neutral peptides may exist in the digest which might not be detected as cations and would have to be analyzed as anions. Taking that into consideration, another set of ACE-UV experiments was performed under basic conditions, using 20 mM TES/Tris electrophoretic buffer at pH 7.5 (Figure 6). Analogous experiments were performed by ACEMS, using 20 mM Tris/acetate buffer at pH 8.0 (at pH 7.5, the Tris/acetate buffer would be highly conductive). This latter buffer was preferred to 20 mM TES/Tris for ACE-MS, since the Tris/ acetate was more compatible with the on-line MS detection. Identical ACE-UV results were obtained for the two buffer systems. Figure 6, however, shows the results for 20 mM TES/ Tris to demonstrate the flexibility in the choice of buffer system. The peptides 1 and 5 were separated as anions in on-line ACE/ MS and detected in the negative ion ESI mode (Figure 7). The antibody binding experiments were performed in the same manner as those at pH 5.1, with the “plug-plug” injection, electrophoretic mixing, and binding in the capillary. The peak corresponding to fragment 1 disappeared upon binding when a sufficient amount of the antibody was injected (Figure 7). After subtraction-screening for the immunoreactive peptide, the binding of the individually synthesized immunoreactive peptide (peptide 1, Table 1) to the antibody was confirmed by the ACEUV experiments at pHs 5.1 and 7.5. Under both pH conditions, the peptide disappeared from the electropherogram upon the addition of the excess antibody (data not shown). Based on these results, the determination of the precise epitope can be accomplished, as described below. Determination of the Precise Epitope. The precise linear epitope was determined by performing binding studies with synthetic truncated peptides derived from the identified immunoreactive sequence. Since the precise epitope of human β-endorphin to its monoclonal antibody is known, the binding studies Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

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A

B

Figure 5. Epitope mapping by ACE-MS in the positive ion ESI mode: (A) tryptic digest, 4.6 pmol/µL, 10 s pressure injection; (B) tryptic digest, 10 s injection followed by 25 s injection of AB (4.7 pmol/µL). Peptides 1 and 2 were detected as doubly charged positive ions. CE conditions: buffer, 50 mM -aminocaproic acid/acetic acid, pH 5.1; 50 µm i.d. capillary, 35 cm total length, 20 kV, 8 µA. TIE, total ion electropherogram. The panels are labeled by the peak numbers (see Table 1); the numbers above the peaks indicate migration times.

Figure 6. Epitope mapping by CE-UV: (A) tryptic digest, 10 s pressure injection, 4.6 pmol/µL; (B) AB, 10 s injection, 4.7 pmol/µL; (C) tryptic digest, 10 s injection followed by 10 s injection of AB, 4.7 pmol/µL; (D) tryptic digest, 10 s injection followed by 15 s injection of AB, 4.7 pmol/µL. CE conditions: buffer, 20 mM TES/TRIS, pH 7.5; capillary, 75 µm i.d., 20 cm effective length, 27 cm total length, 20 kV, 16 µA.

Figure 7. Epitope mapping by ACE-MS in the negative ion ESI mode: (A) tryptic digest, 4.6 pmol/µL, 10 s pressure injection; (B) tryptic digest, 10 s injection followed by 15 s injection of AB (4.7 pmol/ µL) [Note: peak intensity of fragment 1 decreased two times]; (C) tryptic digest, 10 s injection followed by 30 s injection of AB [Note: fragment 1 disappeared]. CE-MS conditions: buffer, 20 mM TRIS/ acetic acid, pH 8.0; capillary, 50 µm i.d., 35 cm total length, 20 kV, 10 µA.

were performed with the epitope, N-terminal pentapeptide, and with lipotropin, N-terminal tetrapeptide of β-endorphin. The structures of both peptides were confirmed by means of MS/MS

experiments. Binding of the precise epitope, Tyr-Gly-Gly-PheMet, to the antibody was demonstrated by CE-UV at pH 7.5, with the disappearance of the peptide upon the addition of the antibody

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Figure 8. Determination of the precise epitope (pentapeptide) and a truncated N-terminal tetrapeptide (lipotropin) by CE-UV: (A) pentapeptide, 8.7 pmol/µl, 5 s pressure injection; (B) pentapeptide, 5 s injection followed by 20 s injection of AB, 4.7 pmol/µL; (C) lipotropin, ∼10 pmol/µl, 5 s injection; (D) lipotropin, 5 s injection followed by 20 s injection of AB, 4.7 pmol/µL. CE conditions: buffer, 20 mM TES/TRIS, pH 7.5; capillary, 75 µm i.d., 20 cm effective length, 27 cm total length, 20 kV, 16 µA.

(Figure 8A,B). Lipotropin, Tyr-Gly-Gly-Phe, was found not to bind tightly to the antibody. The peak of this tetrapeptide broadened (the peak areas of lipotropin in Figure 7C and D are approximately the same) but did not disappear from the electropherogram upon addition of a comparable excess amount of the antibody as in Figure 8B, suggesting a much lower binding affinity. In the case of a more complex system where the precise epitope location is unknown and/or a determined immunoreactive fragment constitutes a much longer (poly)peptide chain, several peptides derived from the found sequence could be synthesized or obtained by further proteolysis for subsequent investigation of the binding affinity.3,17 DISCUSSION A microscale solution strategy has been demonstrated as a new alternative for continuous epitope mapping using on-line ACE-ESI-MS. This technique can be applied to high-affinity systems, when the antibody-antigen complex has a slow off-rate, such that the complex is stable during the course of the CE experiment. The proposed microscale technique is very economical. Due to the limitations of sample handling, at least 10 µL of the 0.7 µg/µL antibody solution and about 0.2 µg of the digest had to be used in the off-line mixing experiments. However, with the “plug-plug” injection procedure, followed by electrophoretic mixing and incubation in-capillary (if necessary), low femtomole quantities of sample were consumed in each experiment. The solution methodology has the following additional advantages compared to solid phase assays: (1) nonspecific interactions with

the adsorbent surface are avoided, (2) the requirements of chemical immobilization to the support are eliminated, and (3) binding kinetics in solution are typically fast. It is to be noted that the subtractive screening technique described here provides an indirect determination of the linear epitope. However, use of synthesized peptide from the presumed tryptic fragment provides strong evidence of the binding sequence and thus the epitope structure. In cases where the molecular weight information is not sufficient for unambiguous epitope determination, direct structural elucidation by a second-order MS/MS experiment can be conducted. Furthermore, prior to the on-line ACE/MS experiments, optimization of the experimental conditions can be done automatically by ACE-UV alone. Before the ACE-UV/MS experiments are performed, the protein is subjected to partial proteolysis by trypsin or another protease, where only a limited number of peptide fragments are obtained, and the digest is analyzed by CE-MS under conditions typical for peptide mapping (pH about 3) in order to identify the peptides present in the digest. The binding experiments must be performed in the limited pH range (roughly 4.5 < pH < 8.5), where the antibody-antigen complex is stable. Under these conditions, some of the proteolytic fragments may be negatively and others positively charged, depending on their pI values. Since EOF is greatly reduced using neutral covalently coated capillaries, the CE-MS experiments have to be performed in both polarity modes at binding pHs in order not to exclude any of the peptides of the diverse digest. First, the digest and the antibody are analyzed by CE-UV/MS individually as cations at a chosen pH value (below pH 7). Then, an excess amount of the antibody is preincubated with the digest sample and analyzed by CE-MS under identical conditions. If an immunoreactive peptide(s) is present in the partial proteolytic digest, it will bind to the antibody and will disappear in the electropherogram at its original position. When using the “plug-plug” approach, the injection order will depend on electrophoretic mobility of the interacting analytes. In a general case where some peptides migrate faster and others slower than the antibody, several options could be considered. For example, two separate “plug-plug” experiments with the injection order inverted can be performed, or, as described above, off-line mixing of the analytes prior to the injection can be used. To analyze more acidic peptides, a similar set of experiments is performed, where the fragments are separated as anions in biological buffers of higher pHs (above pH 7). A set of electrophoretic conditions in the described arrangement (maybe more than one) would allow probing all the peptides for tight binding affinity to an antibody and determination of the linear epitope. Since the mass and charge difference between the epitopeantibody complex and the antibody is small, the electrophoretic behavior of the two species will be similar. The antibody and the complex will also behave similarly in the MS, and neither can be detected by ESIMS in the m/z range monitored under the described experimental conditions. Collision-induced dissociation of the complex in the electrospray ion source was attempted by changing the orifice potential of the mass spectrometer. Unfortunately, this dissociation could not be achieved, presumably for several reasons. First, the rapid dissociation of a high- affinity complex formed by a very large protein may not be easily accomplished. A further complication in our example is the possible initiation of covalent bond cleavage or formation, along with the breakup of noncovalent associations, which could create Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

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ambiguity in the epitope assignment. Also, the complex must be ionized in order to dissociate in the source of the mass spectrometer, which is not readily achievable under the CE conditions used. If the antibody and the complex overlap with a peptide or a group of peptides in a CE analysis, such overlap may cause suppression of the peptide ionization and lead to false positives. To avoid such an occurrence, the same set of experiments may be performed at different pHs. Based on significant differences in electrophoretic behavior of an antibody vs peptides, it is relatively simple to choose appropriate separation conditions. For example, peptides 3 and 4 partially overlap with the antibody at pH 5 (Figure 6B,C), but they are well separated from the antibody at pH 6.2 in the same polarity mode (data not shown). Beyond epitope mapping, the proposed technique can potentially serve as an effective approach for assaying mixtures of compounds for tight binding affinity to biological targets. In this

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Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

solution phase methodology, when proteins are used as targets, the full molecular surface is probed for binding. A solution assay option is very important in cases where locations of binding sites are unknown or immobilization of a biological target-molecule onto a solid support can affect the binding affinity. ACKNOWLEDGMENT The NIH grants GM15847 (B.L.K.) and 1R01CA 69390-01 (P.V.) are gratefully acknowledged. This is Contribution No. 679 from the Barnett Institute. Received for review January 24, 1997. Accepted May 9, 1997.X AC9700944 X

Abstract published in Advance ACS Abstracts, July, 1, 1997.