Kinetics of Antigen−Antibody Interactions Employing a MALDI Mass

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Anal. Chem. 2008, 80, 7720–7726

Kinetics of Antigen-Antibody Interactions Employing a MALDI Mass Spectrometry Immunoassay Bethny Morrissey and Kevin M. Downard* School of Molecular and Microbial Biosciences, The University of Sydney, Australia Time-course MALDI mass spectrometry immunoassays have been shown to be able to detect differences in the relative rates of binding of peptides, both from within and across epitopic domains, with antibodies in non-competitive and competitive experiments. A monoclonal antibody raised to target the HA1 subunit of the hemagglutinin antigen of type A H3N2 influenza strains is found to recognize two epitopic peptides comprising residues 109-125 and 158-166 that likely form part of an extended discontinuous domain. Time-course experiments show the smaller peptide binds antibody at a rate that is 5-fold faster than that for the larger peptide. A shorter segment of this larger peptide, comprised of residues 119-125, is also found to bind at twice the rate of the extended peptide. Studies of modified peptide variants and synthetic variants of HA peptide 119-125 has enabled important contact residues to be identified whose accessibilities in the native protein are in accord with the mass spectrometry results. Antibodies presented on the surface of and secreted from B cells, and soluble antibodies present in the blood and lymphatic fluid, recognize and bind to antigens in a highly specific and directed manner.1 Their production, in response to a foreign pathogen, forms the first line of defense through the activation of the immune response. The binding of antibodies to foreign antigens of bacteria and viruses results in the agglutination or precipitation of antigen-antibody complexes that in turn promotes phagocytosis or blocks viral receptors. In the case of viral infection, B cells are capable of recognizing viral capsid proteins released from an infected cell ahead of viral assembly. The strong associations between antigens and antibodies are stabilized by hydrogen bonds, hydrophobic interactions, electrostatic, and van der Waals forces. These relatively weak noncovalent interactions are strengthened by the participation of multiple sites within the interaction interface of an antigen-antibody complex. The binding of an antigen (Ag) to an antibody (Ab) can be represented by a reversible bimolecular reaction (Ag + Ab a Ag-Ab). It is characterized by an equilibrium affinity constant * To whom correspondence should be addressed. Assoc. Prof. Kevin Downard, School of Molecular and Microbial Biosciences G-08, The University of Sydney, Sydney, NSW 2006, Australia. Phone: +61 (0)2 9351 4140. E-mail: [email protected]. (1) Diamandis, E. P., Christopoulos, T. K. Eds.; Immunoassay; Academic Press: New York, 1996.

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(Ka) that measures the ratio of the rates for the forward association reaction (ka) versus the reverse dissociation (kd) reaction.1,2 Immunoassays take advantage of the specific binding of an antibody to its antigen and are widely used to detect biological markers,3,4 diagnose a disease state,5,6 monitor the progression of a disease,7 or evaluate the success of drug treatments and therapies.8,9 Monoclonal antibodies are frequently employed in such assays due to their specificity for a particular molecule and particular sites within that molecule.10 These epitopes or antigenic determinants reside on the surface of an antigen and are accessible to the Fab portion of an antibody molecule. Immunoassays take many forms from and can be divided into those that employ chemical or biochemical labels and tags and those that do not.1 The label usually consists of an enzyme substrate (in the case of an ELISA),11,12 a radioisotope (RIA),13 or a fluorescence group or molecule.14 In a competitive immunoassay, two or more antigens, or components thereof, compete with one another to bind with an antibody. Surface plasmon resonance enables the detection and measurement of protein interactions including antigen-antibody interactions15 in real time, without the use of labels, though one of the interactants must be first successfully immobilized onto the sensor chip surface. All such approaches can detect and follow antigen-antibody interactions though do not directly identify the site that the antibody binds to the antigen surface. (2) Roitt, I. M. Essential Immunology, 9th ed.; Blackwell Science: Oxford U.K., 1997. (3) Mattoon, D.; Michaud, G.; Merkel, J.; Schweitzer, B. Expert Rev. Proteomics 2005, 2, 879–889. (4) Ling, M. M.; Ricks, C.; Lea, P. Expert Rev. Mol. Diag. 2007, 7, 87–98. (5) Meurman, O.; Ruuskanen, O.; Sarkkinen, H. J. Clin. Microbiol. 1983, 18, 1190–1195. (6) Meriggioli, M. N. Neurologic. Res. 2005, 27, 734–740. (7) Brockhurst, I.; Harris, K. P.; Chapman, C. Nephrol., Dial., Transplant. 2005, 20, 1251–1253. (8) Enomoto, M.; Nishiguchi, S.; Tamori, A.; Kohmoto, M.; Habu, D.; Sakaguchi, H.; Takeda, T.; Kawada, N.; Seki, S.; Shiomi, S. J. Med. Virol. 2005, 77, 77–82. (9) Shaw, L. M.; Korecka, M.; Clark, C. M.; Lee, V. M.-Y.; Trojanowski, J. Q. Nat. Rev. Drug Discovery 2007, 6, 295–303. (10) Goding, J. W. Monoclonal Antibodies: Principles and Practice, 3rd ed.; Academic Press: New York, 1996. (11) Engvall, E.; Perlman, P. Immunochemistry 1971, 8, 871–874. (12) Lequin, R. M. Clin. Chem. 2005, 51, 2415–2418. (13) Edwards, R. In Principles and Practice of Immunoassays; Price, C. P., Newman, D. J. Eds.; Stockton Press: New York, 1997; pp 325-348. (14) Hicks, J. M. Hum. Pathol. 1984, 15, 112–116. (15) Murphy, M.; Jason-Moller, L.; Bruno, J. Curr. Protoc. Protein Sci. 2006, 19.14, 1–17. 10.1021/ac801069q CCC: $40.75  2008 American Chemical Society Published on Web 09/25/2008

A matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) based immunoassay capable of both identifying and localizing epitopic domains within target antigens from single protein samples or protein mixtures was first reported in 1999.16 The approach was developed following the realization that specific antigen-antibody complexes could be preserved on,16 and even ionized from,17 conventional MALDI targets without the immobilization of either component, the recovery or capture of the complex, or the release of epitopic peptides. These are all requirements of other mass spectrometric based immunoassays18-20 where antibodies need to be immobilized prior to their interaction with antigen and the resulting complex must survive the recovery and washing processes ahead of the release the epitopic peptides for MS analysis. The immunoassay employed in this study,6 in contrast, achieves the specific preservation of antigenic peptideantibody complexes on a MALDI target that leads to the subsequent ionization of nonbinding peptides. A comparison of the resultant mass spectrum with that for an antibody-free control sample enables the identity of the antigenic peptides to be established by an absence, or a marked reduction in the relative area, of their ions.16,21-24 Note that there is no requirement that the amount of the peptide be quantified in this approach, only that it is detected in the control sample spectrum so that any reduction in the relative area of its ions can be followed in the spectrum recorded after antibody treatment. The immunoassay has since been successfully applied to monitor the antigenicity of the component antigens of the influenza virus using whole virus21 and protein antigens purified by gel electrophoresis.22-24 Two epitopic peptides within the hemagglutinin antigen of type A H3N2 strains of the influenza virus have recently been identified using this approach.23 These span residues 109-125 and 158-166 that both reside atop the HA1 subunit of the hemagglutinin antigen. These peptide segments are in close spatial proximity and may form part of a larger discontinuous epitope. A smaller peptide within the larger peptide, comprised of residues 119-125, also was found to bind the same monoclonal antibody and thus represents part of the refined epitope.23 The implementation of time-course experiments enables the relative rates that epitopic peptides bind with antibodies to be assessed, in both non-competitive and competitive immunoassays, by measuring the levels of unbound epitopic peptides that remain in the sample over time. In order to establish if the antibody exhibits any higher affinity for either of the two antigenic peptides, and thus whether one is immunodominant, time-course MALDIMS immunoassays have been performed and are described herein. Furthermore, synthetic peptides based on the sequence of the smaller portion of the larger epitopic peptide were also investigated in order to evaluate which of the amino acid residues are most (16) Kiselar, J. G.; Downard, K. M. Anal. Chem. 1999, 71, 1792–1801. (17) Kiselar, J. G.; Downard, K. M. J. Am. Soc. Mass Spectrom. 2000, 11, 746– 750. (18) Papac, D. I.; Hoyes, J.; Tomer, K. B. Protein Sci. 1994, 3, 1485–1492. (19) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153–1158. (20) Peter, J. F.; Tomer, K. B. Anal. Chem. 2001, 73, 4012–4019. (21) Kiselar, J. G.; Downard, K. M. Biochemistry 1999, 38, 14185–14191. (22) Morrissey, B.; Downard, K. M. Proteomics 2006, 6, 2034–2041. (23) Morrissey, B.; Streamer, M.; Downard, K. M. J. Virol. Methods 2007, 145, 106–114. (24) Downard, K. M.; Morrissey, B. Analyst 2007, 132, 611–614.

critical to binding and, in time-course experiments, which amino acid substitution(s) influence the rate of binding. MATERIALS AND METHODS Influenza Strains and Monoclonal Antibodies. The H3N2 influenza type A strain isolate (Panama 2007/99) was purchased from Advanced Immunochemical Inc. (Long Beach, CA). Two other H3N2 isolates, Shangdong 9/93 and Kiev 301/94 (Johannesburg/33/94-like), together with a monoclonal antibody (clone 12/5) derived from the hybridization of myeloma and spleen cells of mice immunized with the H3N2 serotype, were purchased from Research Diagnostics (Concord MA) and used without further purification. Monoclonal antibody Anti-HA (clone HA-7) was purchased from Sigma (Saint Louis, MO) and used without further purification or treatment. Eight peptides with amino acid sequences based on a localized epitopic peptide were synthesized by AusPep Pty. Ltd. (Parkville, Victoria, Australia) and used without further purification. Isolation of Hemagglutinin Antigen (HA) from Influenza Isolates. The hemagglutinin antigens from each of the type A H3N2 strains were separated by SDS-PAGE, digested in-gel with trypsin and the tryptic peptides extracted as previously described.22,23 Briefly, 30 µg of each isolate was added per lane in a 5-fold volume of loading buffer comprising 25 mM TrisHCl, 192 mM glycine,and 0.1% (w/v) of sodium dodecylsulphate at pH 8.3. The final concentration of peptide is estimated based the level of hemagglutinin in type A H3N2 strains representing 35% of the total viral weight.25 Therefore each hemagglutinin band contains 10.5 µg of HA. A systematic study of the recovery of tryptic peptides from gels reports yields of between 70-85%.26 Applying the lowest, most conservative recovery level (70%), the final concentration of total HA peptide was estimated to be 22, 29, and 15 µg in to 30 µL of 50 mM NH4HCO3 pH 8.5 for the Panama, Shangdong, and Kiev strains, where 3, 4, and 2 bands were used respectively. Time-Course MS Immunoassays with Tryptic HA Peptides. A portion (7 µg) of the monoclonal antibody solution (clone 12/5 at a concentration of 1 µg/µL in PBS buffer stabilized with 0.1% sodium azide) was removed and completely dried using the centrivap concentrator. The antibody was than resuspended in 15 µL of PBS buffer (20 mM phosphate, 100 mM NaCl, pH 7.8). A portion of the peptide solution (7.5 µg of total peptide) was added to the 15 µL antibody solution (creating an approximate 1:1 antigen to antibody w/w ratio) and the combined solution incubated at 4 °C for 1, 4, and 24 h. Aliquots (1 µL) of the solution were mixed with matrix solution (3 µL), comprising a saturated solution of R-cyano-4-hydroxycinnamic acid in acetonitrile and water (70:30 v/v) and deposited onto a MALDI target for subsequent analysis. Noncompetitive Time-Course MS Immunoassays with Synthetic Peptides. Stock solutions of each synthetic peptide at a concentration of 1 ng/µL in Milli Q water (Millipore Corporation, MA) were combined with both peptide T and substance P at concentrations of 1 and 3 ng/µL in water, respectively. The peptide solutions were prepared by mixing 13 µL of one synthetic peptide, 10 µL Peptide T, and 3 µL of substance P. A solution of monoclonal (25) Oxford, J. S.; Corcoran, T.; Hugentobler, A. L. J. Biol. Stand. 1981, 9, 483– 491. (26) Speicher, K. D.; Kolbas, O.; Harper, S.; Speicher, D. W. J. Biomol. Tech. 2000, 11, 74–86.

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antibody (4 µL of a solution of clone HA-7 at 1 µg/µL) was then added with 15 µL of PBS buffer to the mixed peptide solution and incubated at 4 °C for 4 h. This equates to a mole excess of monoclonal antibody to test peptide of 3:1. Aliquots of the peptide mixture were removed, mixed with MALDI matrix, and deposited onto the target after 5, 120, and 240 min intervals. Competitive Time-Course MS Immunoassays of Synthetic Peptides against PDYASLR. Peptide solutions were prepared by mixing 6.5 µL of synthetic peptide PDYASLR and 6.5 µL of PDYAALR, PDYASLL, or PDYASLE together with 10 µL Peptide T and 3 µL of substance P from the stock solutions. A solution of monoclonal antibody (4 µL of a solution of clone HA-7 at 1 µg/ µL) was then added with 15 µL of PBS buffer to the peptide solution and incubated at 4 °C for 4 h. This equates to a slight mole excess of monoclonal antibody to total synthetic peptide (excluding peptide T and substance P) of 3:1. Aliquots of the peptide mixture were deposited onto the MALDI target at 5, 120, and 240 min intervals. MALDI Mass Analysis of Antibody-Treated and Untreated (No mAb) Control Samples. MALDI-MS analysis was performed on antibody-treated and non-antibody treated control samples using an Applied Biosystems QSTAR-XL hybrid Q-TOF mass spectrometer operated in the positive ion TOF-MS mode. A portion of the antibody reaction mixture or untreated control (1 µL) was diluted in a saturated solution of R-cyano-4-hydroxycinnamic acid (3 µL) in acetonitrile and water (70:30 v/v) and deposited onto the sample stage (1 µL per spot). MALDI-MS spectra were acquired using Analyst QS (version 1.1) software by averaging data from approximately 100 laser shots per spot using a laser power of approximately 13.2 µJ and an ion extraction delay time of 12 µs. The sample stage was moved relative to the laser beam during acquisition to ensure the spectra were representative of all peptides within the mixture and were not associated with localized deposition and crystallization. The use of an organic solvent in the matrix solution facilitates the faster drying of samples and also ensures that the sample is distributed in a more homogeneous manner across the target. Calculation of the Relative Rates of Antibody Binding. The relative rates of peptide binding were measured from plots of their relative area of peptide ion signals as a function of the time that peptides were incubated with antibody. Peptide areas were based on a sum of the areas across the their isotopic distributions for all [M + H]+ ions in addition to those ion signals associated with salt adducts. The data were best fit to first order exponential decay curves (y ) A e-kx) using Prism v4.0 software (GraphPad Software, La Jolla, CA) and the rate coefficients (k) obtained from the natural log of the quotient. Data points represent the average values from duplicate experiments with three separate MS spectral data sets recorded per experiment. Note that some error bars are too small to be distinguished on the scale shown at some data points. Relative rates across pairs of peptide in competitive and noncompetitive assays were assessed from the ratio of their rate coefficients (kpeptide1/kpeptide2). RESULTS AND DISCUSSION Relative Rates of Antibody Binding Across Epitopic Peptides. The hemagglutinin antigen (HA) was separated from two H3N2 type A strains of the influenza virus (Kiev/301/94 and Shangdong/9/93) as previously described23 with the antigen then 7722

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Figure 1. MALDI mass spectra of the digested hemagglutinin antigen (and some unresolved nucleoprotein) of the type A H3N2 Kiev/301/ 94 influenza strain after treatment with a monoclonal antibody directed to the antigen for up to 24 h. Ions designated * are from matrix cluster ions while those designated # are trypsin autolysis products.

digested in-gel with trypsin. A proportion of the tryptic peptides were treated with a monoclonal antibody to HA and incubated for 24 h. Aliquots of the combined solution were removed after 1, 4, and 24 h and with MALDI mass spectra recorded at each time point and compared with the spectrum for a control (no antibody) sample. Representative mass spectra for the tryptic digests for the Kiev strain are shown in Figure 1. The mass spectra show major ions associated with the HA antigen at m/z 898.4, 1000.5, 1093.5, and 2067.9 corresponding to residues 51-57, 158-166, 99-106, and 109-125, respectively. The ions at m/z 951.5 correspond to residues 383-389 of the nucleoprotein antigen and thus represent a nonbinding peptide that serves as an internal control. Of the ions detected, only the peptide ions at m/z 1000.4 and 2067.8 are seen to progressively decrease in relative intensity following exposure of the peptides to the monoclonal antibody over increased incubation times. Plots of the areas for these peptide ions signals relative to the area of the ions at m/z 1093.5 are shown for the Kiev and Shangdong strains in Figure 2. The areas of the ion signals, over their intensities, are a better measure of the levels of peptide ionized from the target as they accommodate differences in the mass resolution and thus the width of the peaks across the mass scale. The data were best fit to a first order exponential decay curve defined by y ) A e-kx where y represents the relative area of the ion signals and x is the time of antibody incubation in hours. The coefficient k defines the rate of peptide binding and can be expressed in units of h-1.

Figure 2. Time course MALDI mass spectrometry immunoassay data showing the decrease in relative area of the ions of two epitopic peptides comprising residues 109-125 and 158-166 of the hemagglutinin HA1 subunit following their binding with a monoclonal antibody over 24 h. Data points represent the average values from duplicate experiments with three separate MS spectral data sets recorded per experiment.

The rates of binding of tryptic HA peptides comprising residues 158-166 (m/z 1000.5) and 109-125 (m/z 2067.9) from the Kiev strain were found to be 0.24 and 0.05 h-1, respectively. Thus the relative rate of binding in a competitive immunoassay favors the smaller peptide by a ratio of approximately 5:1. Similar results were obtained for the peptides derived from the Shangdong strain with rate coefficients measured to be 0.30 and 0.05 h-1 for the peptide ions at m/z 1000.5 and 2067.9, respectively. Here the relative rate of binding is 6 to 1. In both strains, the peptide aligned to residues 158-166 of the smaller determinant binds 5-6-fold faster than the peptide aligned to residues 109-125 of the larger determinant. Given that the smaller peptide has fewer contact residues available for interaction with the antibody, the favored binding of this epitopic peptide suggests that less steric restrictions, afforded by its smaller size, may assist in its binding in the competitive immunoassay. This smaller peptide of nine residues is consistent with the number of residues of an antigen’s surface usually implicated as constituting an epitopic domain. A second explanation to substantiate the preferential binding of the smaller peptide is that these residues are more prone to substitution through antigenic shift and drift. Several reports27,28 have suggested that dominant epitopes reside in regions prone to high antigenic change since they apply a selective pressure on antibodies to respond to the immunological challenge posed by a mutated antigen. In accord, the region comprising of this epitopic peptide has been shown to undergo significant antigenic change.29,30 This (27) Lee, M. S.; Chen, J. S.; Cho, I. Int. Congr. Ser. 2004, 1263, 626–631. (28) Lee, M. S.; Chen, J. S. Emerging Infect. Dis. 2004, 10, 1385–1390. (29) Jin, H.; Zhou, H.; Liu, H.; Chan, W.; Adhikary, L.; Mahmood, K.; Lee, M.S.; Kemble, G. Virology 2005, 336, 113–119.

Figure 3. Time course MALDI mass spectrometry immunoassay data showing the decrease in relative area of the ions of two peptides comprising residues 109-125 and 119-125 of a common epitope the hemagglutinin HA1 subunit following their binding with a monoclonal antibody over 24 h. Data points represent the average values from duplicate experiments with three separate MS spectral data sets recorded per experiment.

is also evident within the hemagglutinin antigen of the three H3N2 influenza strains examined in this study. In the case of the Shangdong/9/93 and Kiev/301/94 strains, the residues of HA 158-166 have the sequence GSVNSFFSR while in the Panama/ 2007/99 strain two amino acid substitutions results in the sequence being RSNNSFFSR. Conversely, the sequence for residues 109-125 of HA (AYSNCYPYDVPDYASLR) is conserved for all three strains. Relative Rates of Antibody Binding within Epitopic Peptides and the Effect of Peptide Size. Consistent with the importance of the size of epitopic peptides and steric effects impacting on the binding of peptides, a shorter segment of residues 119-125 comprising the C-terminus of the larger binding peptide is also found to bind antibody. The tryptic digestion of the Shangdong and Panama strains gave rise to two peptides with m/z values of 821.4 and 2072.8 comprised of residues 119-125 and 109-125 of the HA1 subunit, respectively. Both have been found to recognize the same monoclonal antibody.23 The binding of these two peptide ions was followed in time-course experiments to explore their relative binding rates as a function of peptide size. The peptide ions signals were monitored relative to those for ions at m/z 1093.5 representing a nonbinding peptide composed of residues 99-106 of the hemagglutinin antigen. Figure 3 shows the exponential decay profiles for the Shangdong and Panama strains reflecting the binding of the two peptides over an increasing incubation time with antibody. In the case of (30) Lindstrom, S. E.; Cox, N. J.; Klimov, A. Virology 2004, 328, 101–119.

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Table 1. Relative Peptide Ion Areas for Unsubstituted Epitopic Peptide and Substituted Peptides with No Antibody and after a 4 Hour Incubation with Antibody relative area (%)a

Figure 4. Time course MALDI mass spectrometry immunoassay data showing the decrease in relative area of the ions of two peptides comprising residues 109-125 of the hemagglutinin HA1 subunit with different modifications at the cysteine side chain (at position 113) following their binding with a monoclonal antibody over 24 h. Data points represent the average values from duplicate experiments with three separate MS spectral data sets recorded per experiment.

the Shangdong strain, the rate coefficients for the peptides HA 119-125 and HA 109-125 were found to be 0.06 and 0.03 h-1, respectively. The same relative rates were observed for those derived from the Panama strain, with rate coefficients measured for the smaller and larger epitopic peptide of 0.10 and 0.05 h-1, respectively. Thus for both strains, the smaller peptide comprised of residues 119-125 of the HA1 subunit binds at a rate twice that for the larger peptide comprised of residues 109-125 in a competitive immunoassay. These results clearly demonstrate the important role of the size of the peptide in the rate of antibody binding. Effect of Amino Acid Modification on Antibody Binding. In the case of the Kiev strain, two forms of the larger epitopic peptide comprised of HA residues 109-125 were detected in the MALDI mass spectra. These originate from the native form of the peptide at m/z 2072 in which the cysteine residue at position 113 supports a carboxyamidomethyl post-translational modification and a second form in which this modification is replaced with a β-hydroxyethyl group. This group is a result of the pretreatment of the antigen with β-hydroxyethanol ahead of digestion. These two peptide forms enable the impact of the cysteine modification on antibody binding to be assessed in a competitive immunoassay. The binding of these two peptide ions was followed in time-course experiments after incubation of the digested hemagglutinin antigen with antibody for 1, 4, and 24 h. The area of each of the two peptide ion signals were plotted relative to the area of the ions at 1093.5 and fit to a first order exponential decay curve (Figure 4). The relative rate coefficients of the peptides at m/z 2066 and 2072 were found to be 0.05 and 0.04 h-1, respectively, and thus the nature of the cysteine side chain has little impact on the binding of these two peptides with antibody. Effect of Amino Acid Substitutions on Antibody Binding. In order to determine the amino acids that are critical to binding within residues HA 119-125 of the refined epitope, eight peptides were synthesized based upon its sequence. These eight peptides successively replace the aspartic acid, tyrosine, serine, and arginine residues through amino acid substitutions. Each residue is replaced with a more hydrophobic residue with a side chain of a comparable size. Thus tyrosine is substituted with a phenyla7724

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peptide number

sequence

without Ab

error

with Ab

error

∆

binds mAb?

1 2 3 4 5 6 7 8

PDYASLR PDYASLE PDYASLL PDYAALR PDFASLR PDFAALR PAFAALR PAYAALR

139 154 56 108 83 68 40 46

25 57 11 30 15 4 11 19

32 65 9 40 74 38 36 24

8 24 8 8 25 11 11 8

107 89 47 68 9 30 4 22

Y Y Y Y N N N N

a Peptide ion areas are measured relative to the sum of the ion areas for peptide T and substance P added to the mixtures as controls. They reflect the total area under all ions of the isotopic distribution for [M + H]+ ions and any salt adducts detected. Results are an average of two separate MS immunoassay experiments from which three spectra were recorded for each sample.

lanine residue, the aspartic acid and serine residues are substituted successively with alanine residues, and the arginine residue was substituted successively with a leucine residue or a glutamic acid residue. The latter changes the charge character of the C-terminal residue from as highly basic to an acidic residue. The binding of each of the peptides was first examined in turn, in the presence of two nonbinding control peptides. These are peptide T and substance P with the amino acid sequences ASTTTNYT and RPKPQQFFGLM(NH2), respectively. Peptide T is a synthetically produced octapeptide derived from the gp120 protein present of the surface of the human immunodeficiency virus while substance P is a 11-amino acid neuropeptide that functions as a neurotransmitter and neuromodulator. Absolute changes in the areas of the peptide ions, relative to the sum of the areas for the ions for peptide T and substance T ions, before and after their individual treatment with the monoclonal antibody are illustrated in Table 1. Four peptides were found to bind the antibody while the other four peptides did not. In addition to the unsubstituted epitopic peptide, ions for the three peptides with sequences PDYASLE, PDYASLL and PDYAALR were found to decrease in relative area by 89%, 47%, 68%, respectively. In contrast, the relative ion areas for the other four peptides decrease by between 4-30%. Although the changes in area for two peptides are greater than 20%, they are within the 10-15% fluctuations associated with experimental errors16,21-24 when the errors from replicate experiments are considered. Noncompetitive time course immunoassays were conducted for each of the four binding peptides, in the presence of peptide T and substance P, and their rates of antibody binding (Figure 5). In these simple mixtures, the refined epitopic peptide and its substituted forms were found to bind rapidly where most peptide bound in the first 2 min. Rate coefficients were measured to range from 0.52-1.0 min-1 and thus the relative rates of binding differ by a factor of 2 or less. Adjusted to the concentration of antibody, this corresponds to an association rate of between (3-6) × 103 M s-1. These rates are in accord with those obtained by surface plasmon resonance31 where the association of a monoclonal (31) Nice, E. C.; McInerney, T. L.; Jackson, D. C. Mol. Immunol. 1996, 33, 659–670.

Table 2. Solvent Accessibility Data at Amino Acid Residue Side Chains of the Segment 109-125 of the HA Antigen Based upon a Reported Structure for the Precursor33 Using the GETAREA Algorithm34

Figure 5. Noncompetitive time course MALDI mass spectrometry immunoassay data showing the decrease in relative area of the ions of an epitopic peptide comprising residue 119-125 of the hemagglutinin HA1 subunit, and three other peptides of related sequence, following their binding with a monoclonal antibody over 240 min. Data points represent the average values from duplicate experiments with three separate MS spectral data sets recorded per experiment. The reduced fit evident in some experiments is associated with the rapid binding of these peptides with antibody within the first 5 min.

Figure 6. Competitive time course MALDI mass spectrometry immunoassay data after showing the decrease in relative area of the ions of an epitopic peptide comprising residue 119-125 of the hemagglutinin HA1 subunit (a) alone, and in the presence of each of the peptides (b) PDYAALR, (c) PDYASLL, and (d) PDYASLE in turn, following their binding with a monoclonal antibody for 0 (unshaded), 2 (light gray), and 4 (dark gray) h.

antibody to an epitopic peptide derived from the H3 hemagglutinin antigen was measured to be 1 × 104 M s-1. The native unsubstituted peptide also resides in a region previously implicated as an antigenic determinant.32 Competitive MS-immunoassays to examine any preferential binding of the unsubstituted peptide with each of the three substituted binding peptides in turn were also performed (Figure 6). The majority of the unmodified peptide PDYASLR binds when not in competition with any of the other peptides. After 2 h with antibody incubation, only 23% of the peptide remains unbound (32) Wilson, I. A.; Niman, H. L.; Houghten, R. A.; Cherenson, A. R.; Connolly, M. L.; Lerner, R. A. Cell 1984, 37, 767–778.

residue

amino acid

side chain surface area (A2)

ratio %

orientation of side chain

109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125

A Y S N C Y P Y D V P D Y A S L R

2 89a 34 58 1 3 4 19 22 1 42 63 83 33 2 0 11

3 49 44 51 1 1 4 10 19 1 40 56 43 51 2 0 5

in out in in in in in in out out in in in

a This is a phenylalanine residue in the structure of the precursor protein.33

according to the relative area of its ion signals. When in competition with three test peptides PDYAALR (b) PDYASLL (c) and PDYASLE (d), the amount of bound PDYASLR is reduced with between 30-51% unbound. However, outside the errors from replicate experiments, the differences are not significant and this suggests that the binding of the unsubstituted peptide does not compete with the binding of other the substituted peptides. Note that the antibody was present at a 1.5-fold mole excess over that for total binding peptide. Location and Accessibility of Substituted Amino Acids in the Hemagglutinin Antigen. The location and solvent accessibility of the amino acid residues of the immunodominant epitope across residues 109-125 of the hemagglutinin antigen is examined in the context 120 of a reported structure for a synthetic construct of the precursor protein.33 Solvent accessibilities of the side chains for all of the amino acid residues in this segment were derived from the based PDB file for the structure using the GETAREA algorithm.34 Their solvent accessibilities are considered in the context of a ratio, expressed as a percentage, that corresponds to the surface area of the residue’s side-chain within the antigen’s structure relative to a “random coil value”. The random coil value measures the average solvent-accessible surface area of the side chain of residue X in the tripeptide Gly-X-Gly across an ensemble of 30 random conformations. Residue side chains are considered to be solvent exposed if the ratio value exceeds 50% and to be buried if the ratio is less than 20%. Others (with ratios between 20-50%) are not defined either way. The results (Table 2) show that the side chains of residues at positions 109, 113-118, 124, and 125 are predicted to reside in the interior of the protein while the side chains of residues at positions 112, 120, and 122 are exposed to solvent with ratios value exceeding 50%. The remaining residues are unclassified as they have ratio lying within 20-50%. Thus of the seven residues within (33) Chen, J.; Lee, K. H.; Steinhauer, D. A.; Stevens, D. J.; Skehel, J. J.; Wiley, D. C. Cell 1998, 95, 409–417. (34) Fraczkiewicz, R.; Braun, W. J. Comput. Chem. 1998, 19, 319–333.

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the smaller C-terminal peptide (HA 119-125), the side chains of residues aspartic acid (at position 120) and alanine (at position 122) should have the greatest contact with antibody when the protein antigen is in its native form. In accord, when the aspartic acid residue is replaced among the amino acid substituted peptides (in peptides 7 and 8, Table 1) the peptide is found no longer to bind antibody. A similar observation is made when the tyrosine residue at position 123 is substituted with phenylalanine (peptide 5, Table 1). No such effect on binding is observed among peptides 2-4 (Table 1) where substitutions are made at residues 123 and 125, both of which are deemed to be buried in terms of the native structure of the antigen. Although the peptides in this study are derived synthetically or from digestion of the antigen (in the case of peptide 1 of Table 1), the directed binding of the antibody raised to whole antigen is nonetheless predetermined. The MS immunoassay results for the larger epitopic peptide comprising residues 109-125 in which the cysteine residue is modified differently is also in accord with the structural data. The cisteine residue side chain that is located toward the interior of the protein’s structure, and thus its modification, is found not to affect the rate of binding of the peptide (Figure 4). Further substitution of the arginine residue at position 125 and serine at position 123 does not alter the binding propensity of the peptide for antibody consistent with the location of these residue side chains in the protein’s interior. Finally, the structure for the hemagglutinin precursor protein shows that residues 109-125 and 158-166 reside in close proximity atop the HA1 subunit (Figure 7) suggesting that they form part of a discontinuous epitope. Thus the mass spectrometry immunoassay is able to identify such epitopes through the binding of their component peptides even where the antigen is digested ahead of its immunochemical treatment with antibody. CONCLUSIONS A time course MALDI mass spectrometry immunoassay has been demonstrated for the first time and is shown to be able to detect epitopic domains and monitor the relative rates of peptide segments within and across them. The role of specific amino acid residue side chains in the binding with antibody can be examined through the study of divergent strains and/or synthetic variants. The assay adds to the repertoire of mass spectrometric approaches for examining antigen-antibody interactions in particular, and protein complexes in general, without the need to immobilize, tag, or recover either component.

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Figure 7. Partial structure of the hemagglutinin antigen of a type A H3N2 strain based on that for a synthetic construct (PDB 1HA0)33 showing the locations of the side chains of residues 109-125 and the proximity of the two epitopic peptide domains. Residues 109-125 are shown in red, residues 159-165 in blue.

ACKNOWLEDGMENT The research was supported by funds from the Australian Research Council through a Discovery Project Grant (Grant DP0449800) to K. Downard. The mass spectrometer used in these studies was purchased with funds provided by Australian Commonwealth Government through the Department of Education, Science and Training and the University of Sydney under the Major National Research Facility (MNRF) scheme.

Received for review May 26, 2008. Accepted August 15, 2008. AC801069Q