Chips from Chips: Application to the Study of Antibody Responses to Methylated Proteins Gae¨lle Piret,† Re´mi Desmet,‡,§,|,∇ Eric Diesis,‡,§,|,∇ Herve´ Drobecq,‡,§,|,∇ Je´rome Segers,§,|,⊥,#,∇ Carine Rouanet,§,|,⊥,#,∇ Anne-Sophie Debrie,§,|,⊥,#,∇ Rabah Boukherroub,† Camille Locht,§,|,⊥,#,∇ and Oleg Melnyk*,‡,§,|,∇ Institut de Recherche Interdisciplinaire (IRI), CNRS USR 3078, Villeneuve d’Ascq Ce´dex, France, CNRS UMR 8161, France, Institut Pasteur de Lille, Lille, France, IFR 142 Molecular and Cellular Medicine, Lille, France, INSERM U1019, Center for Infection and Immunity, Lille, France, CNRS UMR 8204, Lille, France, and Univ Lille Nord de France, Lille, France Received July 9, 2010
Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named “chips from chips”, which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen. Keywords: peptide microarray • chemical modification • desorption/ionization • silicon nanowires • mass spectrometry • Mycobacteria • heparin-binding hemagglutinin (HBHA) • lysine • methylation • reductive alkylation
1. Introduction Peptide microarrays are useful devices for high-throughput studies of biomolecular or peptide-cell interactions. In particular, the parallel detection of antibodies in biological samples has a wide range of potential applications in the diagnosis of allergies, autoimmune and infectious diseases, as well as in epitope mapping and the development of vaccines.1-7 Of particular interest is the design of peptide microarray strategies for deciphering the role of post-translational or other modifications on antigenicity. Usually, post-translational modifications are introduced on peptide chains during their solid phase peptide synthesis and before microarray printing. However, the synthesis of peptide libraries incorporating post* To whom correspondence should be addressed. E-mail: oleg.melnyk@ ibl.fr. † Institut de Recherche Interdisciplinaire (IRI). ‡ CNRS UMR 8161. § Institut Pasteur de Lille. | IFR 142 Molecular and Cellular Medicine. ⊥ INSERM U1019, Center for Infection and Immunity. # CNRS UMR 8204. ∇ Univ Lille Nord de France. 10.1021/pr100707t
2010 American Chemical Society
translational or unnatural modifications can be time-consuming and expensive. We describe here a method named chips from chips for probing the presence of antibodies directed toward modified peptide antigens, starting from unmodified peptide libraries and microarrays (Figure 1). The method requires first the preparation of unmodified peptide microarrays, that is, the parent chip. Then, the peptide microspots are chemically modified by the application of simple chemical reactions. For this, parent chips are covered by the reagent solution, thereby allowing the modification of specific residues to occur, yielding daughter peptide chips. Both parent and daughter chips can then be used for interaction studies. The signal differences between parent and daughter chips give useful information on the importance of a particular modification on antibody recognition. In essence, the chip from chip method shares some similarities with the “libraries from libraries” concept developed by Houghten et al.,8 who used a wide range of selective chemical transformations to derivatize peptide collections in parallel and expend the diversity of chemical libraries. Both strategies rely on the use of unmodified peptide collections that are easy to synthesize. Journal of Proteome Research 2010, 9, 6467–6478 6467 Published on Web 10/16/2010
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Figure 1. “Chips from chip” concept. Chemical treatment of a parent microarray generates a series of daughter microarrays.
The chip from chip concept is illustrated here with lysine as the target residue for modification. Lysine is indeed the subject of various post-translational modifications, such as acetylation; mono-, di-, or trimethylation; glycation;9 and the generation of glycation end products, such as carboxymethylation.10 Lysines within proteins can also be modified by various xenobiotics and generate autoimmune effects.11 A wellknown example is acetylsalicylic acid (aspirin), which acetylates proteins in vivo.12 A similar in vivo N-epsilon acylation by cocaine was observed in human subjects chronically exposed to cocaine.13 Most of the above modifications can be introduced selectively on N-epsilon amino groups using simple chemical reactions, such as acylation or reductive alkylation. Consequently, applications of the chip from chip concept to the study of lysine modifications may be of high biological relevance. We applied in this proof of concept study the chip from chip method to study the role of heparin-binding hemagglutinin (HBHA) methylation on antigenicity. HBHA is a surfaceassociated protein produced by the members of the Mycobacterium tuberculosis complex (for a recent review, see ref 14). It is a 21-kDa protein that binds to heparan sulfate (HS)-containing glycoaminoglycans on the surface of nonphagocytic cells, including alveolar epithelial cells. This binding is thought to be responsible for the extra-pulmonary dissemination of M. tuberculosis, one of the key steps in the pathogenesis of tuberculosis. In addition to being an important virulence factor, HBHA also appears to be a strong immunogen, potentially useful for the diagnosis, especially of latent infection,15 and for the development of novel, acellular vaccines. However, native HBHA (nHBHA-MT) is a methylated protein,16 and both antigenicity in infected humans, as well as the induction of protective immunity rely very strongly on the proper methylation pattern of the antigen.17 The methylation involves the C-terminal domain of HBHA and corresponds to the last 41 amino acids, from L158 to K198.16 This domain is highly basic and contains 15 lysines, 13 of which are mono or dimethylated, resulting in a complex methylation pattern. The methylation of this functionally important domain protects it against proteolytic degradation. The importance of the methylation for HBHA-specific immune responses has been established both at the T cell17 and at the B cell levels.18 However, the humoral response against the methylated domain of HBHA is poorly characterized up to date. In the first part of this study, we characterized the peptide microspot methylation using fluorescence. In particular, the 6468
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rate of methylation was studied using nHBHA-derived peptides, specific antibodies and fluorescence detection. We have also developed an innovative method for characterizing the in situ chemical modification of peptide microspots by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful technique that allows the analysis of a wide variety of compounds and in particular peptides and proteins.19 However, due to a competitive desorption of parasitic ions from the matrix, it is difficult to detect low molecular weight compounds (10 000 A.U.), as compared to the same spots on parent chips (Figure 7A), whereas the signal displayed by the control peptides HA, FLAG, and myc remained below a detectable level. For peptide 3M and, to a lesser extent, for 2M, microspot methylation resulted in an increase in spot intensity (for 3M from 687 to 13 324 A.U.). The microscope glass slide 6476
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substrates used for microarray printing are silanized with 3-aminopropyltrimethoxysilane, that is, present primary amino groups, which can be methylated too. Methylation reduces the density of NH groups on the substrates that can form hydrogen bonds with the adsorbed peptide. In this context, a reorientation of the peptide on the surface induced by the chemical treatment might occur, especially for peptide 3M which is the smallest peptide used in this study (12 amino acids). A small change in the mode of presentation of some amino acids at the solid-liquid interface may result in a large change in the accessibility to antibodies. Apparently, this effect was not observed when methylated microspots were probed with polyclonal Ab7315 (Figure 3B). A larger effect of a conformational change of peptides on antibody binding might be observed for monoclonal antibodies raised against nHBHA-MT, that is, which bind simultaneously to several residues within peptide 3M, than for Ab7315, which binds essentially to dimethyllysine residues. When parent and methylated daughter chips were probed with pooled sera from naı¨ve mice, no significant increase in
Chips from Chips the spot intensities was observed, except for protein A, as expected (Figure 7B). Thus, taken together, these data show that sera from nHBHA-MT-immunized mice bind to methylated peptides derived from the C domain, but not with unmodified peptides. More generally, this part of the work shows the usefulness of the chip from chip method for studying serum reactivity.
4. Conclusion We have developed a novel microarray tool named “chip from chip”, which is based on the chemical modification of peptide microspots to produce daughter chips from parent chips. This microarray can be used to interrogate purified or biological samples and provide useful information on the binding properties of soluble targets. We have also presented a MS method to characterize the chemical modification of peptide microspots. For this, peptide microarrays were prepared on silicon nanowire substrate and chemically modified before surface-assisted LDI-MS analysis. We believe that the chip from chip method and MS characterization technique described in this report will be valuable tools for biological research, and in particular for the study of lysine modifications.
Acknowledgment. We thank the CNRS, Universite´ de Lille Nord de France, Institut Pasteur de Lille and IFR142 for financial support. This work was performed in the Chemistry Systems Biology facility of Institut de Biologie de Lille (http:// csb.ibl.fr), which was financed by CNRS, the Re´gion Nord Pas de Calais, the European Community, and le Ministe`re de l’Enseignement Supe´rieur et de la Recherche. Supporting Information Available: Experimental procedures and characterization data for peptides 1-17 and 1-17M. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Robinson, W. H.; Fontoura, P.; Lee, B. J.; de Vegvar, H. E.; Tom, J.; Pedotti, R.; DiGennaro, C. D.; Mitchell, D. J.; Fong, D.; Ho, P. P.; Ruiz, P. J.; Maverakis, E.; Stevens, D. B.; Bernard, C. C.; Martin, R.; Kuchroo, V. K.; van Noort, J. M.; Genain, C. P.; Amor, S.; Olsson, T.; Utz, P. J.; Garren, H.; Steinman, L. Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis. Nat. Biotechnol. 2003, 21 (9), 1033–9. (2) Robinson, W. H.; Steinman, L.; Utz, P. J. Protein arrays for autoantibody profiling and fine-specificity mapping. Proteomics 2003, 3 (11), 2077–84. (3) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Protein microarray technology. Drug Discovery Today 2002, 7 (15), 815–22. (4) Talapatra, A.; Rouse, R.; Hardiman, G. Protein microarrays: challenges and promises. Pharmacogenomics 2002, 3 (4), 527–36. (5) Duburcq, X.; Olivier, C.; Malingue, F.; Desmet, R.; Bouzidi, A.; Zhou, F.; Auriault, C.; Gras-Masse, H.; Melnyk, O. Peptide-protein microarrays for the simultaneous detection of pathogen infections. Bioconjugate Chem. 2004, 15 (2), 307–16. (6) Duburcq, X.; Olivier, C.; Desmet, R.; Halasa, M.; Carion, O.; Grandidier, B.; Heim, T.; Stievenard, D.; Auriault, C.; Melnyk, O. Polypeptide semicarbazide glass slide microarrays: characterization and comparison with amine slides in serodetection studies. Bioconjugate Chem. 2004, 15 (2), 317–25. (7) Mezzasoma, L.; Bacarese-Hamilton, T.; Di Cristina, M.; Rossi, R.; Bistoni, F.; Crisanti, A. Antigen microarrays for serodiagnosis of infectious diseases. Clin. Chem. 2002, 48 (1), 121–30. (8) Ostresh, J. M.; Husar, G. M.; Blondelle, S. E.; Dorner, B.; Weber, P. A.; Houghten, R. A. Libraries from libraries”: chemical transformation of combinatorial libraries to extend the range and repertoire of chemical diversity. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (23), 11138–42. (9) Johansen, M. B.; Kiemer, L.; Brunak, S. Analysis and prediction of mammalian protein glycation. Glycobiology 2006, 16 (9), 844–53.
research articles (10) Lieuw, A. F. M. L.; van Hinsbergh, V. W.; Teerlink, T.; Barto, R.; Twisk, J.; Stehouwer, C. D.; Schalkwijk, C. G. Increased levels of N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine in type 1 diabetic patients with impaired renal function: correlation with markers of endothelial dysfunction. Nephrol., Dial., Transplant. 2004, 19 (3), 631–6. (11) Rubino, F. M.; Pitton, M.; Di Fabio, D.; Colombi, A. Toward an “omic” physiopathology of reactive chemicals: thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds. Mass Spectrom. Rev. 2009, 28 (5), 725–84. (12) Villanueva, G. B.; Allen, N. Acetylation of antithrombin III by aspirin. Semin. Thromb. Hemostasis 1986, 12 (3), 213–5. (13) Deng, S. X.; Bharat, N.; Fischman, M. C.; Landry, D. W. Covalent modification of proteins by cocaine. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (6), 3412–6. (14) Host, H.; Drobecq, H.; Locht, C.; Menozzi, F. D. Enzymatic methylation of the Mycobacterium tuberculosis heparin-binding haemagglutinin. FEMS Microbiol. Lett. 2007, 268 (2), 144–50. (15) Hougardy, J. M.; Schepers, K.; Place, S.; Drowart, A.; Lechevin, V.; Verscheure, V.; Debrie, A. S.; Doherty, T. M.; Van Vooren, J.-P.; Locht, C.; Mascart, F. Heparin-binding-hemagglutinin-induced IFN-γ release as a diagnostic tool for latent tuberculosis. PLoS One 2007, 2 (10), e926. (16) Pethe, K.; Bifani, P.; Drobecq, H.; Sergheraert, C.; Debrie, A. S.; Locht, C.; Menozzi, F. D. Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (16), 10759–64. (17) Masungi, C.; Temmerman, S.; Van Vooren, J. P.; Drowart, A.; Pethe, K.; Menozzi, F. D.; Locht, C.; Mascart, F. Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis. J. Infect. Dis. 2002, 185 (4), 513–20. (18) Zanetti, S.; Bua, A.; Delogu, G.; Pusceddu, C.; Mura, M.; Saba, F.; Pirina, P.; Garzelli, C.; Vertuccio, C.; Sechi, L. A.; Fadda, G. Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin. Clin. Diagn. Lab. Immunol. 2005, 12 (9), 1135–8. (19) Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 Da. Anal. Chem. 1988, 60 (20), 2299–301. (20) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1988, 2 (8), 151–3. (21) Schu ¨renberg, M.; Dreisewerd, K.; Hillenkamp, F. Laser desorption/ ionization mass spectrometry of peptides and proteins with particle suspension matrixes. Anal. Chem. 1998, 71 (1), 221–9. (22) Dale, M. J.; Knochenmuss, R.; Zenobi, R. Graphite/liquid mixed matrices for laser desorption/ionization mass spectrometry. Anal. Chem. 1996, 68 (19), 3321–9. (23) Sunner, J.; Dratz, E.; Chen, Y.-C. Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions. Anal. Chem. 1995, 67 (23), 4335–42. (24) Wei, J.; Buriak, J. M.; Siuzdak, G. Desorption-ionization mass spectrometry on porous silicon. Nature 1999, 399 (6733), 243–6. (25) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Desorption/ ionization on silicon (DIOS) mass spectrometry: background and applications. Int. J. Mass Spectrom. 2003, 226 (1), 107–16. (26) Peterson, D. S. Matrix-free methods for laser desorption/ionization mass spectrometry. Mass Spectrom. Rev. 2007, 26 (1), 19–34. (27) Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Desorption/ ionization on silicon nanowires. Anal. Chem. 2005, 77 (6), 1641– 6. (28) Piret, G.; Drobecq, H.; Coffinier, Y.; Melnyk, O.; Boukherroub, R. Matrix-free laser desorption/ionization mass spectrometry on silicon nanowire arrays prepared by chemical etching of crystalline silicon. Langmuir 2010, 26 (2), 1354–61. (29) Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 1990, 35 (3), 161–214. (30) Gidley, M. J.; Sanders, J. K. Reductive methylation of proteins with sodium cyanoborohydride. Identification, suppression and possible uses of N-cyanomethyl by-products. Biochem. J. 1982, 203 (1), 331–4. (31) Jentoft, N.; Dearborn, D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 1979, 254 (11), 4359–65.
Journal of Proteome Research • Vol. 9, No. 12, 2010 6477
research articles (32) Duburcq, X.; Olivier, C.; Desmet, R.; Halasa, M.; Carion, O.; Grandidier, B.; Heim, T.; Stievenard, D.; Auriault, C.; Melnyk, O. Polypeptide semicarbazide glass slide microarrays: characterization and comparison with amine slides in serodetection studies. Bioconjugate Chem. 2004, 15 (2), 317–25. (33) Lindmark, R.; Thoren-Tolling, K.; Sjoquist, J. Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. J. Immunol. Meth. 1983, 62 (1), 1–13. (34) Pethe, K.; Aumercier, M.; Fort, E.; Gatot, C.; Locht, C.; Menozzi, F. D. Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin. J. Biol. Chem. 2000, 275 (19), 14273–80. (35) Menozzi, F. D.; Rouse, J. H.; Alavi, M.; Laude-Sharp, M.; Muller, J.; Bischoff, R.; Brennan, M. J.; Locht, C. Identification of a heparinbinding hemagglutinin present in mycobacteria. J. Exp. Med. 1996, 184 (3), 993–1001. (36) Rouse, D. A.; Morris, S. L.; Karpas, A. B.; Mackall, J. C.; Probst, P. G.; Chaparas, S. D. Immunological characterization of recombinant antigens isolated from a Mycobacterium avium lambda gt11 expression library by using monoclonal antibody probes. Infect. Immun. 1991, 59 (8), 2595–600. (37) Pethe, K.; Bifani, P.; Drobecq, H.; Sergheraert, C.; Debrie, A. S.; Locht, C.; Menozzi, F. D. Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (16), 10759–64. (38) Ahn, T.; Yun, C. H. Effects of epitope sequence tandem repeat and proline incorporation on polyclonal antibody production against cytochrome 1A2 and 3A4. BMB Rep. 2009, 42 (7), 418–20. (39) Kim, P.; Pau, C. P. Comparing tandem repeats and multiple antigenic peptides as the antigens to detect antibodies by enzyme immunoassay. J. Immunol. Methods 2001, 257 (1-2), 51–4. (40) Kotera, Y.; Fontenot, J. D.; Pecher, G.; Metzgar, R. S.; Finn, O. J. Humoral immunity against a tandem repeat epitope of human
6478
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Piret et al.
(41)
(42) (43)
(44) (45) (46)
(47)
(48)
mucin MUC-1 in sera from breast, pancreatic, and colon cancer patients. Cancer Res. 1994, 54 (11), 2856–60. Yankai, Z.; Rong, Y.; Yi, H.; Wentao, L.; Rongyue, C.; Ming, Y.; Taiming, L.; Jingjing, L.; Jie, W. Ten tandem repeats of beta-hCG 109-118 enhance immunogenicity and anti-tumor effects of betahCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65. Biochem. Biophys. Res. Commun. 2006, 345 (4), 1365–71. Wilson, I. A.; Niman, H. L.; Houghten, R. A.; Cherenson, A. R.; Connolly, M. L.; Lerner, R. A. The structure of an antigenic determinant in a protein. Cell 1984, 37 (3), 767–78. Brizzard, B. L.; Chubet, R. G.; Vizard, D. L. Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques 1994, 16 (4), 730–5. Evan, G. I.; Lewis, G. K.; Ramsay, G.; Bishop, J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 1985, 5 (12), 3610–6. Host, H.; Drobecq, H.; Locht, C.; Menozzi, F. D. Enzymatic methylation of the Mycobacterium tuberculosis heparin-binding haemagglutinin. FEMS Microbiol. Lett. 2007, 268 (2), 144–50. Menozzi, F. D.; Bischoff, R.; Fort, E.; Brennan, M. J.; Locht, C. Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (21), 12625–30. Nair, D. T.; Singh, K.; Sahu, N.; Rao, K. V.; Salunke, D. M. Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition. J. Immunol. 2000, 165 (12), 6949–55. Mrksich, M.; Whitesides, G. M. Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55–78.
PR100707T
