High-resolution Mapping of Linear Antibody Epitopes Using Ultrahigh-density Peptide Microarrays

Abstract

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level. Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level. The immune system is endowed with a highly diverse repertoire of antibodies capable of targeting virtually any molecular structure. As specific affinity reagents, antibodies have become indispensable tools with a wide range of scientific and diagnostic applications (1Gloriam D.E. Orchard S. Bertinetti D. Bjorling E. Bongcam-Rudloff E. Borrebaeck C.A. Bourbeillon J. Bradbury A.R. de Daruvar A. Dubel S. Frank R. Gibson T.J. Gold L. Haslam N. Herberg F.W. Hiltke T. Hoheisel J.D. Kerrien S. Koegl M. Konthur Z. Korn B. Landegren U. Montecchi-Palazzi L. Palcy S. Rodriguez H. Schweinsberg S. Sievert V. Stoevesandt O. Taussig M.J. Ueffing M. Uhlen M. van der Maarel S. Wingren C. Woollard P. Sherman D.J. Hermjakob H. A community standard format for the representation of protein affinity reagents.Mol. Cell. 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Traditionally, the specificity of an antibody is examined in one or more in vitro assays (ELISA, Western blot, immunohistochemistry, flow cytometry, surface plasmon resonance, and many more (12Brennan D.J. O'Connor D.P. Rexhepaj E. Ponten F. Gallagher W.M. Antibody-based proteomics: fast-tracking molecular diagnostics in oncology.Nat. Rev. Cancer. 2010; 10: 605-617Crossref PubMed Scopus (165) Google Scholar, 13Kurien B.T. Dorri Y. Dillon S. Dsouza A. Scofield R.H. An overview of Western blotting for determining antibody specificities for immunohistochemistry.Methods Mol. Biol. 2011; 717: 55-67Crossref PubMed Scopus (44) Google Scholar, 14Warford A. Flack G. Conquer J.S. Zola H. McCafferty J. Assessing the potential of for systematic Immunol. Methods. 2007; PubMed Scopus Google Ideally, the epitope should be is possible to more a and of the epitope is on the the same antibody and specificity in but not in is a Mol. Biol. 2009; PubMed Scopus Google Scholar). 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Biol. 2009; PubMed Scopus Google epitopes be to be in peptide In of epitopes, to be a peptide be to in of epitopes. In is that have that peptide can a antibody target P. M. protein affinity reagents by peptide on Chem. 2009; PubMed Scopus Google Scholar). In of a epitope can be by peptide on is possible to generate the in either the peptide or A should be possible to many (e.g. that the and specificity of linear peptide epitopes can be identified through a In the all or peptides from the target are which the peptides are selected for with in the A of in the to be and the could be one or a amino can be in the for which an exhaustive analysis of the amino acids at for epitope that the identification of important in a linear epitope can be obtained from single with only one or amino Thus, an even alternative be to a length with a single amino substitution a In a target with a length of amino acids peptide with an of one a single amino of peptide the of peptides be to generate of and such a peptide be to all the peptides for of In ultrahigh-density peptide microarrays to several comprehensive of peptide and analysis to identify a polyclonal distinct peptide epitopes, should even be possible to these peptides to affinity multiple paired antibody to of an thereby one antibody to the results of (12Brennan D.J. O'Connor D.P. Rexhepaj E. Ponten F. Gallagher W.M. Antibody-based proteomics: fast-tracking molecular diagnostics in oncology.Nat. Rev. Cancer. 2010; 10: 605-617Crossref PubMed Scopus (165) Google Scholar). also the for proteome peptide all peptide in the proteome can be by peptides through the proteome a peptide length of and by 12 amino that a peptide the proteome is is the and of and with