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

Søren Buus,Johan Rockberg,Björn Forsström,Peter Nilsson,Mathias Uhlen,Claus Schafer-Nielsen

doi:10.1074/mcp.m112.020800

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.

Highlights

From the ‡Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen N, Denmark; ʈSchool of Biotechnology, AlbaNova University Center, KTH-Royal Institute of Technology, Stockholm, Sweden; **Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden; ‡‡Schafer-N, Copenhagen, Denmark
Ultrahigh-density Peptide Microarrays—Peptide arrays were generated by a combined maskless photolithographic [34] and solid phase peptide synthesis strategy using a digital mirror device (1080P DMD (Digital Light Projections, Digital Light Innovations, Austin, TX) with 1920 ϫ 1080 ϭ 2,073,600 individually addressable micromirrors) to project 365 nm light onto NPPOC-photoprotected [35, 36] amino groups on a glass surface in patterns corresponding to the fields where the amino acid extension should occur
After synthesis of the peptide backbones, all side chain protection groups were removed via TFA treatment, leaving the peptides attached to the matrix through their C-terminals

Summary

Technological Innovation and Resources

High-resolution Mapping of Linear Antibody Epitopes Using Ultrahigh-density Peptide Microarrays*□S. Many other epitope mapping approaches, such as fragmentation [19] or deuterium exchange in the presence or absence of antibody [20], directed mutagenesis, recombinant expression (including arrayed in situ cell-free translation approaches [20, 21]) of protein and peptide arrays, etc., have been suggested [12] Despite this plethora of methods, exact epitope information is lacking for the vast majority of antibodies used in life science research, and there is a significant need for simple and rapid methods to map epitopes. We present the first report on the feasibility of using ultrahigh-density peptide microarrays to address antibody specificities in casu mapping the fine specificity of polyclonal antibodies raised against linear protein epitopes This allowed a fast and exhaustive analysis of the length requirements and a detailed analysis of the fine specificity of these antibodies. We suggest that specificity analysis of linear epitopes using ultrahigh-density peptide microarrays addressing the entire human proteome is within reach

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION