Abstract
Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.
Highlights
From the ‡Science for Life Laboratory, KTH - Royal Institute of Technology, SE-171 21 Stockholm, Sweden; §Department of Proteomics, KTH - Royal Institute of Technology, SE-106 91 Stockholm, Sweden; ¶NimbleGen Systems GmbH, Roche, Beuthenerstr. 2, D-84478 Waldkraiburg, Germany; ʈNimblegen, Roche Applied Science, 500 S
One of the key parameters for antibodies includes the nature of the binding recognition toward the target, involving either linear epitopes formed by consecutive amino acid residues or conformational epitopes consisting of amino acids brought together by the fold of the target protein [8]
We describe the design and use of peptide arrays generated with parallel in situ photolithic synthesis [20] of a total of 2.1 million overlapping peptides covering all human proteins with overlapping peptides
Summary
Bjorn Forsstrom‡, Barbara Bisławska Axnas§, Klaus-Peter Stengele¶, Jochen Buhler¶, Thomas J. We describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. Miniaturization of the peptide arrays [21] has led to improved density of the synthesized peptides and has improved the resolution and coverage of the epitope mapping This has allowed us to study the specificity and cross-reactivity of both monoclonal and polyclonal antibodies across the whole “epitome” with the use of both proteome-wide arrays and focused-content peptide arrays covering selected antigen sequences to precisely map the contribution of each amino acid of the target protein for binding recognition of the corresponding antibodies. The results show the usefulness of proteome-wide epitope mapping, showing a path forward for high-throughput analysis of antibody interactions
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