Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays.
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.
From the ‡Science for Life Laboratory, KTH - Royal Institute of Technology, SE-171 21 Stockholm, Sweden;
Björn
B
Forsström
§Department of Proteomics, KTH - Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
Barbara Bisławska
BB
Axnäs
¶NimbleGen Systems GmbH, Roche, Beuthenerstr. 2, D-84478 Waldkraiburg, Germany;
Klaus-Peter
KP
Stengele
¶NimbleGen Systems GmbH, Roche, Beuthenerstr. 2, D-84478 Waldkraiburg, Germany;
Jochen
J
Bühler
‖Nimblegen, Roche Applied Science, 500 S. Rosa Rd., Madison, Wisconsin 53719.
Thomas J
TJ
Albert
‖Nimblegen, Roche Applied Science, 500 S. Rosa Rd., Madison, Wisconsin 53719.
Todd A
TA
Richmond
§Department of Proteomics, KTH - Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
Francis Jingxin
FJ
Hu
From the ‡Science for Life Laboratory, KTH - Royal Institute of Technology, SE-171 21 Stockholm, Sweden;
Peter
P
Nilsson
§Department of Proteomics, KTH - Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
Elton P
EP
Hudson
§Department of Proteomics, KTH - Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
Johan
J
Rockberg
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; mathias.uhlen@scilifelab.se.
Mathias
M
Uhlen
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D001665:Binding Sites; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D010452:Peptide Biosynthesis / Q000235:genetics* / Q000276:immunology; D020543:Proteome; D014357:Trypsin
D000906:Antibodies; D000939:Epitopes; D020543:Proteome; D014357:Trypsin