Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays

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 massspectrometry-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. Molecular & Cellular

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

“…So far, it was not possible to investigate the binding of antibodies in a proteome-wide manner, but new methods for in situ synthesis of peptides on ultra-dense arrays have made this now achievable. Using a photolithographic approach, about 2 million different peptides can be synthesized on a slide (105), which facilitates a linear epitope mapping of all human proteins (106). With such technologies, much broader MS-specific (auto)antibody patterns will be defined, and this may ultimately yield better biomarkers as well as important insights into the pathogenesis of MS. On the other hand, the antigens and epitopes that have been associated with MS in the past (e.g.…”

The Development and Application of a Quantitative Peptide Microarray Based Approach to Protein Interaction Domain Specificity Space

“…Peptide arrays using physiological ligands do not have these limitations, yet may undersample PID specificity space because of smaller library sizes. Newly emerging ultrahigh density peptide arrays avoid this particular limitation and are capable of sampling the entire proteome (30). However, a common limitation for all of these techniques is their dependence on nonquantitative interaction information.…”

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients

“…Custom high‐density peptide microarrays were obtained in collaboration with Roche‐Nimblegen, and applied for epitope mapping as described before 17. Peptides of 12 amino acid lengths with an 11‐residue overlap were designed to cover the antigen region of AMFR.…”