Comparative Evaluation of Reproducibility of Phage-Displayed Peptide Selections and NGS Data, through High-Fidelity Mapping of Massive Peptide Repertoires

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections.

Automated summary

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides. In repeated selections, only a very small number of common peptides are found, indicating the difficulty of distinguishing between specific peptide binders and background noise. This study compares the NGS data of three peptide repertoires obtained under identical conditions and confirms the reproducibility of phage-displayed peptide selections through peptide-to-peptide mappings.