Peptide barcoding for establishment of new types of genotype–phenotype linkages

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype–phenotype linkages.