Peptide barcoding for one-pot evaluation of sequence-function relationships of nanobodies.

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.