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

Optimization of protein binders relies on laborious screening processes. Particularly slow is investigation of sequence-function relationships of protein binders in which diverse amino-acid-substituted mutants are constructed, purified, and evaluated individually. Here, we developed peptide barcoding 2.0, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining generation of a mutagenized protein binder library fused with unique peptide barcodes, formation of binder–antigen complexes at different ratios, fine fractionation of binder–antigen complexes by size exclusion chromatography, and highly specific quantification of peptide barcodes by mass spectrometry. Applying peptide barcoding 2.0 for evaluation of anti-green fluorescent protein nanobody as a model, we succeeded in identifying various mutant Nbs with decreased affinities at once. Moreover, we successfully discriminated subtle changes in KD at the order of nM to sub-nM. These results showed that peptide barcoding 2.0 is a powerful tool for engineering of protein binders, enabling reliable one-pot evaluation of sequence-function relationships.