Background Binding proteins such as antibodies are useful for basic science, diagnosis, and therapy. Nanobodies are also promising binding proteins that achieve comparable binding affinities and specificities to conventional antibodies, despite comprising only a single 15 kDa variable domain. The generation of functional nanobodies typically involves phage, yeast, or ribosome display, which allows screening billions of nanobodies simultaneously. However, display technologies have some drawbacks, one of which is the immobilization effect. The immobilization of nanobodies on the cell surface is necessary to establish genotype–phenotype linkages, but this can affect nanobody functions. Therefore, we have developed peptide barcoding, a technology with which we can evaluate free nanobodies with low bias . Here, we applied the technology to the alanine scanning of anti-GFP nanobodies and enabled multiplex evaluation of affinities of free nanobodies.
Method We used GFP and anti-GFP nanobody as model antigen and antibody. GFP was produced by E. coli BL21 (DE3) strain. Anti-GFP nanobodies were produced by P. pastoris GS115 strain. To construct the alanine-scanning nanobody library, we substituted each non-alanine residue of the anti-GFP nanobody for alanine and prepared 106 kinds of nanobodies. Each nanobody was attached to a genetically encoded unique peptide barcode. The 106 nanobody–peptide barcode fusion proteins (barcodebodies) were produced in one pot by P. pastoris, digested with trypsin, and applied to LC-MS/MS to confirm the detectability of the peptide barcodes. The peptide barcodes, in this study, were derived from yeast SRM Atlas where tens of thousands of proteotypic peptides are registered . The barcodebody library was mixed with GFP and separated with size-exclusion chromatography depending on the affinity. The fractions containing high-affinity barcodebodies or low-affinity barcodebodies were digested with trypsin and applied to LC-MS/MS.
Results & Discussion We detected more than 95% of the peptide barcode by LC-MS/MS. In the fraction containing low-affinity barcodebodies, we simultaneously identified five peptide barcodes, and in the fraction containing high-affinity barcodebodies, we identified the other barcodes. We measured the affinity of these five nanobodies by surface plasmon resonance (SPR) and confirmed their decrease of affinities. These results demonstrated the practicality and utility of the peptide barcoding for one-pot multiplex evaluation of non-immobilized nanobodies and suggested that this technology could be applied to more high-throughput screening.
 Miyamoto et al., PLOS ONE 14(4): e0215993, 2019
 Picotti et al., Nature 494(7436), 266-270, 2013