Flycodes: barcoding designer peptides for mass spectrometry enable in vivo antibody screening

NestLink enables the simultaneous observation of potentially 1000s of unique proteins via tagging with distinctive genetically-encoded peptides (flycodes), designed for unambiguous detectability via liquid chromatography-mass spectrometry. We demonstrated the utility of NestLink to deep-mine nanobody pools for rare binders that recognize their membrane protein target in the cellular context of pathogenic bacteria. In an attempt to reduce the burden of laboratory animals, we show that flycodes facilitate the simultaneous determination of individual PK/PD parameters and biodistribution in kidney and liver for a pool of over 100 unique nanobody constructs in a single animal. NestLink thus allows a broader range of biologic drug candidates to be assayed in early stages of discovery, thereby diminishing cost, labor, and ethical burdens.