Development of a Translational Assay for Identification and Quantitation of Orthologous Proteins to Aid Drug Development in Preclinical Models

Abstract Unequivocal identification and reproducible quantitation of proteins are key components of research and development activity in drug discovery. In some cases, the target protein might be a protein highly conserved across species; antibody-based assays are not specific enough to discriminate between orthologues, thus forcing drug developers to use multiple analytical platforms across preclinical models. Here, we present a modular mass spectrometry-based assay for the analysis of orthologous proteins across species in the context of translational science using ataxin-2 (ATXN2) as an example. Instead of relying on recombinant proteins, we applied rational peptide design to target human, mouse, human/pig/non-human primates (NHP), or pan-species regions of the full-length protein. Specificity and sensitivity of the method were proven in wild-type and transgenic models. Dose–response upon treatment with different doses of an adeno-associated virus (AAV)-delivering microRNA (miRNA) targeting ATXN2 showed that knock-down at the transcript level was consistent with downregulation at the protein level, confirming the assay suitability for target engagement evaluation. Furthermore, the data presented here indicate that this method is readily transferable to minipigs, ensuring analytical continuity across preclinical studies. More broadly, this work provides a translational blueprint that can be adapted to other orthologues, reducing cost and timeline in drug development.