PROJECT SUMMARY/ABSTRACT Hair cells are the sensory cells of the inner ear that carry out the essential function of mechanotransduction evoked by sound and head movement. It is fundamentally important to characterize global protein expressions and their interactions in hair cells in order to understand the molecular mechanism. However, one of the greatest challenges in protein characterization is the low number of hair cells presented in each inner-ear organ, which urges us to develop sensitive analytical approaches. To this end, our labs develop a microfluidic sample preparation platform, termed as nanoPOTS (nanodroplet processing in one pot for trace samples), for proteomics analysis of low-input biomaterials by downscaling processing volumes to <200 nL. While nanoPOTS is well demonstrated to identify and quantify protein expression from single hair cells, it informs nothing on how proteins interact with each other to implement their functions. The overall objective of this project to develop a sensitive nanoliter droplet-based affinity purification with mass spectrometry (nanoAP-MS) platform to identify protein interacting partners using fewer than 1000 hair cells isolated from utricles or cochleas of the mouse ear. The central hypothesis is that the overall sensitivity of AP-MS assay can be significantly improved by performing affinity purification in nanoliter droplets. Theory suggests this hypothesis should be correct because: 1) Improved protein concentrations by lysing cells in nanoliter volumes will improve protein-bead binding efficiency; 2) Reducing the amounts of affinity beads will reduce non-specific binding, which can otherwise dwarf specific binding; and 3) improved LC-MS will provide sufficient analytical sensitivity to measure low abundance proteins. The central hypothesis will be tested by pursuing two specific aims: 1) To establish a nanoliter droplet-based AP-MS workflow; and 2) To apply this workflow for identification of MYO7A and GIPC3 binding partners in mouse hair cells. We expected the proposed nanoAP-MS platform will increase sensitivity by a factor of 103 or more and allow us to characterize low-abundance protein interaction partners. This research is highly innovative because the nanoAP-MS platform will be the first of its kind to reliably measure protein-protein interactions using a small number of primary cells isolated from physiological environment, including animal models or human biopsies. Statement of Impact: As AP-MS has emerged as powerful technology to discover protein interaction partners and establish protein-protein-interaction networks, the nanoAP-MS technology will enable to examine important protein-protein interactions in small numbers of cells isolated by micropipette, FACS, or laser-capture microdissection, or to examine exceptionally low- abundance interactions like those present in the hair cell's mechanotransduction complex.