The mammalian cochlea houses two distinct mechanosensitive receptor cell types: inner hair cells (IHCs) and outer hair cells (OHCs). Despite their critical role in auditory function, the intricate molecular processes that underlie their unique morphological and functional characteristics remain largely unexplored. Our study aims to fill this gap by constructing an exhaustive proteomic profile of IHCs and OHCs isolated from postnatal day 7 (P7) murine cochlea. This deep proteomic profiling enables the identification of pivotal proteins implicated in precise cellular functionalities or pathological pathways.

Given the limited quantity of hair cells within each cochlea—approximately 3,500 IHCs and 12,000 OHCs—and their inherent fragility, conventional high-throughput collection methods are inadequate for single-cell proteomics. To address this challenge, we employed the suction pipette technique to isolate individual OHCs from enzymatically treated and mechanically dissociated murine cochlea at P7. These isolated OHCs were then cryopreserved in liquid nitrogen. Sample preparation involved a streamlined one-pot method that included lysis, sonication, denaturation, and tryptic digestion in a minimal volume, thereby reducing sample handling and potential contamination. For mass spectrometric analysis, we utilized the TimsTOF SCP, coupled with an Evosep One chromatography system. This method has yielded up to 500 protein groups per OHC and 2200 protein groups for 5 OHCs.

Our primary focus is on OHCs due to their greater abundance and ease of collection. Ultimately, we aim to conduct a comparative analysis between OHCs and the more challenging-to-isolate IHCs. Additionally, we seek to correlate this dataset with established molecular structures in auditory mechanisms and use it to complement affinity-purification mass spectrometry (AP-MS) experiments targeting known components of the hearing machinery, such as protocadherin 15 (Pcdh15). This approach includes utilizing AP-MS with specialized antibodies against Pcdh15 to identify new components of the lower tip-link complex, crucial for mechano-electrical transduction (MET). Protein-protein interactions between potential new and known MET components will be validated using Alphafold2.

These findings promise to provide critical insights into cochlear mechanosensation by elucidating the molecular mechanisms of both IHCs and OHCs. Furthermore, our collection method and subsequent sample preparation approach offer a viable option for single-cell proteomics studies on other challenging-to-collect cell types. This research advances our understanding of auditory biology and lays the groundwork for innovative therapeutic strategies to address hearing impairments, potentially transforming the landscape of auditory health and disease management.