Proteomic Core D

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Abstract Complex biological processes like auditory neural aging are composed of dynamic changes in individual neurons' biochemistry, with various aging process susceptibilities. Direct measures of a single auditory neuron (spiral ganglion neuron (SGN) and cochlear nuclei (CN) neuron subtypes) and their age-related alterations are needed to characterize cellular transitions, regulatory mechanisms, and the microenvironment's contribution to age- related hearing loss (ARHL). A critical unmet need is determining the biochemical makeup of healthy and aged SGNs with single-cell resolution and a depth of coverage extending to thousands of biomolecules. Proteins mediate the bulk of cellular function, and the correlation between RNA and protein abundance is often lacking, and the proteome largely determines cellular structure and function. We hypothesize that the aging SGN undergoes a profound proteomic dynamic that is neuron subtype-specific. The aims of Core D are: 1. To refine liquid chromatograph-mass spectrometry (LC-MS)-based proteomics platform for sensitive and high-throughput SGN proteomics. 2. To apply the improved platform to study aging-related proteome regulations for SGN and CN subtypes and at single-cell resolution. The proposed effort will result in a processing and analysis platform capable of direct and unbiased profiling of SGNs and CN neurons with a depth of coverage of >3000 proteins/cell, thus providing a capability for direct protein measurement analogous to single-cell RNA-seq. Core D serves as a resource to identify ARHL biomarkers for Projects 1-3 to test and perform functional analyses. Outcomes will produce unbiased and in- depth single-cell protein profiles of SGNs and CN to provide a complete picture and identify protein biomarkers associated with ARHL.