Abstract
BackgroundThe sensory epithelium of the inner ear converts the mechanical energy of sound to electro-chemical energy recognized by the central nervous system. This process is mediated by receptor cells known as hair cells that express proteins in a timely fashion with the onset of hearing.MethodsThe proteomes of 3, 14, and 30 day-old mice cochlear sensory epithelia were revealed, using label-free quantitative mass spectrometry (LTQ-Orbitrap). Statistical analysis using a one-way ANOVA followed by Bonferroni’s post-hoc test was used to show significant differences in protein expression. Ingenuity Pathway Analysis was used to observe networks of differentially expressed proteins, their biological processes, and associated diseases, while Cytoscape software was used to determine putative interactions with select biomarker proteins. These candidate biomarkers were further verified using Western blotting, while coimmunoprecipitation was used to verify putative partners determined using bioinformatics.ResultsWe show that a comparison across all three proteomes shows that there are 447 differentially expressed proteins, with 387 differentially expressed between postnatal day 3 and 30. Ingenuity Pathway Analysis revealed ~ 62% of postnatal day 3 downregulated proteins are involved in neurological diseases. Several proteins are expressed exclusively on P3, including Parvin α, Drebrin1 (Drb1), Secreted protein acidic and cysteine rich (SPARC), Transmembrane emp24 domain-containing protein 10 (Tmed10). Coimmunoprecipitations showed that Parvin and SPARC interact with integrin-linked protein kinase and the large conductance calcium-activated potassium channel, respectively.ConclusionsQuantitative mass spectrometry revealed the identification of numerous differentially regulated proteins over three days of postnatal development. These data provide insights into functional pathways regulating normal sensory and supporting cell development in the cochlea that include potential biomarkers. Interacting partners of two of these markers suggest the importance of these complexes in regulating cellular structure and synapse development.
Highlights
The sensory epithelium of the inner ear converts the mechanical energy of sound to electro-chemical energy recognized by the central nervous system
Protein identification and differential expression Cochleae from three biological replicates from three different age groups, Postnatal day 3 (P3), P14, and P30 were solubilized to extract proteins that were first digested with Endoproteinase Lys-C (LysC) endoprotease followed by a second digestion with trypsin
Each digest was separated into 14 fractions using Strong cation exchange (SCX) and analyzed by nano LC-Mass spectrometry (MS)/MS
Summary
The sensory epithelium of the inner ear converts the mechanical energy of sound to electro-chemical energy recognized by the central nervous system. This process is mediated by receptor cells known as hair cells that express proteins in a timely fashion with the onset of hearing. The cochlear sensory epithelium contains specialized receptors known as hair cells, which are responsible for transducing incoming mechanical signals for processing by the brain [1]. The perception and processing of sound are dependent on the expression of many proteins of which many are regulated during the onset of hearing. There have been far fewer studies of the inner ear performed at the proteome level [8,9,10,11,12,13,14], and even fewer that have explored protein pathways [15, 16]
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