BACKGROUND 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. METHODS The 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. RESULTS We 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. CONCLUSIONS Quantitative 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.

中文翻译：

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发育中的耳蜗感觉上皮中差异蛋白表达的无标记定量质谱分析。

背景技术内耳的感觉上皮将声音的机械能转化为中枢神经系统识别的电化学能。这个过程由称为毛细胞的受体细胞介导，毛细胞在听力开始时及时表达蛋白质。方法 使用无标记定量质谱 (LTQ-Orbitrap) 揭示 3、14 和 30 日龄小鼠耳蜗感觉上皮细胞的蛋白质组。使用单向方差分析和 Bonferroni 的事后检验进行统计分析，以显示蛋白质表达的显着差异。Ingenuity Pathway Analysis 用于观察差异表达蛋白质的网络、它们的生物学过程和相关疾病，而 Cytoscape 软件用于确定与选定生物标志物蛋白质的推定相互作用。这些候选生物标志物使用蛋白质印迹进一步验证，而免疫共沉淀用于验证使用生物信息学确定的推定伙伴。结果 我们显示，对所有三种蛋白质组的比较表明，有 447 种差异表达的蛋白质，其中 387 种在出生后第 3 天和第 30 天之间差异表达。Ingenuity Pathway 分析显示，约 62% 的出生后第 3 天下调的蛋白质与神经系统疾病有关。几种蛋白质仅在 P3 上表达，包括 Parvin α、Drebrin1 (Drb1)、分泌蛋白酸性和富含半胱氨酸 (SPARC)、含有跨膜 emp24 结构域的蛋白 10 (Tmed10)。免疫共沉淀表明 Parvin 和 SPARC 分别与整合素连接的蛋白激酶和大电导钙激活钾通道相互作用。结论 定量质谱法揭示了在出生后三天内鉴定出许多差异调节的蛋白质。这些数据提供了对调节正常感觉和支持耳蜗细胞发育的功能途径的见解，包括潜在的生物标志物。其中两个标记的相互作用伙伴表明这些复合物在调节细胞结构和突触发育中的重要性。