Label-free quantitative mass spectrometry analysis of differential protein expression in the developing cochlear sensory epithelium.
Label-free quantitative mass spectrometry analysis of differential protein expression in the developing cochlear sensory epithelium.
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DOI：
10.1186/s12953-018-0144-6
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发表时间：
2018
期刊：
影响因子：
2
通讯作者：
Sokolowski BHA
中科院分区：
文献类型：
--
作者：
Darville LNF;Sokolowski BHA
内耳的感觉上皮细胞将声音的机械能转化为中枢神经系统能识别的电化学能。这个过程是由被称为毛细胞的受体细胞介导的，随着听力的出现，毛细胞会及时表达蛋白质。采用无标记定量质谱法（LTQ-Orbitrap）分析了3、14和30日龄小鼠耳蜗感觉上皮细胞的蛋白质组。采用单因素方差分析和Bonferroni事后检验进行统计分析，以显示蛋白质表达的显著差异。独创性途径分析用于观察差异表达蛋白的网络、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记蛋白的推定相互作用。这些候选生物标记物使用Western blotting进一步验证，而共免疫沉淀用于验证使用生物信息学确定的推定伴侣。我们发现，对所有三种蛋白质组进行比较，发现有447种差异表达蛋白，其中387种在出生后第3天和第30天之间差异表达。匠心途径分析显示，62%的产后第3天下调蛋白与神经系统疾病有关。P3上只表达Parvin α、Drebrin1 （Drb1）、Secreted protein acid and cysteine rich （SPARC）、Transmembrane emp24 domain containing protein 10 （Tmed10）等蛋白。共免疫沉淀表明，Parvin和SPARC分别与整合素连接蛋白激酶和大电导钙活化钾通道相互作用。定量质谱分析显示，在出生后3天的发育过程中，发现了许多差异调节蛋白。这些数据为耳蜗调节正常感觉和支持细胞发育的功能通路提供了见解，包括潜在的生物标志物。这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。本文的在线版本（10.1186/s12953-018-0144-6）包含补充材料，仅供授权用户使用。
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 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. 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. 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. The online version of this article (10.1186/s12953-018-0144-6) contains supplementary material, which is available to authorized users.
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4.4
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