Label-free quantitative mass spectrometry analysis of differential protein expression in the developing cochlear sensory epithelium

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.Electronic supplementary materialThe online version of this article (10.1186/s12953-018-0144-6) contains supplementary material, which is available to authorized users.

Transmembrane emp24 domain proteins in development and disease.

“…In the rat central nervous system, TMED10 expression is found in the cortex, hippocampus and brainstem at postnatal day (P) 7 and P21 (Vetrivel et al, 2008). TMED10 is also expressed in the developing postnatal sensory epithelium of the murine inner ear at P3, but not at P14 and P30, suggesting that TMED10 may play a role in inner ear development (Darville & Sokolowski, 2018). The role, if any, of TMED10 in the pre-and post-natal development of the mammalian brain and inner ear remains to be identified.…”

“…These proteins are evolutionarily conserved, and their localization and levels are tightly regulated by the cellular machinery. As discussed, individual TMED proteins are important for morphogenesis, differentiation and homeostasis in a number of organisms (Carney & Taylor, 2003;Boltz et al, 2007;Vetrivel et al, 2008;Jerome-Majewska et al, 2010;Graveley et al, 2011;Zakariyah et al, 2012;Darville & Sokolowski, 2018). However, although their roles in cargo transport are clearly important and numerous putative cargo proteins are regulated by TMED proteins (Tables 2 & 3), little is understood of the impact of most TMED proteins in development and disease.…”

Evaluation of acquisition modes for semi‐quantitative analysis by targeted and untargeted mass spectrometry

“…There are, however, also limitations to the labeling techniques: they are expensive, can suffer from incomplete labeling efficiencies, and are limited in the number of samples that can be analyzed in parallel within a single experiment. These challenges in quantitation using labeling approaches have given rise to the second family of techniques, termed label‐free quantitation 12–14 . In the case of the label‐free methods no isotopic labels are required; instead the amount of a given analyte is determined by either chromatographic peak area or spectral counting.…”

Development of a bioinformatics platform for analysis of quantitative transcriptomics and proteomics data: the OMnalysis

“…Omics is composed of multiple layers (genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, and phenomics), however, transcriptomics and proteomics remain the most commonly used omics ( Gomez-Cabrero et al, 2014 ; Hasin, Seldin & Lusis, 2017 ; Yan et al, 2018 ). Transcriptomics and proteomics experiments require high-cost instrumental setup, labor-intensive sample preparation, and technical skills ( Darville & Sokolowski, 2018 ; Hrdlickova, Toloue & Tian, 2017 ). As a result, researchers often outsource samples to core facilities and obtain log-transformed or normalized expression data, which require to be interpreted into biological relevance using bioinformatic tools.…”

“…The output usually depends on the type of statistical package and function used, for example, EdgeR ( Robinson, McCarthy & Smyth, 2010 ), DESeq2 ( Love, Huber & Anders, 2014 ) and Cuffdiff ( Trapnell et al, 2012 ). On the other hand, in the case of quantitative proteomics the data analyzed for quality check, peptide identification, protein quantification, and normalization ( Darville & Sokolowski, 2018 ) deliver a table containing columns of identified proteins, the magnitude of change in abundance against control and FDR-adjusted P -value. Such data matrices can be analyzed using different bioinformatic tools depending on the study type and level of regulation.…”