PMC:3886832 / 36542-43499 JSONTXT

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Proteomic Analysis of the Inner Ear\nmiRNAs can affect their targets either by degrading the mRNA or by repressing their translation [70]. If miRNAs target their mRNAs at the translational level by causing repression, it is not possible to identify those targets by profiling their gene expression, as the mRNA levels will remain the same, while the protein levels change. The reason why proteomics is necessary can be easily understood by comparing the ~30,000 human coding genes and the ~500,000 proteins resulting from post-translational modifications (PTMs), splicing variants and proteolysis products resulting from these genes [30,50]. Furthermore, the relative differences between mRNA levels and protein turnover, differences in the function of translational machinery and complex interactions cannot be understood from gene array studies [50]. Proteomic profiling done in the inner ears of mouse have identified that the targets of miR-135 and miR-205 only change at the protein level, but not at the mRNA level [87]. A comparison of mRNA and protein profiling did not show complete correlation between transcript and protein levels, indicating that protein expression levels cannot always be extrapolated from mRNA levels [57,93].\n\n5.1. Antibody Microarrays\nProteins can be separated and characterized via a number of different techniques (reviewed in [30]). One of these techniques is the use of antibody microarrays, which are similar to DNA microarrays, except that antibodies are printed on the chip instead of DNA probes [52]. Antibody microarrays were first used by Jamesdaniel et al. [53] to study hair cell damage in the inner ears of rats treated with cisplatin. This study identified the dynamic changes of 19 proteins that were either up- or downregulated and 15 of those were novel proteins that belong to either cell death or survival pathways [53]. Differential expression of proteins in the organ of Corti, lateral wall and modiolus of chinchilla showed enrichment of cell death pathways in both the sensory epithelia and modiolus [54]. Immunolabeling experiments confirmed the antibody microarray results by identifying the expression of E2F3 in nuclei and WSTF and FAK-p-Tyr577 in the stereocilia in the organ of Corti. Detection of FAK-p-Tyr577 supports the idea that post-translational modification of proteins can be identified using antibody microarrays, but cannot be detected using gene arrays [54]. It is necessary to have a large amount of tissue to study proteomics, and this problem was circumvented by an approach called “Subtractive Strategy Using Mouse Mutants” that can detect large number of proteins with only a small amount of starting material. Using the Pou4f3 mutant mouse model, the association of Pou4f3 in the inner ear with other genes, such as Notch1-4, gata3, p27Kip1, Eya1, S100a1, Tbx3, Shh, Fgfr3, Bmp5, Jag2 and Fkh10, were able to be identified [50].\n\n5.2. Liquid Chromatography Coupled with Mass Spectrometry\nRecently, a study by Peng et al. [55] demonstrated the ability of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to detect hundreds of proteins in the normal cochlea of mice. LC-MS/MS can be used to detect protein expression and PTMs, and this study detected 628 proteins, the largest number of proteins identified in the organ of Corti, so far. Using the same LC-MS/MS technique, another study identified 620 and 134 proteins in the embryonic stage (E20–E21) chicken utricle and cochlea, respectively [57]. Organ of Corti from the normal mice showed expression of many proteins, such as cochlin, isoform-1-α-tectorin, gap junction β6 protein and myosin VI, which are all involved in hearing impairment. Both phosphorylated and acetylated forms of proteins, including both mono- and di-phosphorylated states and N-termini and lysine acetylated states, could be detected using LC-MS/MS [55]. Calcium buffers were abundant in the cochlea, while histones and nuclear lamins were abundant in the utricle. Heat shock proteins were found to be of equal abundance in both cochlea and utricle of chicken. Validation of mass spectrometry (MS) data was done on a subset of proteins using immunoblots, and it showed similar trends in the differential abundance between utricle and cochlea, as was shown by MS. Differential expression of glycolytic and gluconeogenesis enzymes were high in cochlea, while citric acid cycle and electron transport chain enzymes were high in utricle. In addition to this, glucose and lactate transporters were upregulated in cochlea identified by MS. Immunoblot and immunocytochemistry validated the MS data, and moreover, glycolytic rates measured using tritiated hydrogen also confirmed the high energy demand in the cochlea [57]. \nAnother study using LC-MS/MS identified many presynaptic proteins in the ribbon synapses [56] that are present in hair cells that exhibit fast kinetics and are optimized to release large amounts of neurotransmitters [94]. The presynaptic proteins can be categorized into vesicle and membrane transport proteins, proteins that regulate synaptic exocytosis, ion channels, transporters, pumps and calcium binding proteins. Immunoblots and immunofluorescence labeling confirmed the expression of SNAP25, NSF, syntaxin1, syntaxin6, VAMP2, alpha α-SNAP, β-SNAP and VAP33 proteins in the chicken cochlear hair cells. The proteomic profile of the synaptic fraction showed that otoferlin, synaptotagmin7, alpha-synuclein, syntaphilin, piccolo, synaptojanin2 and SCAMP1 were only identified in the cochlea of chicken, but not in the retina or brain, suggesting these proteins have specific function in hair cells and that there are compositional differences between hair cell and retinal ribbon synapses [94]. The proteome of hair cell bundles in chicken utricles detected 59 proteins, representing a large fraction of cytoskeletal, energy metabolism and stress response proteins and other proteins, such as calcium buffers and transmembrane proteins. Both actin and creatine kinase B were found to be abundant, and using quantitative immunoblot, actin was shown to be eight-fold higher than creatine kinase B (B-CK). Immunolabeling confirmed B-CK expression in chicken and bullfrog utricles and in mouse inner and outer hair cells [56]. \nIn conclusion, these studies show the potential of proteomics to identify proteins that cannot be identified from mRNA expression levels alone. Antibody microarrays can be customized to include antibodies to detect target proteins of interest that can have specific PTMs. The use of proteomics to investigate biological questions in the inner ear has been limited, due to the requirement of a large amount of tissue, and inner ears are very small and often encased in bony structures. This problem was solved by the development of the subtractive strategy [49] and LC-MS/MS methods [55,56,57], which can use minute quantities of starting materials and detect hundreds of proteins.\n"}