5.2. Liquid Chromatography Coupled with Mass Spectrometry
Recently, 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]. 
Another 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]. 
In 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.