Proteome Analysis of Human Perilymph Using an Intraoperative Sampling Method.

The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. This study aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph was established during inner ear surgeries (cochlear implantation, vestibular schwannoma surgeries), and safety of the sampling method was determined by checking hearing threshold with pure-tone audiometry postoperatively. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and the individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 µL was analyzed by data-dependent acquisition, resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped by patient's age and surgery type, leading to the identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might serve as biomarkers to develop tools for noninvasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

In Silico Localization of Perilymph Proteins Enriched in Menier̀e Disease Using Mammalian Cochlear Single-cell Transcriptomics.

Hypothesis: Proteins enriched in the perilymph proteome of Menier̀e disease (MD) patients may identify affected cell types. Utilizing single-cell transcriptome datasets from the mammalian cochlea, we hypothesize that these enriched perilymph proteins can be localized to specific cochlear cell types. Background: The limited understanding of human inner ear pathologies and their associated biomolecular variations hinder efforts to develop disease-specific diagnostics and therapeutics. Perilymph sampling and analysis is now enabling further characterization of the cochlear microenvironment. Recently, enriched inner ear protein expression has been demonstrated in patients with MD compared to patients with other inner ear diseases. Localizing expression of these proteins to cochlear cell types can further our knowledge of potential disease pathways and subsequent development of targeted therapeutics. Methods: We compiled previously published data regarding differential perilymph proteome profiles amongst patients with MD, otosclerosis, enlarged vestibular aqueduct, sudden hearing loss, and hearing loss of undefined etiology (controls). Enriched proteins in MD were cross-referenced against published single-cell/single-nucleus RNA-sequencing datasets to localize gene expression to specific cochlear cell types. Results: In silico analysis of single-cell transcriptomic datasets demonstrates enrichment of a unique group of perilymph proteins associated with MD in a variety of intracochlear cells, and some exogeneous hematologic and immune effector cells. This suggests that these cell types may play an important role in the pathology associated with late MD, suggesting potential future areas of investigation for MD pathophysiology and treatment. Conclusions: Perilymph proteins enriched in MD are expressed by specific cochlear cell types based on in silico localization, potentially facilitating development of disease-specific diagnostic markers and therapeutics.

Personalized Proteomics for Precision Diagnostics in Hearing Loss: Disease-Specific Analysis of Human Perilymph by Mass Spectrometry.

Despite a vast amount of data generated by proteomic analysis on cochlear fluid, novel clinically applicable biomarkers of inner ear diseases have not been identified hitherto. The aim of the present study was to analyze the proteome of human perilymph from cochlear implant patients, thereby identifying putative changes of the composition of the cochlear fluid perilymph due to specific diseases. Sampling of human perilymph was performed during cochlear implantation from patients with clinically or radiologically defined inner ear diseases like enlarged vestibular aqueduct (EVA; n = 14), otosclerosis (n = 10), and Ménière's disease (n = 12). Individual proteins were identified by a shotgun proteomics approach and data-dependent acquisition, thereby revealing 895 different proteins in all samples. Based on quantification values, a disease-specific protein distribution in the perilymph was demonstrated. The proteins short-chain dehydrogenase/reductase family 9C member 7 and esterase D were detected in nearly all samples of Ménière's disease patients, but not in samples of patients suffering from EVA and otosclerosis. The presence of both proteins in the inner ear tissue of adult mice and neonatal rats was validated by immunohistochemistry. Whether these proteins have the potential for a biomarker in the perilymph of Ménière's disease patients remains to be elucidated.