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

ABSTRACT: 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.

SUBMITTER: Schmitt HA

PROVIDER: S-EPMC8387986 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The outcome of cochlear implantation depends on multiple variables including the underlying health of the cochlea. Brain derived neurotrophic factor (BDNF) has been shown to support spiral ganglion neurons and to improve implant function in animal models. Whether endogenous BDNF or BDNF-regulated proteins can be used as biomarkers to predict cochlear health and implant outcome has not been investigated yet. Gene expression of BDNF and downstream signaling molecules were identified in tissue of human cochleae obtained from normal hearing patients (n = 3) during skull base surgeries. Based on the gene expression data, bioinformatic analysis was utilized to predict the regulation of proteins by BDNF. The presence of proteins corresponding to these genes was investigated in perilymph (n = 41) obtained from hearing-impaired patients (n = 38) during cochlear implantation or skull base surgery for removal of vestibular schwannoma by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Analyzed by mass spectrometry were 41 perilymph samples despite three patients undergoing bilateral cochlear implantation. These particular BDNF regulated proteins were not detectable in any of the perilymph samples. Subsequently, targeted analysis of the perilymph proteome data with Ingenuity Pathway Analysis (IPA) identified further proteins in human perilymph that could be regulated by BDNF. These BDNF regulated proteins were correlated to the presence of residual hearing (RH) prior to implantation and to the performance data with the cochlear implant after 1 year. There was overall a decreased level of expression of BDNF-regulated proteins in profoundly hearing-impaired patients compared to patients with some RH. Phospholipid transfer protein was positively correlated to the preoperative hearing level of the patients. Our data show that combination of gene expression arrays and bioinformatic analysis can aid in the prediction of downstream signaling proteins related to the BDNF pathway. Proteomic analysis of perilymph may help to identify the presence or absence of these molecules in the diseased organ. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

Project description:BACKGROUND:Due to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, these types of investigations are needed to better understand the etiology, pathophysiology and progression of several inner ear disorders. Moreover, the fluid from the inner ear cannot be sampled for micro-chemical analyses from healthy individuals in vivo. Therefore, in the present paper, we studied patients with vestibular schwannoma (VS) undergoing trans-labyrinthine surgery (TLS). Our primary aim was to identify perilymph proteins in patients with VS on an individual level. Our second aim was to investigate the proteins identified at a functional level and our final aim was to search for biological markers for tumor-associated hearing loss and tumor diameter. METHODS AND FINDINGS:Sixteen patients underwent TLS for sporadic VS. Perilymph was aspirated through the round window before opening the labyrinth. One sample was contaminated and excluded resulting in 15 usable samples. Perilymph samples were analyzed with an online tandem LTQ-Orbitrap mass spectrometer. Data were analyzed with MaxQuant software to identify the total number of proteins and to quantify proteins in individual samples. Protein function was analyzed using the PANTHER Overrepresentation tool. Associations between perilymph protein content, clinical parameters, tumor-associated hearing loss and tumor diameter were assessed using Random Forest and Boruta. In total, 314 proteins were identified; 60 in all 15 patients and 130 proteins only once in 15 patients. Ninety-one proteins were detected in at least 12 out of 15 patients. Random Forest followed by Boruta analysis confirmed that alpha-2-HS-glycoprotein (P02765) was an independent variable for tumor-associated hearing loss. In addition, functional analysis showed that numerous processes were significantly increased in the perilymph. The top three enriched biological processes were: 1) secondary metabolic processes; 2) complement activation and 3) cell recognition. CONCLUSIONS:The proteome of perilymph in patients with vestibular schwannoma has an inter-individual stable section. However, even in a cohort with homogenous disease, the variation between individuals represented the majority of the detected proteins. Alpha-2-HS-glycoprotein, P02765, was shown to be an independent variable for tumor-associated hearing loss, a finding that needs to be verified in other studies. In pathway analysis perilymph had highly enriched functions, particularly in terms of increased immune and metabolic processes.

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

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