Date Published: June 1, 2018
Publisher: Public Library of Science
Author(s): Jesper Edvardsson Rasmussen, Göran Laurell, Helge Rask-Andersen, Jonas Bergquist, Per Olof Eriksson, Jon M. Jacobs.
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.
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.
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.
Due to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, such investigations are needed in order to gain a better understanding of the etiology, pathophysiology and progression of several inner ear disorders. The inner ear harbors the membranous system, consisting of the cochlear and semicircular ducts with ampullas, the otolith organs (saccule and utricle) and the endolymphatic duct and sac. The labyrinth membrane separates the two extracellular inner ear fluids, referred to as perilymph and endolymph. Endolymph is rich in potassium, and in the cochlea, is also associated with an electric field potential (the endo-cochlear potential or EP) which is essential for hair cell transduction and hearing [1,2].
Modern surgical approaches, combined with advances in analytical techniques, have provided us with new opportunities with which to explore perilymph in the inner ears of humans affected by different pathological conditions. This study was performed exclusively on patients with VS to characterize the perilymph in these patients at an individual level. We did this by applying MS-technology for proteomic analysis. The individual approach provided new insight of the large variations in the protein content of perilymph in VS disease. With a uniform diagnosis, it was also possible to identify inter-individual differences related to clinical outcome. No sampling technique is currently available that allows sampling of perilymph in vivo from individuals with normal inner ear function. Therefore, studies of the normal perilymph proteome are not yet possible.
Using MS to analyze perilymph from 15 individual patients with VS, we found a stable section of the proteome consisting of 91 proteins which were detected in the majority of patients. The stable section was validated by comparison to earlier proteomic studies on perilymph and 89 proteins were confirmed. There was also a highly individual section of the proteome consisting of 184 proteins only detected in a minority of patients. This showed an important variation in the proteome between individuals, even in a cohort with a homogenous diagnosis.