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Proteome Analysis of Human Perilymph using an Intraoperative Sampling Method Heike Andrea Schmitt, Andreas Pich, Anke Schröder, Verena Scheper, Giorgio Lilli, Günter Reuter, and Thomas Lenarz J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00986 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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Journal of Proteome Research

Proteome Analysis of Human Perilymph using an Intraoperative Sampling Method Heike A. Schmitt1,3,*, Andreas Pich2, Anke Schröder2, Verena Scheper1,3, Giorgio Lilli1,3, Günter Reuter1,3, Thomas Lenarz1,3 1

Department of Otolaryngology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

2

Core Facility Proteomics, Hannover Medical School, Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 3

Cluster of Excellence of the German Research Foundation (DFG; “Deutsche Forschungsgemeinschaft”) “Hearing4all”

Keywords: perilymph, inner ear, diagnostic proteomics, data dependent acquisition

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Abstract:

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 has been 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 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 non-invasive inner ear diagnostics and to elucidate molecular profiles of SNHL.


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Introduction: Sensorineural hearing loss (SNHL) is caused by functional damage of various structures of the cochlea, which includes the perilymph-filled scalae tympani and vestibuli and the endolymph-filled cochlear duct as well as different cell types (inner and outer hair cells), all responsible for signal transduction.1 Inner ear hearing loss can be induced by noise,2 different diseases (e.g., viral infections), can be age-related3 or genetic,4 and leads to impaired signal transduction to the auditory nerve. The knowledge about the exact etiology and pathophysiology of SNHL currently remains limited.5 One approach to improve the knowledge base can be given by the investigation of inner ear fluids. Assessing inner ear structures or fluid (perilymph or even endolymph) requires invasive procedures, making the testing of hypotheses derived from clinical observations highly challenging. These invasive procedures especially in humans might have the risk to induce further damage to the sensory organ. As a consequence, knowledge about numerous biochemical and physical parameters such as chemical composition of the human inner ear fluid perilymph or perfusion of the cochlear tissue is insufficient up to now. Due to the progress in developing atraumatic surgical techniques using atraumatic electrodes, hearing preservation in cochlear implantation (CI) can be reliably achieved with minimal risk of further damage to the inner ear by inflammatory reactions.6-9 Therefore, the sampling of human perilymph for diagnostics of inner ear diseases by a minimal invasive sampling method during inner ear surgeries should be possible and an applicable method to investigate disease-specific changes in perilymph composition5 and identify physiologic relevant biomarkers. The pathophysiological alteration of the composition of the perilymph due to or in progress of inner ear diseases is largely unknown and not described in detail up to now. Some groups studied the composition of perilymph or other parts of the inner ear like the sensory epithelium in humans and animals.10, 11 For human perilymph analysis, postmortem samples were used in some studies.12 Thalmann and Arrer were pioneers in the investigation of perilymph composition performed by gel 3 ACS Paragon Plus Environment


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electrophoresis, resulting in first preliminary perilymph protein profiles for humans.12,

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13

A

specialized sampling technique for perilymph of animals was established by Salt et al..14, 15 A review summarizing proteomic inner ear studies concludes that further studies in the field of auditory science are essential.5 Sampling perilymph from human subjects (besides postmortem) demands highest medical experience and carefulness. To develop and validate an invasive method for the analysis of the composition of the perilymph and its changes in different types of sensory hearing loss is desirable. The possibilities of analytics for perilymph are limited due to the small volume of samples, which can be taken during surgery. Protein analytics showed rapid progress over the last decades, especially in terms of the sensitivity of the measurements. Mass spectrometry (MS)-based protein analysis is the method of choice for protein analysis due to its high specificity and sensitivity, particularly if only small sample amounts are available. Proteins in the sub-femtomol-range can be detected by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS)16 and data dependent acquisition. Due to the high sensitivity especially for sample-limited analysis,17 this method can be also applied to examine the small samples of perilymph being taken during CI. In the present study, we aimed to a) develop a valid sampling method of human perilymph b) analyze the protein composition of human perilymph in comparison to human serum and cerebrospinal fluid (CSF) samples and c) compare the perilymph protein composition of patients with different etiologies of hearing loss.

Material and Methods: Sampling Method A sampling method for human perilymph with modified micro glass capillaries was developed (Fig. 1). Micro glass capillaries (VWR, Mikropipetten ringcaps 5+10 µl VE 250) with an inner diameter of 0.47 mm and an outer diameter of 1.2 mm were modified by means of a puller (P-97, Flaming/Brown Micropipette Puller, Sutter Instrument Company) resulting 4 ACS Paragon Plus Environment

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in a very fine tip normally used for patch clamp experiments. The tip was broken off manually to reach a stable but very fine tip with a sufficient capacious inner diameter at the tip for sampling perilymph by capillary forces. The outer diameter at the tip of the capillary amounts to 170 µm. The tip of the capillary was polished manually to reach a beveled sharped tip for puncture of the round window membrane. Additionally the capillary was scaled with 1 µl markers for controlling of perilymph volume during sampling process. Sampling of human perilymph was mainly performed during CIs. Samples from 37 ears were taken for MS-analysis. At the time of data analysis perilymph was sampled from additional 47 ears, resulting in 84 ears. The audiometric data of all 84 ears being manipulated for perilymph sampling were used for the evaluation of sampling method safety. The 47 perilymph samples not analyzed up to now will be evaluated by MS in future. The 37 samples analyzed by MS were derived from 34 patients undergoing a CI, including 3 patients (children) with bilateral CI. After surgical exposure the round window was punctured manually under microscope with the modified micro glass capillaries using a special forceps (Fig. 1). In order to avoid contamination by other body fluids the surgical area was dried using suction and careful application of hemostatics. The capillary was hand-held by the surgeon with the forceps and left in place for up to 60 sec to sample volumes from 1 to 12 µl. Perilymph sampling time was varying from some seconds to one minute due to the different celerity for filling the capillary with a sufficient volume (>1 µl) of perilymph necessary for MS-analysis. Filling time is most likely dependent from the pressure existing in the inner ear and the surgical procedure especially the connection of the capillary tip to the inner ear fluid and possible blockage of the capillary by microparticles from the round window membrane. At least sampling time was kept as short as possible to ensure a careful sampling. Perilymph sampling was also performed during vestibular schwannoma (VS) surgeries with translabyrinthine approach via the semicircular canal, collected from 4 patients. The samples were directly after sampling (i.e., within minutes) cooled on ice in the operating room and 5 ACS Paragon Plus Environment


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stored at -80 C until MS analysis to avoid changes of protein composition. The samples were macroscopically controlled for possible blood contamination resulting in a slight red colored fluid and additionally by centrifugation of the sample. In the case of a minor red pellet containing red blood cells the pellet was discarded, the supernatant was used for analysis and the sample was marked as slightly contaminated with blood. Human CSF samples were collected by puncture of the dura mater with a syringe or micro glass capillaries during the 4 translabyrinthal VS-surgeries. The CSF sampling during CI was not feasible. Three human blood serum samples were taken from CI-patients and VS-patients in order to compare the protein composition of perilymph with the protein composition of blood serum to examine the perilymph samples for possible blood contamination. The CSF and blood serum samples were also analyzed mass spectrometrically as reference for perilymph analytics and to define perilymph-specific, CSF-specific and blood-specific proteins. Protocols for collection of specimens were approved by the Hannover Medical School Ethics Committee for perilymph during CI (approval no. 1883-2013) and for perilymph and CSF during translabyrinthal VS-surgeries (approval no. 2403-2014) in compliance with all federal and institutional regulations concerning the use of human subjects in research.

Figure 1. Sampling of human perilymph. (a) Left image: Modified micro glass capillary with an inner diameter of 0.47 mm, outer diameter at the tip of 170 µm and scaled with 1 µl-markers appropriate for perilymph sampling. (b) magnification of the tip. (c) Exposure of the round window (black arrow) during an inner ear surgery (left ear) in the context of a CI, (#) Facial nerve canal, (◊) chorda tympani, (*) CI-electrode (prepared for insertion). (d) The micro glass capillary punctures the 6 ACS Paragon Plus Environment

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round window membrane directly before insertion of the CI-electrode. The micro glass capillary was inserted into the perilymphatic space of the scala tympani and human perilymph was taken in by capillary forces, depicting a very gentle sampling method. (Photo courtesy ad: H.A. Schmitt)

Evaluation of Sampling Method Safety To evaluate the safety of the sampling method on the residual hearing in patients, hearing threshold changes after perilymph sampling and CI were compared to hearing threshold changes of randomly chosen CI-patients before and after being implanted with a MEDEL FLEX 24 cochlear implant (n=28). For the control group CI-patients with a CI-electrode length of 24 mm were chosen, because 24 mm depicts the medium electrode length of the CIelectrodes used in the perilymph sampled group (16-32 mm length). Data of 84 ears undergoing a CI with perilymph sampling were used for this comparison. Children and already deaf patients were excluded, resulting in n=46 ears to be included in analysis. Before surgery, at first fitting and 3 month after first fitting pure tone audiometry was performed using a calibrated audiometer according to DIN EN 60318 to detect the acoustic hearing threshold. The test method follows DIN ISO 8253 with headphones for air conduction and headset for bone conduction in both ears. The mean hearing loss (hearing threshold difference before and after implantation) was analyzed for the low frequency region of 125, 250, 500, 750, 1000 and 1500 Hz. The hearing threshold at 500 Hz was exemplarily analyzed for all cases to determine the sampling effect on a cochlear region with residual hearing but without being directly manipulated by the sampling method or the implant insertion. Analyses were performed with GraphPad Prism 5 (Version 5.02, GraphPad Software Inc., La Jolla, CA, USA) using a 5 % criterion for statistical significance. Since data passed the KolmogorovSmirnov normality test 2-way ANOVA with Bonferroni post-test was performed to assess the



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effects of perilymph sampling over time or to detect group differences compared to the nonsampled control group.

Measurement of perilymph protein content The protein content of the human perilymph samples was analyzed by the dotMETRICTM

Assay (G-Biosciences) immediately before mass spectrometric analysis. 2 µl of the gel loading samples consisting of the perilymph sample diluted 7-fold with Laemmli buffer were used for protein content measurement. In case of perilymph volume 18 years; n = 25) and children (< 18 years; n = 12) were selected and compared. In both groups were found 624 proteins, of which 12 are significantly higher quantified in the adults group and 12 significantly higher in the children group. 142 proteins were identified only in adults. 41 proteins were solely found in children perilymph samples of which 1 protein is significantly higher compared to adult patients.


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The identified proteins with significantly increased abundances in children were mainly found to be involved in immune response like the Ig proteins lambda and kappa chains. Proteins with a significant higher abundance were vasorin, alpha-1-acid glycoprotein 2, ganglioside GM2 activator, carboxypeptidase N subunit 2, fibrinogen alpha chain, and also collagen alpha-2(XI) chain, which is known to cause sensorineural hearing loss (stickler syndrome 3, autosomal dominant deafness 13 and 53) by mutations affecting the gene.34-36 In the adult group, the proteins beta-Ala-His dipeptidase, dickkopf-related protein 3, monocyte differentiation antigen CD14, Ig alpha chains, angiotensinogen, and complement component proteins showed an increased abundance. Additionally, protein Ig lambda chain V-VI region WLT was found solely with a higher abundance in perilymph samples of children (Tab. 2). The UniProt entry of this protein characterizes an antigen binding molecular function, location in the plasma membrane or extracellular space and an involvement in the biological function immune response and complement activation.37

Table 2. Significant perilymph proteins in comparison adults vs children. Uniprot ID

Protein name

p< 0.05

At least 9 in e.g.

Protein identified in

Q96KN2

Beta-Ala-His dipeptidase

↓

Q9UBP4

Dickkopf-related protein 3

↓

X

Ad, Ch

P08571

Monocyte differentiation antigen CD14

↓

X

Ad, Ch

P01877

Ig alpha-2 chain C region

↓

X

Ad, Ch

P01876

Ig alpha-1 chain C region

↓

X

Ad, Ch

P01019

Angiotensinogen

↓

X

Ad, Ch

P0C0L5

Complement C4-B

↓

X

Ad, Ch

Q5JNX2

Complement C4 beta chain

↓

X

Ad, Ch

B0V2C8

Complement C4 beta chain

↓

X

Ad, Ch

A2BHY4

Protein LOC100293534

↓

X

Ad, Ch

Q6U2E9

C4B1

↓

X

Ad, Ch

P01859

Ig gamma-2 chain C region

↓

X

Ad, Ch

P06318

Ig lambda chain V-VI region WLT

↑

P23083

Ig heavy chain V-I region V35

↑

P22792

Carboxypeptidase N subunit 2

↑

Ad, Ch

Ch X

Ad, Ch Ad, Ch



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Q6ZNN4

Collagen alpha-2(XI) chain

↑

Ad, Ch

P02671

Fibrinogen alpha chain

↑

P17900

Ganglioside GM2 activator

↑

P06312

Ig kappa chain V-IV region

↑

X

Ad, Ch

P01613

Ig kappa chain V-I region Ni

↑

X

Ad, Ch

P83593

Ig kappa chain V-IV region STH

↑

X

Ad, Ch

P01781

Ig heavy chain V-III region GAL

↑

X

Ad, Ch

P01714

Ig lambda chain V-III region SH

↑

X

Ad, Ch

Q6EMK4

Vasorin

↑

X

Ad, Ch

P19652

Alpha-1-acid glycoprotein 2

↑

X

Ad, Ch

X

Ad, Ch Ad, Ch

Significantly (p

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