FAKULTAS KEDOKTERAN UNIVERSITAS BRAWIJAYA

RSUD DR SAIFUL ANWAR MALANG

MKDU PS PDS JULI 2022
KELOMPOK 3
1. dr. Setia Budi (Neurologi)
2. dr. Ferdian Musthafa (Kedokteran Fisik dan Rehabilitasi)
3. dr. Dyah Anisa A. (Ilmu Kesehatan Mata)
4. dr. Erna Yulida (Ilmu Kesehatan Mata)
5. dr. Lanisa Hapsari (Ilmu Kesehatan Mata)
Introduction

Human inner ear is relatively Samples of inner ear fluid Proteomic analysis in
inaccessible and difficult to cannot be obtained for micro- perilymph analyzed post-
reach for cellular and chemical analyses from mortem perilymph (Arrer et
molecular analyses healthy individuals in vivo al) and perilymph fistula

Aim:
1. To identify perilymph proteins in patients with VS on an individual level.
2. To investigate identified proteins at a functional level and
3. Search for biological markers for tumor-associated hearing loss and tumor diameter.
Patients and Method
● Patients

Sixteen patients undergoing TLS for sporadic VS, with varying degree of
sensorineural hearing loss

Patients with previous neurosurgery

Patients with comorbidity exclude
Contaminated sample

In total, 16 patients were included, but the sample from one patient was excluded
after MS, resulting in the final study involving 15 patients.
Patients and Method
● Sampling of perilymph

The RWM was perforated with a sharp needle and an Eppendorf Research -Plus
Variable pipette (5–10 μL, Eppendorf AG, Hamburg, Germany) with a 20 μL tip
was used to aspirate a maximum of 10 μL perilymph during a period of
approximately 10 seconds.

The sample was immediately frozen in liquid nitrogen and later transferred to -
80 ̊C.

All samples were collected using sterile technique, including sterile vials for the
fluid samples.
 Patients and Method
● Preparation for mass spectrometry
Samples were freeze-dried over night to remove aqueous content and dissolved in Sodium
Dodecyl Sulfate lysis buffer (4% SDS in 100 mM Tris/HCl pH 7.6) at 70 ̊C for 5 minutes.

Solubilized proteins were separated from sample debris by centrifugation at 16,000 g for 5
minutes

Enzymatic fragmentation of proteins was performed by filter-aided sample preparation

Reverse-phase liquid chromatography for peptide separation was performed using an Easy
Nano Flow System coupled to an LTQ-Orbitrap Velos Pro mass spectrometer

After separation, peptides were ionized using a nano-electrospray ionization source and
transferred into the mass spectrometer.
Patients and Method
● Proteomic analysis

o MS data were analyzed using Proteome Discovery 2.1.0.81 to identify the

total number of proteins. MaxQuant 1.5.6.5
o MaxQuant 1.5.6.5 was used to quantify the proteins in individual samples
o Only peptides with a minimum of 7 amino acids, as well as at least one
unique peptide, were required for protein identification
o Only proteins with at least two peptides, and at least one unique peptide,
were considered as being identified and used for further data analysis.
Patients and Method

● Pathway analysis

o The PANTHER Overrepresentation Test was used to identify all up- or

down-regulated pathways
o The reference used was the whole Homo sapiens genome
o We choose to present only significant (p<0.05) up- or down-regulated
pathways in our results.
 Patients and Method
● Statistical methods

o Analysis was designed to assess the associations between perilymph protein content and
clinical parameters, tumor-associated hearing loss and tumor diameter.
o Only proteins detected in at least 12 out of the 15 samples were included in the final
analysis  This cut off was decided upon after analyzing the detection frequency for
each protein in the samples, to ensure that there was consistency
o First, we applied univariate linear regression with hearing loss or tumor diameter as
dependent variables, and protein levels as explanatory variables
o Random Forest was used to deter- mine the variable importance of all variables in the
data followed by the Boruta method, which is based on repeated Random Forest
analyses, to evaluate if the result was independent from random variations.
Patients and Method

● Ethic statements

This study was approved by the regional ethics committee in Uppsala. Oral
and written consent was obtained from all patients prior to surgery and the study
complied with the rules of the Declaration of Helsinki.
RESULTS
 Quantification of perilymph proteins

• Total 314 proteins

• 60 proteins were identified in all 15
patients
• 91 proteins were detected in 12
patients (cut-off 12 out of 15
patients)
• These 91 proteins  frequently
identified proteins
 Perilymph protein and tumor associated hearing loss

Univariate Linear Regression

4 proteins significantly correlated to tumor-associated hearing loss

1. Ig gamma-4 chain C region (p=0.005)
2. Ig kappa chain C region (p=0.015)
3. Complement C3 (p=0.016)
4. 4. Ig heavy constant gamma 3 (p=0.023)
 Perilymph protein and tumor associated hearing loss

Random Forest and Boruta

Alpha-2-HS-glycoprotein was confirmed as

independent variable for tumor –associated
hearing loss
 Perilymph proteins and tumor diameter

UNIVARIATE LINEAR RANDOM FOREST & BORUTA

REGRESSION
Complement component C6 as
Tumor diameter was correlated with the ”tentative” independent variable for
expression of tumor diameter
N-acetylmuramoyl-L-alanine amidase
95%CI:0,46-8,94; p=0.049
 Pathway Analysis of Perilymph
Using PANTHER overrepresentation test
279 unique proteins were classified according to Gene Ontology (GO) combined with
2 PANTHER subsets of ontologies
1. Biological process
2. Molecular function
3. Cellular component
4. PANTHER protein class
5. PANTHER pathways

Results are shown in logarithmic graphs

 1. Biological Process
● Describes the biological system that
a protein contributes to within the
cell or organism.
● The three most up-regulated
processes were secondary metabolic
processes, complement activation
and cell recognition
● Alpha-2-HS-glycoprotein (P02765)
is part of the acute-phase response
(GO:0006953), neutrophil
degranulation (GO:0043312) and
pinocytosis (GO:0006907), which
all have ancestry links to highly up-
regulated biological processes
 2. Molecular Function
● Refers to the functional interaction a
protein carries out with its
molecular targets
● The most highly up-regulated
molecular functions were antigen
binding, serine-type endopeptidase
inhibitor activity, antioxidant
activity and peptidase inhibitor
activity
● Alpha-2-HS-glycoprotein (P02765)
is a direct part of endopeptidase
inhibitor activity (GO:0004866) and
cysteine-type endopeptidase
inhibitor activity (GO:0004869).
 3. Cellular Component
● Includes subcellular structures, such
as the nuclear membrane, and refers
to the location of a protein, and
where it performs its functional
activity
● The most highly enriched cellular
components found in perilymph
were tubulin complex, intermediate
filament cytoskeleton and
immunoglobulin complex. Only the
`integral to membrane' group was
down-regulated
● Alpha-2-HS-glycoprotein (P02765)
was related to the extracellular
region (GO:0005576)
 4. PANTHER Pathways

● Similar to biological process

analysis, but also specifies the
relationship between interacting
molecules
● 7 significantly up- or down-
regulated pathways
● Enriched pathways were blood
coagulation, the plasminogen
activity cascade, and glycolysis
 5. PANTHER Protein Class
● Includes several protein classes that
are not included in the GO
molecular function ontology
● Twenty protein classes were up-
regulated and only one was down-
regulated
● The most highly enriched
PANTHER protein classes were
complement component, tubulin,
actin and actin-related proteins.
Only nucleic acid binding was
down-regulated
 Discussion
• The total number of identified proteins in the present study (314). 91
proteins were identified in 12 to 15 samples. 89 proteins were also found
in samples from VS patients in two earlier studies and represent a stable
part of the perilymph proteome in patients with VS.

• 51 out of 71 protein could be found among the frequently detected

proteins in perilymph from VS patients than in normal proteome . This
discrepancy could be explained by the large inter individual variation
demonstrated here with 184 out of 314 proteins detected in 4 patients or
less, even with a homogenous diagnosis group.
 Discussion
• Alpha-2-HS-glycoprotein (P02765) was confirmed by nonlinear multivariate
methods Random forest and Boruta as an independent variable for tumor-
associated hearing loss.

• Alpha-2-HS-glycoprotein is part of the acute-phase response, an ancestor to

response to stress and also part of neutrophil degranulation an ancestor to
immune response. It may be excreted from the VS into the extracellular fluid
perilymph where it exerts pro inflammatory activity that contributes to
sensorineural hearing loss.

• Alpha-2-HS-glycoprotein was part of the stable section of the proteome, an

interesting candidate as possible biomarker.
 Discussion
• The use of pathway analysis, such as the PANTHER overrepresentation test,
provides us with an informative presentation of protein-related activities in
the inner ear.

• 10 enriched biological processes, three immune responses were present,

three metabolic processes were identified and the other four processes
identified were cell recognition, phagocytosis, blood coagulation and
response to biotic stimulus.

• Perilymph is much more active from an immunological and metabolic aspect

than previously expected.
 Conclussion

● We found a stable section of the proteome which were detected in the

majority of patients. There was also an important variation in the proteome
between individuals, even in a cohort with a homogenous diagnosis.

● Alpha-2-HS-glycoprotein, P02765, was an independent variable for tumor-

associated hearing loss. This needs to be confirmed by future studies.

● Pathway analysis, showed that perilymph exhibited numerous functions

which were highly enriched. Particularly noteworthy was the highly
significant increase in the activity of immune and metabolic processes.
CRITICAL APPRAISAL
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1. Were the criteria for inclusion in the sample clearly defined?

□ □ □ □
The authors should provide clear inclusion and exclusion criteria that they developed prior to recruitment of
the study participants. The inclusion/exclusion criteria should be specified (e.g., risk, stage of disease
progression) with sufficient detail and all the necessary information critical to the study.
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2. Were the study subjects and the setting described in detail?

□ □ □ □
The study sample should be described in sufficient detail so that other researchers can determine if it is
comparable to the population of interest to them. The authors should provide a clear description of the
population from which the study participants were selected or recruited, including demographics,
location, and time period.
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3. Was the exposure measured in a valid and reliable way?

□ □ □ □
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4. Were objective, standard criteria used for measurement of the
condition?
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5. Were confounding factors identified?

□ □ □ □
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6. Were strategies to deal with confounding factors stated?

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7. Were the outcomes measured in a valid and reliable way?

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8. Was appropriate statistical analysis used?

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Overall appraisal: Include □

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Comments (Including reason for exclusion) :
• 2 out of 8 parameters confirmed this study as included and eligible for cross sectional study

• The weaknesses of this study were that it was not clearly describe sample and confounding. The author
did mention sampling but is limited to inclusion criteria. furthermore, there was not information regarding
the background characteristics of the sampling population. In addition, the authors explained how this
study used 2 filters to get the sampling before using MS. However, there was not further explanation
regarding the details of filter measurements and to what extent sampling contamination was minimized.

• Despite its weaknesses, detailed inclusion criteria, valid and reliable exposure and outcome
measurement, and appropriate statistical analysis provide a solid bases for this study