International Journal of

Molecular Sciences

Article
Comparative Proteomic Profiling of Blood Plasma Revealed
Marker Proteins Involved in Temporal Lobe Epilepsy
Yury E. Glazyrin 1,2, * , Dmitry V. Veprintsev 1 , Elena E. Timechko 3 , Zoran Minic 4 , Tatiana N. Zamay 1,2 ,
Diana V. Dmitrenko 3 , Maxim V. Berezovski 4 and Anna S. Kichkailo 1,2

1 Laboratory for Digital Controlled Drugs and Theranostics, Federal Research Center “Krasnoyarsk Science
Center of the Siberian Branch of the Russian Academy of Science”, Akademgorodok 50,
660036 Krasnoyarsk, Russia; [email protected] (D.V.V.); [email protected] (T.N.Z.);
[email protected] (A.S.K.)
2 Laboratory for Biomolecular and Medical Technologies, Prof. V.F. Voino-Yasenetsky Krasnoyarsk State
Medical University, Partizana Zheleznyaka 1, 660022 Krasnoyarsk, Russia
3 Department of Medical Genetics and Clinical Neurophysiology, Prof. V.F. Voino-Yasenetsky Krasnoyarsk State
Medical University, Partizana Zheleznyaka 1, 660022 Krasnoyarsk, Russia; [email protected] (E.E.T.);
[email protected] (D.V.D.)
4 Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie-Curie,
Ottawa, ON K1N 6N5, Canada; [email protected] (Z.M.); [email protected] (M.V.B.)
* Correspondence: [email protected]

Abstract: Temporal lobe epilepsy has various origins, involving or not involving structural changes
in brain tissue. The mechanisms of epileptogenesis are associated with cell regulation and signaling
disruptions expressed in varied levels of proteins. The blood plasma proteomic profiling of temporal
lobe epilepsy patients (including magnetic resonance imaging (MRI)-positive and MRI-negative
ones) and healthy volunteers using mass spectrometry and label-free quantification revealed a list
of differently expressed proteins. Several apolipoproteins (APOA1, APOD, and APOA4), serpin
protease inhibitors (SERPINA3, SERPINF1, etc.), complement components (C9, C8, and C1R), and a
total of 42 proteins were found to be significantly upregulated in the temporal lobe epilepsy group. A
Citation: Glazyrin, Y.E.; Veprintsev,
classification analysis of these proteins according to their biological functions, as well as a review
D.V.; Timechko, E.E.; Minic, Z.; Zamay,
of the published sources, disclosed the predominant involvement of the processes mostly affected
T.N.; Dmitrenko, D.V.; Berezovski,
during epilepsy such as neuroinflammation, intracellular signaling, lipid metabolism, and oxidative
M.V.; Kichkailo, A.S. Comparative
stress. The presence of several proteins related to the corresponding compensatory mechanisms
Proteomic Profiling of Blood Plasma
Revealed Marker Proteins Involved in has been noted. After further validation, the newly identified temporal lobe epilepsy biomarker
Temporal Lobe Epilepsy. Int. J. Mol. candidates may be used as epilepsy diagnostic tools, in addition to other less specific methods such
Sci. 2024, 25, 7935. https://doi.org/ as electroencephalography or MRI.
10.3390/ijms25147935
Keywords: biomarkers; temporal lobe epilepsy; proteomics; mass spectrometry
Academic Editor: Henry Hing
Cheong Lee

Received: 12 June 2024

Revised: 15 July 2024 1. Introduction
Accepted: 19 July 2024
Epilepsy affects more than 70 million people and is one of the most common neuro-
Published: 20 July 2024
logical disorders in the world [1]. Temporal lobe epilepsy (TLE) is often accompanied by
histological changes in the brain tissue, such as hippocampal sclerosis (HS), characterized
by the loss of pyramidal neurons and gliosis in the C1 and C3 regions, as well as in the
Copyright: © 2024 by the authors.
areas of the Ammon’s horn [2]. HS is one of the most common causes of drug-resistant
Licensee MDPI, Basel, Switzerland. epilepsy in adults [1]. However, according to magnetic resonance imaging (MRI), some
This article is an open access article patients do not have any structural changes in the brain. Despite advances in research
distributed under the terms and on the neuronal mechanisms underlying epileptogenesis and the spread of new anticon-
conditions of the Creative Commons vulsants, there are no pharmacological agents for the prevention of epileptogenesis; the
Attribution (CC BY) license (https:// mechanisms of epileptogenesis do not coincide with the mechanisms of ictogenesis, the pro-
creativecommons.org/licenses/by/ cess leading to seizure [3]. Along with the lack of effective pharmacological approaches and
4.0/). disease-modifying therapies, the diagnosis of epilepsy also remains a clinical problem [4].

Int. J. Mol. Sci. 2024, 25, 7935. https://doi.org/10.3390/ijms25147935 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 7935 2 of 15

Today, diagnostics of epilepsy mostly depend on clinical examination, history, and elec-
troencephalography (EEG) monitoring—a method that is neither specific nor sensitive [4],
and also, in some cases, leads to an erroneous diagnosis.
Accordingly, there is considerable interest in the discovering and testing of new
biomarkers for the earlier and more reliable diagnosis for timely treatment. Information
on the specific protein expression will lead to a deeper insight into the mechanism of
epileptogenesis. The proteomic profiling, characterization, and comparison of the different
types of epilepsy in terms of proteins present in the blood is extremely important for the
development of clinical diagnostic tools. A relevant model for the study of epilepsy is TLE,
as the most common form of focal epilepsy.
Several studies evaluated the levels of the selected candidate biomarker proteins
in blood or cerebrospinal fluid (CSF) [5,6]. The whole proteomic quantitative analysis
offers an unbiased screening approach—and gives a general idea of the changes in protein
levels [7]. Plasma proteomic studies have provided information on the potential biomarkers
for several neurological disorders [8], but there are few proteomic studies of epilepsy.
Biomarker discovery performed on animal and human tissue models [9] revealed the
aberrant expression of markers of neurodegeneration and neuroinflammation, as well as
changes in the energy metabolism systems. Only a few studies evaluated patient plasma
biomarkers, where the authors found several differentially expressed proteins associated
with the immune response in children with Rolandic epilepsy [10]. In a recent study [11],
some proteins associated with neuroinflammation and neurodegeneration were tested in
plasma as predictive markers of the course of TLE in relation to drug resistance.
The majority of TLE patients took antiepileptic drugs (AEDs) in monotherapy or
polytherapy. It is quite likely that the use of AEDs can affect protein expression. It would
certainly be important to identify potential protein markers of epilepsy without taking
into account the influence of AEDs, but epilepsy is a disease that requires the long-term
use of AEDs. Thus, the involvement in studies of large patient groups without drug
therapy seems quite difficult. In this research, we compared the proteomic profiles of the
blood plasma of AED-taking patients with TLE (including MRI-positive and MRI-negative
ones) and healthy volunteers to search for new markers of the disease. The proteins
implicated in early studies as being associated with AEDs were rejected. The proteins
involved in neuroinflammation were proposed as epileptogenesis marker candidates. The
different expression of the identified proteins indicates multiple disturbances in intracellular
signaling, lipid metabolism, inflammation, and impaired processes of neuronal excitability.

2. Results
2.1. Manifestation of Proteomic Differences in the Groups
A mass spectrometry analysis of plasma samples was performed for 27 temporal
lobe epilepsy patients and 7 volunteers. The data analysis led to obtaining quantitative
information about the distribution of 192 proteins in plasma samples of 34 participants
made in triplicates. Triple repetitions were averaged without significant loss, as described
in Section 4.
Principal component analysis (PCA) is a way to reduce the dimensionality of the
complex dataset by diminishing the least amount of information. A multi-dimensional
matrix of proteins and their quantitative indicators is reduced by projecting them onto
the new axes of the principal components. At the same time, the presence of the most
distinctive proteins affects the relative positions of the observations in the new co-ordinates,
clearly highlighting their differences.
The manifestation of differences in proteomic profiles between MRI-negative and MRI-
positive TLE groups, and also the healthy group, are illustrated in principal component
co-ordinates by 3D PCA (Figure 1). The MRI-positive group is separated from the MRI-
negative group, which allows us to justify the comparison of proteomic profiles of the
groups to identify the most distinguishing proteins.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 16

Int. J. Mol. Sci. 2024, 25, 7935

MRI-negative group, which allows us to justify the comparison of proteomic profiles of
3 of 15
the groups to identify the most distinguishing proteins.

The3D
Figure1.1.The
Figure 3Dprincipal
principalcomponent
componentanalysis
analysisdiagram
diagramofofprotein
proteindistributions
distributionsfor
forthe
theMRI-positive
MRI-positive
epilepsypatients,
epilepsy patients,MRI-negative
MRI-negativeepilepsy
epilepsypatients,
patients,and
andthe
thehealthy
healthygroup.
group.Point
Pointlabels
labelsare
aredescribed
described
in
inthe
thefootnote
footnoteto
toTable
Table1.1.

Epilepsycohort.
Table1.1.Epilepsy
Table cohort.

Freq. of Freq.
Last
of Duration
Last of
Duration of Antiepileptic Focal Seizure
Focal Seizure Duration
Duration ofof
Age
Age MRI MRI Antiepileptic Drug,
NN ID
ID GG over4040
GD GD DR FBTC
DR FBTC,
FBTCper FBTC,
FBTC, per Epilepsy,Epilepsy,
FBTC, Drug, mg/per Freq., per
Freq., per Remission,
Remission,
over Status Status mg/per Day
Month Month Year Year
Years Years Day Month
Month Years
Years
LTG 200 mg +
11 A1
A1 FF No
No TLE TLE
Neg. Neg. Yes R
R Yes
7 72021 2021 29 29LTG 200 mg + TPM 100 77 No
No
TPM 100 mg
mg
LCS 150 mgLCS+ 150
LEV mg +
1500
2 A2 M Yes TLE Neg. R Yes 3 2021 11 No No
2 A2 M Yes TLE Neg. R Yes 3 2021 11 LEV 1500 mg No No
mg
3 A3 M No TLE Neg. R Yes 12 2021 10 LTG 150 mg No No
3 A3 M No TLE Neg. R Yes 12 2021 10 LTG 150 mg No No
44 V1 FF
V1 Yes
Yes TLE TLE
Neg. Neg.
NR YesNR Yes
2 22021 2021 1 1 OXCOXC 600 mg
600 mg No
No No
No
55 V2 FF
V2 No
No TLE TLE
Neg. Neg.
R RE R RE 2015 2015 24 24 No No No
No 66
66 V3
V3 FF No
No TLE Neg.
TLE NR
Neg. RE NR RE 2017 2017 13 13 LCS LCS
100 mg
100 mg No
No 33
77 V4
V4 FF No
No TLE Neg.
TLE R
Neg. RE R RE 2018 2018 3 3 LCS LCS
75 mg75 mg No
No 33
88 V5
V5 MM No
No TLE Neg.
TLE NR
Neg. RE NR RE 2015 2015 25 25 CBZ CBZ
400 mg
400 mg No
No 22
CBZ 800 mg + LEV 1000
99 V6
V6 FF No
No TLE TLE
Neg. NR
Neg. No NR No 2019 2019 27 27
CBZ 800 mg + No
No 22
mg 1000 mg
LEV
TLE TLE
& &M
1010 VA
VA FF No
No Neg. Neg. No R
R No 2020 2020 18 18 CBZ CBZ 800 mg
800 mg No
No 11
M
LTG 150 mg +
TLE & 6LTG 150 mg
VPA+450
VPAmg450
1111 V7
V7 FF No
No TLE Neg.
&S Neg. Yes R
R Yes
1 12020 2020 6 + <1
<1 No
No
S PMP44mg
mg + PMP mg
TLE & LTG 200 mg +
1212 VG1
VG1 FF Yes
Yes Pos.
TLE & S RPos. RE R 12
RE 2020
12 2020 48 LTG 200 mgPMP
48 + PMP 4 mg
4 mg
66 No
No
S
13 VG2 F No TLE &
TLE & S Pos. R No 12 LCS 350 mg 5 No
13 VG2 F No Pos. R No 12 LCS 350 mg 5 No
14 VG3 M No S TLE & S Pos. NRt Yes 1 2020 4 LCS 200 mg 1 No
TLE &
1415 VG3
VG4 MF No
No TLE & S
Pos. Pos. Yes R
NRt No
1 2020 4 9 LCS CBZ 600 mg
200 mg 11 No
No
S
16 VG5 F No TLE & S Pos. NR Yes 2019 32 CBZ 300 mg 2 No
TLE &
15 VG4 F No Pos. R No 9 CBZVPA
600200
mgmg + 1 No
17 VG6 F No S TLE & S Pos. NR RE 2017 22 LTG 200 mg + 4 No
TLE & ZNS 300 mg
16 VG5 F No Pos. NR Yes 2019 32 CBZ 300 mg 2 No
S
Int. J. Mol. Sci. 2024, 25, 7935 4 of 15

Table 1. Cont.

Freq. of Last Duration of Antiepileptic Focal Seizure Duration of

Age MRI
N ID G GD DR FBTC FBTC, per FBTC, Epilepsy, Drug, mg/per Freq., per Remission,
over 40 Status
Month Year Years Day Month Years
PB 175 mg +
18 VG7 M Yes TLE & S Pos. R RE 2018 46 LCS 200 mg + 2 No
VPA 600 mg
LEV 2000 mg +
19 VG8 M Yes TLE & S Pos. R No 5 1 No
OXC 2100 mg
CBZ 1000 mg +
20 VS1 M No TLE & VA Pos. NR Yes 1 2020 11 2 No
LTG 200 mg
VPA 900 mg +
21 VS2 F Yes TLE & VA Pos. R RE 2013 28 6 No
OXC 600 mg
LTG 150 mg +
22 VS3 M Yes TLE & VA Pos. R RE 2017 46 VPA 900 mg + 2 No
LEV 1500 mg
23 VP1 F Yes TLE & M Pos. NR No 15 OXC 600 mg 2 No
24 VP2 M No TLE & M Pos. R No 8 OXC 600 mg 1 No
25 VP3 F Yes TLE & M Pos. NR No 2020 3 No 1 No
TLE & OXC 600 mg +
26 VPG1 M Yes Pos. R RE 2018 4 2 No
M&S BRV 100 mg
OXC 1500 mg +
TLE &
27 VPG2 M No Pos. R RE 2010 17 LEV 1500 mg + 1 No
M&S
LCS 400 mg
G—gender (M—male; F—female). GD—general diagnosis (TLE—temporal lobe epilepsy; TLE & M—temporal
lobe epilepsy with malformations; TLE & S—temporal lobe epilepsy with hippocampus sclerosis; TLE &
VA—temporal lobe epilepsy vascular anomaly; TLE & M & S—temporal lobe epilepsy with malformations and
hippocampus sclerosis). DR—drug-resistance (R—resistant; NR—non-resistant). FBTC—focal bilateral tonic
clonic seizure (RE—registered earlier). Antiepileptic Drugs: BRV—Brivaracetam; CBZ—Carbamazepine;
LCS—Lacosamide; LEV—Levetiracetam; LTG—Lamotrigine; OXC—Oxcarbazepine; PB—Phenobarbital;
PMP—Perampanel; TPM—Topiramate; VPA—Valproate; ZNS—Zonisamide.

PCA was also used to search for proteomic differences between the groups by sex, age,
and drug resistance. No distinct clusters were found, which shows that these features do
not have a significant influence on the proteomic profiles of the samples.
The different arrangement of patient groups in the planes of the principal components
gives grounds to compare their proteomic profiles statistically for the identification of
significant protein markers of these groups. The proteomic profiles of the selected pa-
tient groups were compared in pairs, and statistically different proteins (corrected by the
Benjamini–Hochberg procedure Welch’s t-test p-value < 0.01) were noted as significantly
changed based on their label-free quantification (LFQ) values.

2.2. Proteomic Profile Comparison of the Whole Epilepsy Group (TLE) versus the Healthy Group
When comparing the whole group of TLE patients with healthy controls, 42 proteins
were identified as significantly upregulated in epilepsy (Table S1). An analysis of the tissue
protein expression using Human Protein Atlas (HPA) demonstrated the presence of mainly
liver proteins. A Gene Ontology (GO) analysis performed using ShinyGo 0.76 demonstrated
the involvement of overexpressed proteins in the regulation of the following biological
processes: lipid metabolism, immune response, catalytic activity, and coagulation (Figure 2).
The proteins upregulated in the epilepsy group (Table 2) are involved in the processes
of the response to external stimuli, stress, and the immune response, which indicates the
presence of a neuroinflammatory process that accompanies and maintains epileptogenic
activity. However, the question of their specificity is still open. Proteins that regulate
the process of intracellular signaling have also been identified, which demonstrates the
dysregulation of the cascade of intracellular events. The altered expression of proteins that
remodel the levels of plasma lipoproteins indicates a lipid metabolism disorder in patients
with epilepsy.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 of 16
Int. J. Mol. Sci. 2024, 25, 7935 5 of 15

Plot of
Figure 2. Plot of biological
biological processes
processes for
for upregulated
upregulated proteins
proteins when comparing the overall epilepsy
epilepsy
group with the healthy group.

TableThe proteins upregulated

2. Upregulated in the
epilepsy proteins epilepsy
involved group biological
in different (Table 2) processes.
are involved in the pro-
cesses of the response to external stimuli, stress, and the immune response, which indi-
Number of Involved Proteins High-Level Gene Ontology Category Protein’s Names
cates the presence of a neuroinflammatory process that accompanies and maintains epi-
leptogenic activity. However, the questionCPB2, ATRN,specificity
of their SERPIND1, isHPX, APOA4,
still open.C9, SERPINC1,
Proteins that
APOA1, CLU, C4BPB, PROZ, SERPINF1, AGT, GSN,
26 Response to stress
regulate the process of intracellular signaling haveALB,
alsoPRDX2,
been FGG,
identified, which demon-
SERPING1, A2M, APOD, SERPINA3,
strates the dysregulation of the cascade of intracellular events.
SERPINA1, ORM1, HPR, The alteredSERPINA10
SERPINF2, expression of
proteins that remodel the levels of plasma lipoproteins indicatesHPX,
PON1, CPB2, SERPIND1, a lipid metabolism
APOA4, C9, BCHE, dis-
20 Responsewith
order in patients to external stimulus
epilepsy. SERPINC1, APOA1, CLU, C4BPB, SERPINF1, AGT, GSN,
SERPING1, ALB, PRDX2, FGG, A2M, SERPINF2

Table 2. Upregulated epilepsy proteins involvedPON1, ITIH1, biological

in different SPP2, SERPIND1, APOA4, SERPINC1,
processes.
APOA1, CLU, SERPINF1, AGT, GSN, SERPING1, PRDX2,
20 Regulation of molecular function
A2M, CPN2, SERPINA3, SERPINA1, APOC2,
Number of
High-Level Gene Ontology SERPINF2, SERPINA10
Involved Protein’s Names
Category CPB2, HPX, APOA4, C9, SERPINC1, APOA1, CLU, C4BPB,
19 Proteins Immune system process GSN, SERPING1, PRDX2, FGG, A2M, CPN2, SELL, APOD,
SERPINA3,
CPB2, ATRN, SERPINA1,HPX,
SERPIND1, ORM1APOA4, C9,
CPB2, HPX, C9, SERPINC1,
SERPINC1, APOA1,
APOA1, CLU, CLU, C4BPB,
C4BPB, PROZ,
18 Regulation of response to stimulus SERPINF1, AGT, SERPING1, PRDX2, FGG, A2M, CPN2,
SERPINF1, AGT, GSN, SERPING1,
SELL, APOD, VASN, SERPINF2 ALB,
26 Response to stress
PRDX2, FGG, A2M, APOD, SERPINA3,
CPB2, HPX, APOA4, C9, SERPINC1, APOA1, CLU, C4BPB,
17 Immune response SERPINA1,
GSN, ORM1,
SERPING1, FGG, A2M,HPR,
CPN2,SERPINF2, SER-
SELL, SERPINA3,
SERPINA1, ORM1
PINA10
CPB2, HPX, C9,
PON1, APOA1,
CPB2, CLU, C4BPB,
SERPIND1, SERPING1,
HPX, APOA4,PRDX2,
C9,
14 Regulation of the immune system process
FGG, A2M, CPN2, SELL, APOD, ORM1
BCHE, SERPINC1, APOA1, CLU, C4BPB,
11
20 Response to external stimulusHPX, BCHE, APOA1, CLU, AGT, PRDX2, FGG, A2M,
Regulation of signaling SERPINF1, AGT,
APOD, GSN,
VASN, SERPING1, ALB,
SERPINF2
PON1, APOA4, APOA1, CLU, SERPINF2
PRDX2, FGG, A2M, C4BPB, ALB,
8 Catabolic process
PON1, ITIH1, SPP2,
PRDX2, SERPIND1, APOA4,
APOC2
7 Activation of immune response SERPINC1,
CPB2, C9, CLU,APOA1, CLU, SERPINF1,
C4BPB, SERPING1, A2M, CPN2AGT,
Regulation of molecular func-
20 GSN, SERPING1,
CPB2, PRDX2, A2M,
SERPINC1, SERPING1, PRDX2,CPN2,
FGG, SER-
7 tion
Regulation of hemostasis
A2M, SERPINF2
PINA3, SERPINA1, APOC2, SERPINF2,
Regulation of plasma lipoprotein particle
6 APOA4, APOA1,SERPINA10
AGT, ALB, LCAT, APOC2
levels
CPB2, HPX, APOA4, C9, SERPINC1,
APOA1, CLU, C4BPB, GSN, SERPING1,
2.3. Proteomic
19 Profile
ImmuneComparison
systemofprocess
MRI-Negative and MRI-Positive Epilepsy Groups
PRDX2, FGG, A2M, CPN2, SELL, APOD,
Next, we compared the proteomic profiles of two subgroups of epilepsy, which differ
SERPINA3, SERPINA1, ORM1
on the PCA diagram—MRI-negative and MRI-positive.
Regulation of response to stim- CPB2, HPX, C9, SERPINC1, APOA1, CLU,
The
18 MRI-positive TLE patients are characterized by MRI-visible affected areas of the
ulus C4BPB, SERPINF1, AGT, SERPING1,
epileptogenic focus. The MRI-negative group is presented by TLE patients with recurrent
 2.3. Proteomic Profile Comparison of MRI-Negative and MRI-Positive Epilepsy Groups
Next, we compared the proteomic profiles of two subgroups of epilepsy, which differ
on the PCA diagram—MRI-negative and MRI-positive.
Int. J. Mol. Sci. 2024, 25, 7935 The MRI-positive TLE patients are characterized by MRI-visible affected areas of 6 of
the15
epileptogenic focus. The MRI-negative group is presented by TLE patients with recurrent
unprovoked seizures, originating from the temporal lobe based on electroclinical data, in
unprovoked seizures, originating from the temporal lobe based on electroclinical data, in
the absence of an epileptogenic lesion on a visual examination of the MRI, and without
the absence of an epileptogenic lesion on a visual examination of the MRI, and without
signs of hippocampal sclerosis, vascular malformations, tumors, hamartomas, etc.
signs of hippocampal sclerosis, vascular malformations, tumors, hamartomas, etc.
For these groups, a list of 46 significantly altered proteins was obtained (Table S2).
For these groups, a list of 46 significantly altered proteins was obtained (Table S2).
Eleven proteins were found to be upregulated in the MRI-positive group. The GO analysis
Eleven proteins were found to be upregulated in the MRI-positive group. The GO analysis
revealed the involvement of these proteins in the list of biological processes (Figure 3).
revealed the involvement of these proteins in the list of biological processes (Figure 3).
Most of the overrepresented MRI-positive group proteins are involved in the regulation
Most of the overrepresented MRI-positive group proteins are involved in the regulation of
of the immune response.
the immune response.

Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 16

Figure 3.
Figure Plot of
3. Plot of biological
biological processes
processes for
for upregulation
upregulation in
in MRI-positive
MRI-positive group
group proteins.
proteins.
The group of MRI-negative patients is characterized by the upregulation of 35 pro-
The group of MRI-negative patients is characterized by the upregulation of 35 proteins.
teins. The GO analysis revealed the involvement of these proteins in a number of biologi-
The GO analysis revealed the involvement of these proteins in a number of biological
cal processes (Figure 4).
processes (Figure 4).

Figure4.
Figure Plotof
4.Plot ofbiological
biologicalprocesses
processesfor
forupregulation
upregulationin
inMRI-negative
MRI-negative group
group proteins.
proteins.

For both groups, most of the overrepresented proteins are involved in the regulation
For both groups, most of the overrepresented proteins are involved in the regulation
of the immune response, which is typical for the neuroinflammatory process. Neuroinflam-
of the immune response, which is typical for the neuroinflammatory process. Neuroin-
mation is one of the leading mechanisms of epileptogenesis. However, the set of differently
flammation is one of the leading mechanisms of epileptogenesis. However, the set of dif-
expressed proteins for the MRI-positive and MRI-negative groups was different. Currently,
ferently expressed proteins for the MRI-positive and MRI-negative groups was different.
an additional examination for the MRI-negative group reveals the autoimmune nature of
Currently, an additional examination for the MRI-negative group reveals the autoimmune
the disease, which explains the widespread involvement of inflammatory proteins in the
nature of the disease, which explains the widespread involvement of inflammatory pro-
pathogenesis of the disease.
teins in the pathogenesis of the disease.
Plasma proteomic profiles dependent on drug response status, age, and sex of the
Plasma proteomic profiles dependent on drug response status, age, and sex of the
TLE patients were tested. No statistically significant difference was found between the
TLE patients were tested. No statistically significant difference was found between the
drug-resistant and non-drug-resistant epilepsy groups, as well as between the two age
groups (under 40 and over 40), or based on gender.

3. Discussion
 Int. J. Mol. Sci. 2024, 25, 7935 7 of 15

drug-resistant and non-drug-resistant epilepsy groups, as well as between the two age
groups (under 40 and over 40), or based on gender.

3. Discussion
3.1. Association of Plasma and Brain Proteins in Epilepsy
According to the Human Protein Atlas available online: https://www.proteinatlas.
org/ (accessed on 18 July 2024), some of the proteins we discovered in plasma are detected
in the brain: HPX, CP, SERPINA3, APOA1, APOD, HPR, APOC2, BCHE, SELL, AGT,
VASN, IGKV4-1, ORM1, ALB, and SERPINF2. However, blood immunological inflamma-
tory substrates have been found to play a role in the pathogenesis of many neurological
disorders. In particular, some studies have demonstrated the induction of plasma inflamma-
tory and neurotrophic markers [12]. This was also observed in animal models of temporal
lobe epilepsy [13]. Moreover, a number of studies discussed the epileptogenicity of the
activation of the peripheral immune system [14]. It is known that prolonged seizures are
associated with an increase in the permeability of the blood–brain barrier (BBB), as well as
with multiple changes in the properties of the BBB [15], which was demonstrated by the
infiltration of the brain parenchyma by peripheral immune cells [16]. In addition, plasma
inflammation persists long-term after an attack [17]. These data suggest the presence
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 16 of a
systemic response involved in epileptogenic processes, indicating that the changed plasma
protein profile with epilepsy reflects changes in brain proteins.
The majority of the proteins with an altered expression found by the comparison
evidenced by the aberrations of proteins involved in various mechanisms. Epilepsy does
of epilepsy and healthy plasma proteomic profiles are involved in immune response
not cause strictly specific dysregulations. Biochemical cascades triggered by an attack lead
processes, lipid metabolism, and the regulation of peptidase activity (Figure 5). The process
to changes in various biological and molecular pathways, that are not direct factors in the
of epileptogenesis causes massive changes in cellular and molecular processes, as evidenced
development of the disease.
by the aberrations of proteins involved in various mechanisms. Epilepsy does not cause
Some of the proteins with an altered expression found in earlier studies are compiled
strictly specific dysregulations. Biochemical cascades triggered by an attack lead to changes
in Table S3.
in various biological and molecular pathways, that are not direct factors in the development
of the disease.

Figure 5. Network of biological processes of proteins with altered expression, registered by comparing
plasma
Figure proteomic
5. Network profiles ofprocesses
of biological epilepsy patients withwith
of proteins healthy control
altered plasma. registered by com-
expression,
paring plasma proteomic profiles of epilepsy patients with healthy control plasma.
Some of the proteins with an altered expression found in earlier studies are compiled
in Table S3.
3.2. Structural Hippocampal Changes in Epilepsy
Hippocampal sclerosis is not considered to be a distinct disease, but likely consists of
several subtypes, and the causes of this condition are currently poorly understood [2]. In
addition, according to histological studies, in patients with temporal lobe epilepsy, the
loss of neuronal cells and pronounced astrocytic gliosis in the hippocampus are recorded,
in contrast to epilepsy not associated with sclerosis, when neurons are preserved and glio-
Int. J. Mol. Sci. 2024, 25, 7935 8 of 15

3.2. Structural Hippocampal Changes in Epilepsy

Hippocampal sclerosis is not considered to be a distinct disease, but likely consists of
several subtypes, and the causes of this condition are currently poorly understood [2]. In
addition, according to histological studies, in patients with temporal lobe epilepsy, the loss
of neuronal cells and pronounced astrocytic gliosis in the hippocampus are recorded, in
contrast to epilepsy not associated with sclerosis, when neurons are preserved and gliosis
has no features. A decrease in synaptic proteins is a manifestation of the loss of neuronal
cell bodies and dendrites, whereas an increase in glial-associated proteins is a manifestation
of astrocyte proliferation and hypertrophy. It is believed to be a result of sclerosis of the
hippocampus.
Traditionally, epileptogenesis is the process by which the neural network of the brain
functionally changes to increase the susceptibility to epileptic seizures, which increases the
likelihood of spontaneous recurrent seizures (SRSs) [18]. Therefore, the process of epilepto-
genesis was considered exclusively in the context of the “latent period”, i.e., as the period of
time between the epileptogenic event and the onset of the first clinically detectable seizure.
However, a number of studies have demonstrated that the frequency and severity of SRSs
continues to increase after the first unprovoked or spontaneous attack [19,20]. Therefore,
there are data that allow us to consider the process of epileptogenesis as a continuous
and long-term process. It is also demonstrated that the processes of the reorganization of
brain tissue, leading to the formation of the first SRS, also continue after it [18,21], which
contributes to the further progression of the disease and the induction of subsequent attacks.
At the same time, there are opinions that epilepsy is a progressive disease, which includes
the death of neurons, gliosis, the reorganization of neural networks and the development
of pharmacoresistance, which is associated directly with epileptomorphic brain activity ac-
cording to the “seizures-beget-seizures” theory [22]. Therefore, the frequency and severity
of attacks, as well as structural changes in the hippocampus, may be a consequence of the
ongoing process of epileptogenesis.

3.3. Oxidative Stress Regulation

Several groups of proteins identified in our study and previously associated with
epilepsy exhibit a similar pattern. Among them are apolipoproteins involved in lipid
metabolism that reduce the oxidative stress caused by lipid peroxidation: APOA1, APOD,
APOE, and APOA4.
APOA1 regulates the functions of neutrophils and reduces the synthesis of reactive
oxygen species in brain cells. An increased content of APOA1 was found in the hippocam-
pus tissues of mesial TLE patients [23]. Our study found APOA1 overexpression in TLE
patients compared to controls, regardless of their MRI status. APOD has a high affinity for
arachidonic acid (AA), which is a regulator of many inflammatory processes associated
with neuronal damage [24]. APOD and its transcript are overexpressed during the acute
inflammatory phase, reducing the secretion of pro-inflammatory cytokines and interferons.
The early accumulation of the APOD protein in hippocampal neurons after limbic seizures
has been reported [25]. A selective increase in APOD mRNA was observed in hippocam-
pal tissues in a kainic model of epilepsy in rats [26]. It was associated with Alzheimer’s
and Parkinson’s diseases [27], as well as related to oxidative stress [28], and multiple
sclerosis [29]. In our study, APOD was found to be overexpressed in the plasma of TLE
patients compared to controls. A high level of APOE was found to be associated with an
earlier onset of temporal lobe epilepsy and was observed in TLE patients’ plasma [30]. In
our study, APOE overexpression was found in the group of MRI-positive TLE patients.
APOA4 is a potent inhibitor of lipid oxidation and an anti-inflammatory agent inhibiting
P-selectin-mediated adhesive interactions between leukocytes and platelets. Its concentra-
tion increases in the plasma of refractory epilepsy patients [31]. In our study, an increased
expression of APOA4 was found in the epilepsy group. The overall overexpression of
proteins involved in lipid metabolism was established in our study, which is in agreement
with previous studies of TLE.
Int. J. Mol. Sci. 2024, 25, 7935 9 of 15

During the study, overrepresented proteins with antioxidant activity were identified,
which may be a compensatory reaction for oxidative stress. Among them are CP, AFM,
PON1, PRDX2, RBP4, BCHE, HPX, SELENOP, and CFHR4. The attenuation of oxidative
damage in models of acquired epilepsy appears to protect against cognitive malfunction,
as well as provide neuroprotection [32].

3.4. Neuroinflammation
The impaired activation and regulation of inflammatory cells and molecules in dam-
aged nervous tissue is one of the factors in the development and maintenance of epilep-
togenic activity. Status epilepticus can activate micro- and astroglia, as well as cells of
the peripheral immune system, to release several pro-inflammatory mediators, thereby
initiating a cascade of inflammatory processes in brain tissue, which, in turn, can change
the excitability of neurons and affect the physiological functions of glia.
Patients with both MRI-positive and MRI-negative forms of epilepsy had clinical
and electroencephalographic manifestations of TLE, according to the International League
Against Epilepsy (ILAE) criteria. Inflammation and other immune-mediated processes are
essential in epileptogenesis [33]. Clinically, brain injury activates innate immunity, and the
disruption of the blood–brain barrier allows adaptive immunity and peripheral immune
responses, as well as specific antibody production, to influence the brain. These mechanisms
contribute to epileptogenesis in many acquired epilepsies. Processes of neuroinflammation
are equally identified in many acquired models of epilepsy (e.g., after status epilepticus,
stroke, and traumatic brain injuries) [34,35].
Several proteins that induce inflammatory processes were found to be upregulated
in epilepsy in our study. These are C9, C1R, FGG, ITIH1, ITIH2, ITIH4, ATRN, SPP24, F5,
C8A, SELL, AGT, CFD, CNDP1, FCN3, and FCN2.
The neuroinflammatory process is accompanied by oxidative stress. Oxidative stress
is mediated by the formation of reactive oxygen species (ROS), which plays an important
role in the pathogenesis of epilepsy [36]. Recurrent epileptic seizures can increase the
concentration of reactive oxygen species and superoxides in the brain [37]. Free radicals
can directly induce seizure activity through the inactivation of glutamine synthase, which
leads to glutamate excitotoxicity [38], as well as the inhibition of glutamate decarboxylase.
In patients with TLE, antioxidant systems such as glutathione and superoxide dismutase
(SOD) are altered, indicating ongoing oxidative stress [39].
The complement system is involved in the epileptogenesis of TLE in the chronic
phase, both in experimental models and in humans [40]. Persistent complement activation
can promote a sustained inflammatory response and destabilize the neural networks
involved. The sequential administration of five membrane attack pathway proteins into the
hippocampus of awake and free-moving rats induced both behavioral and electrographic
seizures, as well as cytotoxicity. The onset of seizures occurred during or shortly after
the C8/C9 infusion [41]. The overexpression of C8B was found in the plasma of patients
with Rolandic epilepsy [10]. The overexpression of C9 was observed in the hippocampal
tissues of patients with TLE [40]. Our study found increased C9 expression in the epilepsy
group. Elevated levels of C8 and C8A were also found in the group of MRI-positive
patients in comparison with the MRI-negative ones, which indicates a more intensive
neuroinflammatory process in MRI-positive patients.
Angiotensin (AGT) promotes an inflammatory response causing oxidative stress.
AGT receptor activation has been demonstrated in the cortex and hippocampus of mesial
temporal sclerosis [42]. AGT mediates a predisposition to seizures [43]. It has an inhibitory
effect on K+ -induced GABA release, as demonstrated by the analysis of hippocampal
sections [44]. Our study demonstrated AGT overexpression in the epilepsy group compared
to the healthy group.
The inflammatory process is compensated by the overexpression of proteins, in one
way or another, inhibiting inflammatory cascades. Our study found several proteins that
compensate for the inflammatory response, including SPP24, A2M, CLU, CPN2, CPB2,
Int. J. Mol. Sci. 2024, 25, 7935 10 of 15

FETUB, VASN, C4BPB, IGLC1, IGKV3, IGKV4-1, IGKC, PROZ, PROC, VTN, IGFALS, BTD,
AFM, and AAG.
Alpha-2-macroglobulin (A2M) is a glycoprotein that can inhibit proteases without
directly blocking their active site. It suppresses complement activation. An increased content
of A2M in the CSF was found in children with encephalitis [45]. Moderately elevated levels
of A2M were also observed in the CSF of patients with drug-resistant epilepsy [46]. In our
study, the increased expression of A2M was observed in the MRI-negative group compared
to the MRI-positive group.
Clusterin (CLU or APOJ) exhibits a protective effect against brain damage by stabiliz-
ing stress proteins and inhibiting apoptosis. Traumatic brain injuries (TBIs) induce massive
CLU mRNA expression and immunoreactivity in neurons and astroglia [47]. Clusterin
knockout leads to increased proapoptotic activity in neurons [48]. Induced status epilepti-
cus leads to a sharp increase in clusterin mRNA in glial cells. The increased expression of
this protein may be a compensatory response aimed at slowing down the neurodegenera-
tive cascade controlled by the complement system [49]. An increased expression of CLU
was observed in the TLE patients, where it may be a response to medication by valproic
acid [50].
Vitronectin (VTN) is a multifunctional glycoprotein of the hemopexin family, present in
the blood and extracellular matrix. It binds glycosaminoglycans, collagen, and plasminogen,
which are involved in neurodegenerative processes. It is an indicator of the reactive gliosis
of the hippocampus, which was demonstrated in kainic models of epileptogenesis [51].
An increased VTN expression was observed in MRI-positive patients compared to MRI-
negative patients, which was noted in previous studies.
Gelsolin (GSN) is associated with the reorganization of actin in microglia, which
contributes to the restoration of the processes of the nervous tissue after damage and
inflammation. Studies of GSN knockout mice [52] showed that gelsolin protects neurons
from excitotoxic Ca2+ overload in vitro and in vivo. A marked decrease in GSN levels is
observed in CSF and tissue in patients with TLE [53]. In our study, on the contrary, an
increased expression of GSN was observed in the whole group of epilepsy in comparison
with the healthy group, which may be caused by compensatory mechanisms.

3.5. Signal Transduction

Neuroinflammation and lipid peroxidation can disrupt the membrane structure, lead-
ing to a change in cell permeability, by increasing the viscosity of the membrane, which, in
turn, can trigger a cascade of events leading to a change in the functioning of membrane
proteins and the dysregulation of intracellular signaling proteins, which further results in
the increased excitability of neurons. Our study revealed an upregulation in epilepsy of
a significant number of serpin proteins involved in the control of intracellular signaling.
These are SERPINA3, SERPINF1, SERPINA10, SERPING1, SERPIND1, SERPINC1, and
SERPINF2, which confirms the hypothesis of the massive dysregulation of intracellular pro-
cesses. The neuroinflammatory process and the accompanying cascade of molecular events
ultimately lead to disturbances in the structure of cell membranes and membrane proteins,
which contribute to the spread and maintenance of epileptogenic activity. In the aberrant
expression of proteins involved in various biological, cellular, and molecular processes, the
systemic nature of the disease is observed. The presence of similar aberrations in several
other neurological disorders indicates the association of TLE with other brain diseases.

3.6. Antiepileptic Drugs’ Influence

The majority of studied patients (88.9%) received antiepileptic drugs (AEDs) recom-
mended for the treatment of focal seizures and focal bilateral tonic clonic seizures (FBTCs).
Antiepileptic therapy should obviously affect the protein expression. Thus, an increased
expression of some specific proteins was found in the epilepsy group. These are HPX, CP,
BCHE, PON1, CLU (APOJ), FGG, APOA1, BTD, and immunoglobulins, as well as proteins
of the coagulation cascade. According to the previous research, some of the protein-level
Int. J. Mol. Sci. 2024, 25, 7935 11 of 15

alterations that we have been discovered may be a consequence of taking antiepileptic

drugs. In particular, HPX expression can be induced by phenobarbital [54]; the expression
of CP, PON1, APOA1, and FGG can be influenced by valproic acid [55–57]. Therefore, these
proteins cannot be considered to be potential biomarkers. Additional data on the possible
effect of drugs on protein expression are presented in Table S4.

3.7. Limitations and Future Directions

This study was focused on the primary comparison of proteomes of blood plasma
from epilepsy patients, including patients with structural TLE and focal TLE of unspecified
etiology (MRI-negative), and healthy individuals. This selection of study groups initially
has a number of limitations. The main restriction is the limited sample set available at one
time. The complications were related to the sample collection from the patients meeting
the inclusion criteria, laborious sample preparation, and reproducibility limitations during
the analysis and processing of the sample set. Moreover, most of the involved patients
were taking AEDs, which certainly can influence the protein expression. We were unable to
overcome the difficulty in identifying proteins that are strictly unaffected by drugs due to
the low availability of untreated patients.
Our study had the general purpose of a primary search. The focus on studying
individual proteins suggested as biomarker candidates and revealing their functions in the
mechanism of epilepsy development requires special targeted research, which was beyond
our purpose. Future work should include targeted proteomic studies of large patient
cohorts for the validation of the presented results in terms of reliability and generalizability.

4. Materials and Methods

4.1. Patient Cohort
All participants signed an informed consent. The study was approved by the Local
Ethics Committee of Prof. V.F. Voino-Yasenetsky Krasnoyarsk State Medical University
Ministry of Health of Russia (protocol No. 85/2018 dated 27 September 2018). Blood
samples were collected in the Neurological Center for Epileptology, Neurogenetics, and
Brain Research of the University Clinics, Prof. V.F. Voino-Yasenetsky Krasnoyarsk State
Medical University, Krasnoyarsk, Russia.
The entire study cohort included 34 individuals. Plasma samples were collected from
27 patients with temporal lobe epilepsy of various origins and 7 healthy volunteers. The
epilepsy cohort was divided into MRI-negative and MRI-positive groups, including three
types of lesions: hippocampus sclerosis, vascular anomaly, and malformations (Table 1).
Patients met the following criteria for inclusion in the study: (1) absence of acute
infectious diseases at the time of the study and within 1 month before blood sampling,
(2) absence of somatic and mental illnesses in the stage of decompensation, (3) absence
of any comorbidities potentially affecting the plasma proteome, (4) no hereditary history
of epilepsy, (5) taking antiepileptic drugs (AEDs) not exceeding the average therapeutic
daily dose, (6) absence of clinical signs of side effects of AEDs at the time of the study, and
(7) absence of focal bilateral tonic clonic seizures (FBTCs) within 4 weeks before the study.
We found 25 out of 27 (88.9%) patients took AEDs, and 12 out of 25 (48%) patients
received AED monotherapy. The patients predominantly received sodium channel blockers,
both in monotherapy and in combination with other AEDs. The patients were divided into
drug-resistant and drug-responsive groups according to the International League Against
Epilepsy (ILAE) criteria (failure of adequate trials of two tolerated and appropriately chosen
AED schedules (whether as monotherapies or in combination) to achieve seizure freedom).
The median duration of epilepsy was 13 (7–26) years. In 6 out of 27 (22.2%) cases,
remission of epileptic seizures was observed, lasting from 1 to 6 years, with an average of
2.8 years, against the background of ongoing therapy with anticonvulsants. Remission and
without-study-limitation FBTCs were recorded during the last year in 8 out of 27 (29.6%)
patients, with an average frequency of 4.9 attacks per year. FBTCs were absent from patients
within 1 month before inclusion in the study. In 18 patients, seizures with a focal onset,
Int. J. Mol. Sci. 2024, 25, 7935 12 of 15

motor and non-motor, with and without impaired awareness, were recorded, with an
average frequency of 2.6 months.

4.2. Blood Sample Treatment

The blood samples were collected into heparin vacuum tubes (BD, Franklin Lakes,
NJ, USA) and centrifuged for 10 min at 3000 rpm; collected plasma was stored at −80 ◦ C.
The thawed plasma samples were depleted from albumin and immunoglobulin using
ProteoPrep Blue Albumin & IgG Depletion Kit (Millipore, Darmstadt, Germany) according
to the manufacturer protocol. Sample preparation for mass spectrometry was carried out
as described earlier [58]. Briefly, the depleted plasma samples containing 4 micrograms
of protein were reduced by dithiothreitol, alkylated by iodoacetamide, and digested by
trypsin according to the manufacturer protocol (Thermo Scientific, Waltham, MA, USA).
Then, the samples were desalted by C18 pipette tips from the same manufacturer under its
protocol and dried. All plasma samples were prepared in triplicates.

4.3. Mass Spectrometry

The samples were dissolved in water containing 0.1% of formic acid (Sigma-Aldrich,
St. Louis, MO, USA), and injected into the Dionex UltiMate 3000 RSLC nano liquid chro-
matographer (Thermo Scientific, Waltham, MA, USA). Peptides were separated on an
in-house packed reversed-phase C18 column (Polymicro Technology, Phoenix, AZ, USA),
15 cm × 70 µm ID, Luna C18, 3 µm, 100 Å (Phenomenex, Torrance, CA, USA), using a stan-
dard 0–38% water/acetonitrile/0.1% formic acid gradient at the flow rate of 300 nL/min.
The Orbitrap Fusion mass spectrometer (Thermo Scientific, Waltham, MA, USA) was op-
erated in data-dependent mode with scans of precursor and fragment ions changing in a
cycle of 3 s. Precursor ions were detected at a resolution of 60,000 by the Orbitrap. The
fragment ions were generated by collision-induced dissociation (CID) at 35% of collision
energy and were registered by an ion trap detector.

4.4. Data Analysis

The full set of RAW data files obtained by mass spectrometry was processed using
MaxQuant version 2.0 proteomic software. The label-free quantification (LFQ) parameter
was enabled. A protein search was performed using the actual annotated UniProt database
available online: https://www.uniprot.org/help/downloads/ (accessed on 18 July 2024)
with the false discovery rate (FDR) set to 0.01. The obtained LFQ values for each identified
protein were considered as relative quantitative measures of protein distribution among
the plasma samples.
The tripled LFQ data for each plasma sample were averaged using weights corre-
sponding to the fraction of proteins that lead in a certain triplicate. Principal component
analysis (PCA) was applied to the obtained LFQ dataset to assess primary proteomic
differences between the groups using the SIMCA version 13 program (Sartorius AG, Goet-
tingen, Germany). A general comparison of protein distributions among the selected groups
to identify specific biomarker candidates was carried out using Welch’s t-test with the
Benjamini–Hochberg correction for multiple comparisons. FDR was set to 0.01. Analysis
was performed using Anaconda Distribution Python version 3.8. The analysis of biological
functions, as well as the drawing of the corresponding diagrams, was carried out using
the SninyGo version 0.76 program available online: http://bioinformatics.sdstate.edu/go/
(accessed on 18 July 2024).

5. Conclusions
Epilepsy can be caused by various reasons, but, finally, they all lead to the onset and
persistence of the inflammatory process caused and maintained by several proteins found in
our study, which, in turn, triggers a molecular cascade of events that contribute to the spread
and preservation of epileptogenic activity. Neuroinflammation and the accompanying
oxidative stress can be compensated to some extent by the induction of inflammation-
Int. J. Mol. Sci. 2024, 25, 7935 13 of 15

inhibiting proteins and antioxidants. However, uncompensated processes ultimately lead

to the dysregulation of the functions of the membrane and membrane proteins, which
results in a decrease in the activation threshold of channels and the spread of excitation.
Plasma proteome profiling may be a way to identify potential biomarkers of epileptoge-
nesis and/or seizure activity. While some of the most differentially regulated proteins found
in our study are new, such as protein transport agent afamin (AFM); phosphatidylcholine-
sterol acyltransferase (LCAT), which is responsible for the extracellular metabolism of
plasma lipoproteins; and some protease inhibitors from the serpin family, most of the others
have previously been reported in the studies of epilepsy. These proteins may ultimately
help to explore pathways of epileptogenesis, while the further validation of new potential
candidates in large patient cohorts versus other neurological disorders will confirm their
use as potential biomarkers for clinical diagnosis.

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/ijms25147935/s1.
Author Contributions: Conceptualization, Y.E.G., D.V.V., T.N.Z. and D.V.D.; methodology, Y.E.G. and
Z.M.; investigation, Y.E.G., D.V.V., T.N.Z. and D.V.D.; data curation, Y.E.G. and D.V.V.; writing—original
draft preparation, Y.E.G. and E.E.T.; writing—review and editing, Z.M., T.N.Z., D.V.D., M.V.B. and A.S.K.;
visualization, D.V.V.; supervision, M.V.B. and A.S.K. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Ministry of Science and Higher Education of the Russian
Federation, project numbers: FWES-2022-0005 for Y.E.G., D.V.V., T.N.Z. and A.S.K.; and REYC-2021-
0002 for E.E.T. and D.V.D.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and approved by the Local Ethics Committee of Prof. V.F. Voino-Yasenetsky Krasnoyarsk
State Medical University (protocol No. 85/2018, dated 27 September 2018).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The original data are available upon reasonable request from the
corresponding authors.
Acknowledgments: The authors are grateful to all the patients participating in this research. Technical
and instrumental support was provided by Krasnoyarsk Regional Center for Collective Use at the
Federal Research Center KSC SB RAS, Shared Core Facilities of Molecular and Cell Technologies
at Krasnoyarsk State Medical University, and John L. Holmes Mass Spectrometry Facility at the
University of Ottawa.
Conflicts of Interest: The authors declare no conflicts of interest.

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