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A Serum Analysis Before and After Antidepressant
Treatment in Major Depression: A Pilot Study
Murielle Girard1, Karine Vuilliers-Devillers2, Emilie Pinault2, Barbara Bessette3, Brigitte Plansont1
and Dominique Malauzat1
1
Département Recherche et Développement, Centre Hospitalier Esquirol, Limoges, France. 2FR 3503 GEIST, Service Commun de Recherche
et d’Analyse de Biomolécules de Limoges (SCRABL), Faculté des Sciences et Techniques, Limoges, France. 3EA 3842 Homéostasie
Cellulaire et Pathologies, Faculté de Médecine, Limoges, France.
ABSTR ACT: We investigated the serum protein profiles of subjects with major depressive disorder (MDD), with (n = 4) and without clinical
improvement (n = 4), at the initiation of antidepressant treatment (venlafaxine) (T0) and 4 weeks later (T28), by difference gel electrophoresis in
two dimensions (2D-DIGE) and mass spectrometry. The nine proteins displaying differences in composition between the two time points in the
group with clinical improvement between T0 and T28 included gelsolin, clusterin, and the activated fragment of complement C3 (C3a). We then
analyzed serum samples from MDD subjects receiving different antidepressants between T0 and T28. Subjects were classified into two groups,
with (n = 17) or without (n = 14) clinical improvement (50% decrease in baseline Hamilton Depression Rating Scale score), at T28. Clusterin
levels did not differ between groups at either time point. Gelsolin and C3a levels differed between T0 and T28 only in the group presenting clinical improvement. A comparison with serum samples from controls suggested that the levels of these two proteins changed during MDD and were
potentially modified after successful antidepressant treatment. Despite the small sample size, the results of this pilot study suggest that several
changes in the expression of some serum proteins occur in association with the clinical relevance of the treatment, and indicate changes to general
pathways requiring further study.
KEY WORDS: proteome, major depressive disorder, C3, gelsolin, clusterin, antidepressant
CITATION: Girard et al. A Serum Analysis Before and After Antidepressant Treatment
in Major Depression: A Pilot Study. Clinical Medicine Insights: Psychiatry 2015:6 1–12
doi:10.4137/CMPsy.S20765.
CORRESPONDENCE: murielle.girard@ch-esquirol-limoges.fr
FUNDING: Authors disclose no funding sources.
Paper subject to independent expert blind peer review by minimum of two reviewers.
All editorial decisions made by independent academic editor. Upon submission
manuscript was subject to anti-plagiarism scanning. Prior to publication all authors
have given signed confirmation of agreement to article publication and compliance
with all applicable ethical and legal requirements, including the accuracy of author
and contributor information, disclosure of competing interests and funding sources,
compliance with ethical requirements relating to human and animal study participants,
and compliance with any copyright requirements of third parties. This journal is a
member of the Committee on Publication Ethics (COPE).
COMPETING INTERESTS: Authors disclose no potential conflicts of interest.
Published by Libertas Academica. Learn more about this journal.
RECEIVED: October 2, 2014. RESUBMITTED: February 26, 2015. ACCEPTED FOR
PUBLICATION: March 17, 2015.
ACADEMIC EDITOR: Jaswinder Kaur Ghuman, Editor in Chief
TYPE: Original Research
COPYRIGHT: © the authors, publisher and licensee Libertas Academica Limited.
This is an open-access article distributed under the terms of the Creative Commons
CC-BY-NC 3.0 License.
Introduction
Proteomic analysis provides an inventory and a means
of identifying the proteins involved in specific and wellcharacterized clinical states, opening up new opportunities for
the identification of novel disease biomarkers. This approach
has already been explored in mental disorders such as schizophrenia,1 bipolar disorder, Alzheimer’s disease, 2 substance
abuse,3,4 and neurodegenerative diseases.5,6 Most such studies
in humans are based on postmortem analysis or cerebrospinal
fluid examination, but blood-based candidate biomarkers of
mental diseases are required to facilitate noninvasive test procedures on blood samples. Changes to peripheral protein profiles
(metabolome, cytokines, proteins) have been observed in several
psychiatric diseases, 2 including schizophrenia,7,8 Alzheimer’s
disease,9 and alcohol dependence.10,11 These changes result in
modifications to the cholesterol system, immunological state,
or metabolic pathways.12 The occurrence of changes to serum
protein profiles during major depressive disorder (MDD) has
not been studied in detail. MDD is a widespread public health
problem, but there are still no reliable biological markers for
diagnosis and treatment monitoring, and our understanding of
the pathogenesis of this condition and of the mechanisms of
action of antidepressants remains incomplete.13–16
Studies have essentially focused on the mechanisms
occurring in the central nervous system, providing evidence
of changes to neurogenesis and synaptic plasticity.17 The
levels of some molecules accessible in plasma or serum are
known to vary during major depression, and peripheral biochemical variations are known to occur during antidepressant
treatment.18 These changes concern several regulatory pathways that are probably involved in MDD pathogenesis or its
consequences16,19: energy homeostasis and metabolic disorder
molecules, such as leptin and ghrelin;20 immunity molecules,
such as cytokines21–23 and steroids; neuromediators, such as
homovallinic acid 24 and hydroxyindolacetic acid;25,26 molecules involved in neurogenesis or neurotropism, such as brainderived neurotrophic factor, glial-derived neurotrophic factor,
nerve growth factor, 27–30 TrkB, 31 neopterin, 32 nesfatin, 33 and
vascular endothelial growth factor.34 However, these factors
have been assessed separately, are difficult to use in practice,
particularly due to their lack of specificity, and are involved
in pathways that are affected differently in different clinical
Clinical Medicine Insights: Psychiatry 2015:6
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Girard et al
contexts. However, serum is a complex biological fluid and
its composition is not well known and difficult to determine.
Nevertheless, serum analysis can provide a broad range of
information about a subject and a disease. Little is currently
known about the occurrence of modifications to serum protein profiles during MDD and antidepressant treatment in
humans.
We hypothesized that peripheral molecular modifications occur in relation to symptoms and clinical changes on
antidepressant treatment. We therefore investigated the serum
protein profiles of subjects with MDD, at the initiation of
antidepressant treatment (venlafaxine) (T0) and 4 weeks later
(T28), as a function of clinical improvement, and assessed
them on the Hamilton Depression Rating Scale (HDRS).
Difference gel electrophoresis in two dimensions (2D-DIGE)
and mass spectrometry were used to screen for modifications
to the broadest possible spectrum of proteins in the serum.
Several variant proteins of interest were identified and studied
further by enzyme-linked immunosorbent assay (ELISA) in
controls and in other subjects with MDD treated with various
antidepressants and grouped according to clinical improvement after treatment.
Methods
Population.
Patients with major depression. All subjects admitted to
our hospital (Centre Hospitalier Esquirol, Limoges, France)
with a major depressive episode, diagnosed on the basis of
the Diagnostic and Statistical Manual of Mental Disorders
(DSM-IV-TR) criteria and beginning a new course of antidepressant treatment, were asked to participate in this study.
All participants were between the ages of 18 and 60 years, had
health insurance coverage, and gave written informed consent for participation in the study. The referring psychiatrists
checked that their patients did not meet the exclusion criteria:
an inability to understand the tests or the French language, an
HDRS score 24, the presence of any psychiatric or somatic
comorbid condition that might modify serum protein profiles
(eg, acute inflammatory disorders, schizophrenia), the use of
treatments with potential side effects including the induction
of a major depressive episode or the aggravation of its clinical
symptoms (antiviral drugs), and menopause or pregnancy in
the case of women. The study was approved by the local ethics committee (Persons’ Protection Committee from France
South-West and Oversea IV). This research complied with
the principles of the Declaration of Helsinki.
Depression intensity was evaluated with the HDRS.35,36
Patients were considered to have responded to treatment if
the HDRS score after treatment, at 28 days, was 50% that
at baseline or lower.37 In total, 63 patients were recruited for
the study, 24 of whom were excluded due to a revised diagnosis (7), not following the antidepressant treatment correctly
(3), an HDRS score 24 (4), non-attendance of the T28 visit
(7), or other reasons (3 patients; eg, incorrect sampling). The
2
Clinical Medicine Insights: Psychiatry 2015:6
remaining patients, for whom follow-up at 28 days, with the
collection of complete data, was possible, were treated with
venlafaxine (n = 16), mirtazapine (n = 8), escitalopram (n = 5),
citalopram (n = 4), paroxetine (n = 3), fluoxetine (n = 1), sertraline (n = 1), or duloxetine (n = 1).
Analyses were carried out with serum samples from subjects treated with venlafaxine, the most frequently prescribed
antidepressant. Five subjects presented a clinical improvement
(HDRS T0/HDRS T28 = 2.2 ± 0.58), and six did not (HDRS
T0/HDRS T28 = 1.45 ± 0.24). We aimed to carry out the
analysis with the participants with the most pronounced profiles in each clinical subgroup. We therefore retained, for the
analysis, the four participants in the group without clinical
improvement with the HDRS ratio values closest to 1, and the
four participants from the group with clinical improvement
with the highest HDRS ratios (2).
Controls. Control serum samples were obtained from
blood donors between the ages of 18 and 60 with no chronic or
psychiatric disease, with no medical treatment. The controls
agreed to supply a blood sample for research purposes. Whole
blood (5 mL) was collected just before the blood donation and
was immediately centrifuged. The only data collected for this
group were age and sex. An agreement was signed with the
French blood transfusion agency for the testing of the blood
samples from donors for pathogens and pathological conditions after the centrifuging of the samples. Twenty-four controls (9 men and 14 women) were matched with the members
of the two MDD groups for age and sex. The mean age of the
controls was 37.5 ± 9.4 years.
Two-dimensional fluorescence difference gel electrophoresis (2-D-DIGE) analysis.
Serum preparation. Blood samples were collected from
fasting MDD patients. These samples were collected into
tubes without additives on the day immediately after admission (T0), corresponding to the start of a new course of
antidepressant treatment, and 28 days later (T28). Samples
were allowed to clot at room temperature for a minimum of
120 minutes. Serum was then obtained by centrifugation at
900 g for five minutes at room temperature. Aliquots of the
serum (0.5 mL) were taken and stored at -40°C until use. The
time from collection to freezing did not exceed four hours.
Inhibitors of serine, cysteine, and calpain proteases (10 µL/mL;
GE Healthcare) were added to the serum samples, and aliquots of 40 µL of serum were then depleted of albumin and
G immunoglobulins according to the kit manufacturer’s
instructions (Vivapure anti HSA/IgG kit for human albumin and IgG depletion, Vivascience), subjected to precipitation, and desalted (ReadyPrep 2-D Cleanup kit, Bio-Rad
Laboratories). Pellets were redissolved in the solubilization
buffer (8 M urea/2 M thiourea/4% w/v CHAPS). Protein
content was determined with the PlusOne 2D Quant Kit
(GE Healthcare).
2-DE and image analysis. The experimental strategy for
2-D DIGE was based on minimal labeling with dye swapping.
Serum proteomic analysis in major depression
We labeled 50 µg of serum collected on T0 or T28 with Cy3
or Cy5. The internal standard, which was prepared by mixing
equal amounts of all samples, was labeled with Cy2. Labeling reactions were carried out according to the manufacturer’s instructions. Each sample was labeled with 400 pmol of
CyDye (GE Healthcare) by incubation on ice for 30 minutes in
the dark, and reactions were stopped by adding 1 µL of 10 mM
lysine. The CyDye-labeled samples (sera collected at T0 and
T28, and internal standard) were mixed and added to an equal
volume of the solubilization buffer (containing 130 mM DTT,
1% v/v IPG buffer, and a trace of bromophenol blue).
Analytical 2-DE was performed as follows: the CyDyelabeled protein samples were focused in the first dimension
on IPG 18 cm pH 4–7 gels. The gels were rehydrated at room
temperature for 12 hours in a reswelling tray (GE Healthcare),
and isoelectric focusing (IEF) was carried out for 89 kVh in
the dark. The gel strips were then equilibrated by incubation
in 5 mL of equilibration buffer A (50 mM Tris-HCl pH 8.8,
6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM DTT) for
15 minutes, with gentle shaking, followed by 5 mL of equilibration buffer B (50 mM Tris-HCl pH 8.8, 6 M urea, 30% v/v
glycerol, 2% w/v SDS, 2.5% w/v iodoacetamide, with a trace of
bromophenol blue). The equilibrated strips were loaded on to
the top of a 10% polyacrylamide SDS-PAGE gel (24 × 20 cm),
which was then sealed with 1% w/v agarose. Separation in
the second dimension was carried out in Tris-glycine buffer.38
After 2-DE, the gels were scanned with a Typhoon TRIO
scanner (GE Healthcare), using appropriate filters for the
excitation and emission wavelengths of each dye. We obtained
sufficient quantities of the proteins from the individual spots
for identification by running 450 µg T0 serum and 450 µg of
T28 serum separately on 2-DE gels and staining them by a
colloidal CBB G-250 procedure.39 Preparative gels were run
in the same electrophoresis conditions and scanned with an
Image Scanner II (GE Healthcare).
The images were scanned and the Nonlinear Dynamics
Progenesis Samespots software was used for differential gel
analysis.
We compared the 2-D images of T0 serum samples
with those of T28 serum samples, using the internal standard
sample for the group displaying clinical improvement. The
final values for the expression ratio of specific protein spots
between T0 and T28 serum samples were determined for differences of ±1.2-fold.
The statistical significance of differences in protein levels between the two time points was calculated by applying
ANOVA to the log ratio, with an alpha risk of 5% (P  0.05).
Mass spectrometry analysis.
Protein digestion. Protein spots were excised from 2-DE
gels stained with Colloidal blue G-250. The excised spots
were destained by washing in milliQ distilled water, then
dehydrated by incubation in 50 µL of acetonitrile (ACN), and
rehydrated by incubation in 50 µL of 100 mM ammonium
bicarbonate for 15 minutes at 37°C. An equal volume of ACN
was then added to the mixture, which was incubated for an
additional 15 minutes at 37°C. Samples were then dried in a
Speed Vac. Sequencing-grade modified trypsin was prepared
from a 0.1 µg/µL stock solution, by dilution in 25 mM ammonium bicarbonate to give a final concentration of 10 ng/µL.
Dehydrated spots were incubated overnight at 37°C in 25 µL
of 10 ng/µL trypsin solution (a total of 250 ng per spot). The
supernatant was then collected in a 0.5-mL microfuge tube
and the digested peptides were extracted sequentially in 50 µL
of 40% ACN/1% FA, followed by 10 µL of 25% ACN/1% FA
and, finally, 25 µL of 60% ACN. All the samples were then
dried in a Speed Vac.
Mass spectrometry. After trypsin digestion and evaporation, the peptides were resolubilized in 6 µL of Switchos solvent (2% ACN, 0.05% TFA) for analysis by nano-LC MS/
MS with a Packings liquid nano-chromatography system
(Dionex) coupled to a QTRAP mass spectrometer (Applied
Biosystems). We injected 5 µL of each sample onto a precolumn (C18 Pepmap 300 µm ID × 5 mm) with the Switchos
unit. The precolumn was desalted for three minutes with the
Switchos solvent, the precolumn was switched online with
the analytical column (C18 Pepmap 75 µm ID × 150 mm)
pre-equilibrated with 100% solvent A (ACN 2%/FA 0.1%).
Peptides were eluted from the precolumn onto the analytical
column and then onto the mass spectrometer, with a linear
gradient of 0% to 50% of solvent B (90% ACN, 0.1% FA) over
65 minutes, at a flow rate of 300 nL/min.
Data were acquired with the IDA (InformationDependent Acquisition) software of Analyst 1.4.2 (Applied
Biosystems). MS and MS/MS data were recorded continuously, with a cycle duration of three seconds. For each MS
scan, two precursors were selected for fragmentation on
the basis of their intensity (greater than 20,000 cps), their
charge state (2+, 3+), and whether the molecule concerned
had already been selected for fragmentation (dynamic exclusion). The collision energies were adjusted automatically as
a function of the charge state and ionic mass of the selected
precursors.
Peptide identification. For protein identification, the
results of the nano-LC MS/MS analysis were used to search
the SwissProt database with Mascot software (version 2.2,
Matrix Science) using the following criteria: species Homo sapiens (database version 2011_04 containing 20233 sequences),
0.5 Da tolerance for peptide and peptide fragment mass,
a single missed cleavage site allowed during trypsin digestion
and carbamidomethylation of cysteine residues (due to the
alkylation of -SH groups by iodoacetamide), and methionine
oxidation as variable modifications. Protein identification was
validated if at least two peptides had a score greater than 25, or
if one peptide had a score greater than 50 at a confidence level
of at least 95%.
Western blotting. For confirmation of the 2D-DIGE
results and of the production of the proteins identified as
differentially expressed, we carried out western blotting for
Clinical Medicine Insights: Psychiatry 2015:6
3
Girard et al
three target proteins. We used depleted serum from all eight
patients for whom 2D-DIGE analysis was carried out.
The proteins present in serum samples depleted of IgG
and albumin were quantified with the Bradford Quick Start
Protein Assay (Bio-Rad), by measuring absorbance at 595 nm,
with bovine serum albumin (BSA) as the protein standard.
The protein (5 µg) diluted 1/10 in Laemmli buffer (Bio-Rad)
or a standard protein mix (Precision Plus Protein Standards,
Bio-Rad) was loaded onto a polyacrylamide gel with a 4%
acrylamide stacking gel (Criterion TGX Stain-Free, Any kD,
Bio-Rad) and subjected to electrophoresis at 120 V in a Mini
Protean 3 electrophoresis system (Bio-Rad) in the presence
of Tris/glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3;
Bio-Rad).
The separated proteins were transferred onto poly
(vinylidene difluoride) (PVDF) membranes (Bio-Rad) by blotting in 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol.
Protein transfer was carried out at 20 V, for 90 minutes, with a
Bio-Rad Transblot SD cell (Bio-Rad). Transfer efficiency was
evaluated by staining the membranes in Ponceau-S-Red and
then destaining in PBS (50 mM sodium phosphate, 0.9% w/v
NaCl, pH 7.4). Membranes were blocked by incubation for
one hour in 5% w/v fat-free milk powder in PBS containing
0.5% v/v Tween 20, and then incubated overnight at 4°C with
the primary antibody diluted in the same buffer: mouse antiactivated C3 1/500, mouse anti-clusterin 1/200, mouse antigelsolin 1/200 (Santa Cruz Biotechnology). Membranes were
washed with 0.5% (v/v) Tween 20 in PBS, and incubated with
a goat peroxidase-conjugated anti-mouse IgG antibody at a
dilution of 1/5000 and Precision Protein StrepTactin-HRP
conjugate (dilution of 1/20,000) for the lane containing the
protein standards, for one hour at room temperature, in 0.5%
(v/v) Tween 20 and 5% (w/v) fat-free milk powder in PBS.
The membrane was washed at least six times in 0.5% (v/v)
Tween 20 in PBS, and immunostaining was detected by chemiluminescence (Immun-Star WesternC chemiluminescence
kit, Bio-Rad). The membranes were scanned with a GS-800
densitometer (Bio-Rad). Quantification was performed with
Image J software (NIH), and immunodetection was carried
out twice in each case.
Enzyme-linked immunosorbent assay. ELISA-based
validation experiments were carried out with serum samples
from the original pilot cohort and from an independent
serum series. The crude nondepleted sera were diluted
1/2000 for clusterin (Quantikine, R&D Systems), 1/1000 for
gelsolin and 1/100 for activated complement component 3a
(Uscn, Life Science Inc.), each tested twice, in duplicate. All
assays were carried out according to the kit manufacturer’s
instructions.
Statistical analysis. Quantitative variables are expressed
as medians and interquartile ranges because of their nonnormal distribution. Qualitative variables are presented as
frequencies and percentages. Analyses were performed with
Systat software version 11.0 for Windows. Nonparametric
Mann–Whitney tests were used to compare protein levels,
age, and HDRS scores between groups (clinical improvement vs no clinical improvement). The significance of differences between T0 and T28 for each group was determined
by carrying out nonparametric Wilcoxon’s signed rank tests.
Spearman’s correlation tests were carried out to assess the correlation between HDRS scores and the protein level determined by ELISA.
A P-value of less than 0.05 was considered statistically
significant.
Results
Subjects. The mean age of the participants retained for the
2D analysis (7 women and 1 man) was 41.4 ± 5 years and there
was no significant difference between the groups (Table 1).
HDRS scores for the group with clinical improvement and
the group without clinical improvement were similar at T0
(P = 0.235), whereas these scores differed significantly between
these two groups at T28 (P = 0.004) (Table 1).
2D-DIGE analysis of the immunodepleted serum
proteome. Differences in the serum protein profiles between
T0 and T28 were analyzed in the groups with and without
clinical improvement.
We checked the integrity of the serum profiles after
depletion, and all serum samples presented the same protein
profiles after one-dimensional electrophoresis and colloidal
blue staining.
We initially detected 1452 protein spots for both groups.
Image analysis revealed no evidence of differential expression,
for any of the proteins, in the group without clinical improvement between T0 and T28. By contrast, in the group with
clinical improvement, 147 protein spots were considered to
Table 1. Age and HDRS scores characteristics of the subjects retained for 2D-DIGE analysis (n = 8) in a group with clinical improvement (n = 4)
and a group without clinical improvement (n = 4).
ALL
WITH CLINICAL IMPROVEMENT
WITHOUT CLINICAL IMPROVEMENT
Age
38.5 [30.1, 44.6]
38.5 [34, 45.5]
36.5 [15.7, 54.3]
HDRS score at T0
25 [26.4, 24.1]
25 [27.6, 22.4]
25.5 [23.4, 27.6]
HDRS score T28
15 [10.8, 20.1]
11 [14.9, 6.6]
20.5 [16.3, 24.2]
Mean % decrease
(HDRS T0/HDRS T28)
41.3 [20.7, 56.8]
56.6 [40.5, 73.5]
16.3 [5.5, 35.5]
4
Clinical Medicine Insights: Psychiatry 2015:6
Serum proteomic analysis in major depression
display differential expression. Protein abundance changed by
a factor of more than 1.2 between T0 and T28, as shown by
Student’s t-tests, for which values of P  0.05, considered significant, were obtained for 11 spots. Only nine spots were of
sufficient intensity to be picked and subjected to MS (Fig. 1).
Identification of the spots identified as differentially
expressed by 2D-DIGE. The nine spots of potential interest
were picked and analyzed by MS. The identities of these spots
are listed in Table 2.
We attributed the presence of the Ig alpha-1 chain C region
and haptoglobin to contamination arising from the depletion process or from blood collection (possible unnoticed mild
hemolysis, for example). The C3 component of complement was
found in five of the nine spots. A comparison of the sequence
covered by the identified peptides and molecule processing
(Fig. 2A) identified these five spots as one of the complement
C3 chains obtained after cleavage. These assignments were
confirmed by comparing the experimental isoelectric points
and molecular weights with the theoretical parameters of the
complement C3 component fragments (Fig. 2B). The sequence
coverage percentages of the assigned fragments were related to
the theoretical C3 fragment sequences rather than the entire
protein sequence, providing support for our hypothesis and an
explanation of the origin of these experimental fragments.40
We checked the localization of the other proteins identified on the Swiss-2DPAGE plasma reference gel (http://
world-2dpage.expasy.org/).
The proteins found to be more abundant in T28 sera
than in T0 sera (T28/T0 ratio 1.2) were the complement
C3 component (C alpha) and gelsolin. The other compounds
Figure 1. Representative 2D-DIGE image of internal standard master
labeled with Cy2. Indicated numbers correspond to identified spots
(refer to Table 2). 2-DE was performed using a pH range 4–7 in the first
dimension and SDS-PAGE (10%) in the second.
identified had a negative ratio (-1.2): clusterin, other C3
fragments corresponding to cleaved activated C3 (Calpha’ F1
and Calpha’ F2), zinc alpha-2-glycoprotein (Table 2). The C3
alpha subcomponent was more abundant at T28 than at T0,
whereas C3c alpha’ F2, C3dg, C3c alpha’ F1, C3dg (all obtained
from C3 alpha subcomponent processing) proteins were less
abundant at T28 than at T0. All these observations suggest that
processing of the complement C3 component, particularly that
of the C3 alpha chain, decreased between T0 and T28.
These findings provided some indication of the functional
relevance of these proteins. Literature searches revealed that
most of the proteins found to display differential expression
between T0 and T28 had previously been associated with
apoptosis and inflammation processes. On this basis, we
selected three proteins of interest for further validation: gelsolin, clusterin, and the activated fragment of complement C3, as
representative of the activation of the C3 component pathway.
Confirmation of protein production. We carried out
western blotting on depleted serum samples to check whether
the proteins were present. The proteins were separated electrophoretically and transferred to membranes, which were then
probed with antibodies. The results confirmed that all three
proteins were present in all serum samples (Fig. 3).
We then carried out ELISA to evaluate protein levels
more accurately for DIGE analysis. The changes in protein
levels differed between individuals, for all three proteins, in
both groups. Serum clusterin concentrations did not change
between T0 and T28 in the group with clinical improvement
(343 [289; 403] µg/mL to 337 [204; 504] µg/mL), or in that
without clinical improvement (508 [223; 920] µg/mL to 440
[327; 521] µg/mL). However, serum clusterin concentration
decreased in two of the four subjects in each group (Fig. 4).
Gelsolin levels followed a similar pattern in both
groups: from 95 [58; 135] µg/mL to 81 [55; 11] µg/mL for
the group without improvement versus 74 [34; 125] µg/mL
to 74 [52; 95] µg/mL for the group with improvement. Three
subjects with clinical improvement displayed a tendency
toward a decrease in C3a levels (60 [41; 78] ng/mL to 55 [20;
89] ng/mL), whereas such a tendency was observed in only
one subject in the group without clinical improvement (58 [15;
101] ng/mL at T0 to 62 [21; 106] ng/mL at T28) (Fig. 4).
Protein quantification and clinical improvement.
Analyses were carried out on serum samples not subjected to
2D-DIGE analysis, from subjects treated with various antidepressants, and classified according to clinical outcome at T28.
Seventeen showed clinical improvement, whereas 14 did not
(Table 3).
No differences were found between the clinical groups
in terms of age (P = 0.308), sex (P = 0.293), and the type of
antidepressant (P = 0.096). HDRS scores were similar in the
two groups at T0 (P = 0.131), but they were significantly different at T28 (P  0.001) (Table 3), consistent with the clinical significance of the group definitions.
Clinical Medicine Insights: Psychiatry 2015:6
5
6
Clinical Medicine Insights: Psychiatry 2015:6
-1.5
-1.5
-1.5
1.2
1.4
1.2
1.3
-1.4
–1.5
1
2
3
4
5
6
7
8
9
4.67/44910
4.67/43180
5.39/33050
6.14/91620
5.79/118280
6.08/91620
5.45/132880
4.80/41240
5.51/65920
EXP
pI/MWc
Ig alpha-1 chain C region OS = Homo sapiens
GN = IGHA1 PE = 1 SV = 2
Complement C3 OS = Homo sapiens GN = C3 PE = 1
SV = 2
Clusterin OS = Homo sapiens GN = CLU PE = 1 SV = 1
IGHA1_HUMAN
CO3_HUMAN
CLUS_HUMAN
Complement C3 OS = Homo sapiens GN = C3 PE = 1
SV = 2
Zinc-alpha-2-glycoprotein OS = Homo sapiens GN =
AZGP1 PE = 1 SV = 2
Clusterin OS = Homo sapiens GN = CLU PE = 1 SV = 1
Complement C3 OS = Homo sapiens GN = C3 PE = 1
SV = 2
Zinc-alpha-2-glycoprotein OS = Homo sapiens GN =
AZGP1 PE = 1 SV = 2
Haptoglobin OS = Homo sapiens GN = HP PE = 1 SV = 1
ZA2G_HUMAN
CLUS_HUMAN
CO3_HUMAN
ZA2G_HUMAN
HPT_HUMAN
Not identified
Gelsolin OS = Homo sapiens GN = GSN PE = 1 SV = 1
Complement C3 OS = Homo sapiens GN = C3 PE = 1
SV = 2
Gelsolin OS = Homo sapiens GN = GSN PE = 1 SV = 1
CO3_HUMAN
GELS_HUMAN
CO3_HUMAN
GELS_HUMAN
Ig alpha-1 chain C region OS = Homo sapiens GN =
IGHA1 PE = 1 SV = 2
Complement C3 OS = Homo sapiens GN = C3 PE = 1
SV = 2
CO3_HUMAN
IGHA1_HUMAN
PROTEIN DESCRIPTIONd
ACCESS
NUMBERd
(C3c α’ chain fragment 2)
(C3c α’ chain fragment 2)
(C3α chain)
(C3dg fragment)
(C3c α’ chain fragment 1
+ C3dg fragment)
123
345
892
147
149
557
211
2108
592
90
164
372
297
734
SCOREe
4
12
31
3
5
19
6
64
18
2
5
9
5
23
MATCHED
PEPTIDESf
14%
42%
61%
11%
29%
46%
9%
64%
25%
7%
10%
31%
14%
44%
SEQUENCE
COVERAGE
6.13/45177
5.71/34237
4.79/39488
5.89/52461
5.71/34237
4.79/38488
5.9/85644
5.55/113028
5.90/85644
6.08/37631
5.89/52461
5/38905
6.08/37631
5.45/62477
THEOR
pI/MASSg
Notes: aSpot number refers to numbering in Figure 1. bRatio refers to the ratio of normalized spot intensities of T0 and T28 sera. A positive ratio indicates overexpression in T28 sera, whereas a negative ratio indicates
a reduced expression in T28 sera. cExperimental isoelectric point and molecular weight (Da). dAccession numbers of proteins and protein description from Swiss-Prot databases. eMascot score for the identified proteins
based on the peptide ion score with a confidence level of at least 95% (http://www.matrixscience.com). fNumber of peptides that match with the protein sequence. gTheoretical isoelectric point and molecular weight
obtained from Swiss-Prot and the ExPASy databases (http://www.expasy.org).
RATIOb
SPOTa
Table 2. List of overlap proteins that were identified using mass spectrometry.
Girard et al
Serum proteomic analysis in major depression
B Complement C3 processing
Positions
pI
MW (kDa)
C3
23–1663
6.00
184.9
C3 β chain
23–667
6.82
71.3
C3 α chain
672–1663
5.55
113
C3a anaphylatoxin
672–748
9.69
9.1
C3b α’ chain
749–1663
5.18
103.9
C3f fragment
1304–1320
10.83
2.0
C3c α’ chain fragment 1
749–954
6.79
39.5
C3dg fragment
955–1303
5.00
38.9
C3c α’F1 + C3dg
749–1303
5.45
62.5
C3c α’ chain fragment 2
1321–1663
6.89
23.6
Figure 2. (A) Cascade of human complement component C3 cleavages and the resulting chains and fragments. The disulfide bridges are indicated
between chains. (B) Isoelectric point (pI) and molecular weight (MW) associated to the complement C3 processing.
7 7
7 7
7 7
7 7
7 7
7 7
7 7
7 7
0ROHFXODU:HLJKW
$
N'D
&OXVWHULQ
Įȕ±N'D
N'D
%
N'D
N'D
*HOVROLQN'D
N'D
&
N'D
N'D
N'D
N'D
:LWKFOLQLFDOLPSURYHPHQW
&N'D
&DOSKDFKDLQ
N'D
&DOSKD¶)&GJ
N'D
:LWKRXWFOLQLFDOLPSURYHPHQW
Figure 3. Immunodetection by chemiluminescence of depleted sera with (A) anti-clusterin antibodies, (B) anti-gelsolin antibodies, and (C) anti-activated
C3 antibodies.
Clinical Medicine Insights: Psychiatry 2015:6
7
Girard et al
A
1
2
1000
900
100
800
700
80
600
60
500
400
40
300
200
20
100
0
B
1
1
T0
0
T28
140
2
120
80
80
60
60
40
40
20
20
0
T28
1000
2
900
800
700
600
600
500
500
400
400
300
300
200
200
100
100
T28
T28
900
700
T0
T0
1000
800
0
T28
120
100
T0
T0
140
100
0
C
120
0
T0
T28
Figure 4. Levels of (A) clusterin, (B) gelsolin, and (C) C3a at T0 and T28 in each sera of venlafaxine-treated MDD subjects submitted to 2D-DIGE
analysis. 1. Subjects without clinical improvement (n = 4). 2. Subjects with clinical improvement (n = 4).
No change between T0 and T28 was observed for serum
concentrations of clusterin, gelsolin, or C3a in the whole
MDD group or in the group without clinical improvement.
In the group with clinical improvement, a very slight trend
toward a change in these concentrations was observed for gelsolin, but not for clusterin. Only C3a levels differed between
T0 and T28 in this group (Table 4).
There were no differences between the two groups
of MDD subjects in terms of serum C3a, gelsolin, or clusterin concentrations either at T0 (P = 0.905, 0.311, and
0.552, respectively) or at T28 (P = 0.105, 0.868, and 0.430,
respectively).
8
Clinical Medicine Insights: Psychiatry 2015:6
The T28/T0 concentration ratio did not differ between
groups (P = 0.868 for clusterin, P = 0.183 for gelsolin, and
P = 0.212 for C3a).
The number of participants with a T28/T0 ratio 
1.2did not differ, according to the presence or absence of clinical improvement, for clusterin (P = 0.818), C3a (P = 0.431), or
gelsolin, despite a slight trend (P = 0.063). However, the T28/
T0 ratio differed from 1, indicating a change between T0 and
T28 for C3a (P = 0.025) and gelsolin (P = 0.003), only in the
group with clinical improvement.
The controls and the group without clinical improvement
tended to have different C3a levels at T0 (P = 0.061), but not at
Serum proteomic analysis in major depression
Table 3. Characteristics of the controls, the whole MD group, and the subgroups with or without clinical improvement (median [interquartile
ranges]).
CONTROLS (n = 24)
MD SUBJECTS
ALL (n = 31)
WITHOUT CLINICAL
IMPROVEMENT (n = 14)
WITH CLINICAL
IMPROVEMENT (n = 17)
Age (years)
38 [35.3; 43.1]
43.5 [37.9; 45.6]
43.5 [36.8; 44.8]
43 [35.9; 49.3]
Sex ratio (male/female)
10/14
12/19
4/10
8/9
T0
–
25 [23.2; 26.5]
24 [19.8; 25.9]
25 [24.8; 28.2]
T28
–
15 [13.6; 16.7]
18 [17.1; 20.5]
12 [10.3; 13.5]
T28/T0
–
0.6 [0.537; 0.720]
0.407 [0.272; 0.515]
0.6 [0.496; 0.871]
HDRS score
Discussion
T28 (P = 0.109). Their gelsolin concentrations differed at T0
(P = 0.046) and at T28 (P = 0.049).
The controls and the group with clinical improvement had
different C3a levels at T0 (P = 0.004) and T28 (P  0.001),
suggesting a trend toward a decrease in these levels during
MDD, potentially due to modifications on successful antidepressant treatment. These two groups had different gelsolin
concentrations at T0 (P = 0.005), but not at T28 (P = 0.269),
indicating a trend toward a decrease in gelsolin levels in MDD
subjects, with these concentrations approaching those of the
controls after successful antidepressant treatment (Table 4).
No correlation was observed between HDRS score and
the concentration of any of the compounds considered at
either T0 or T28.
In this pilot study, we used 2D-DIGE to investigate differences in the protein profiles of sera from MDD subjects before
and after 28 days of antidepressant treatment, and confirmed
some of the results with ELISA. Our observations suggest
that several changes in serum concentrations were associated
with the clinical relevance of the treatment rather than the
precise type of antidepressant prescribed.
The use of 2D-DIGE for comparisons of the proteome
before and after antidepressant treatment revealed a number
of proteins for which changes in levels during treatment differed between the two groups (those with and without clinical improvement). The changes in serum protein profiles were
analyzed further by mass spectrometry identification of the
Table 4. Median serum concentrations and interquartile ranges at T0 and T28 for C3a, gelsolin, and clusterin, in controls, the whole group of
MDD subjects, and the groups of patients with and without clinical improvement after 28 days of antidepressant treatment.
T0
T28
T28/T0
NUMBER WITH T28/T0  │1.2│
Clusterin
304 [295; 391]
–
–
–
Gelsolin
50 [44; 65]
–
–
–
C3a
44 [38; 50]
–
–
–
Clusterin
309 [290; 366]
296 [286; 362]
1.020 [0.927; 1.120]
7
0.644
Gelsolin
38 [29; 55]
43 [37; 59]
1.155 [0.980; 2.145]
14
0.271
C3a
60 [53; 73]
71 [61; 86]
1.120 [1.006; 1.487]
13
0.199
P*
Controls (n = 24)
MD subjects (n = 31)
All
Without clinical improvement (n = 14)
Clusterin
311 [264; 353]
290 [261; 335]
1.013 [0.875; 1.127]
3
0.975
Gelsolin
41 [23; 78]
45 [30; 61]
0.900 [0.741; 1.461]
4
0.73
C3a
58 [45; 86]
56 [46; 81]
1.002 [0.837; 1.286]
5
0.778
With clinical improvement (n = 17)
Clusterin
309 [282; 406]
310 [280; 412]
1.020 [0.886; 1.200]
4
0.535
Gelsolin
27 [25; 44]
40 [34; 68]
1.369 [0.902; 3.032]
10
0.098
C3a
61 [51; 71]
79 [63; 101]
1.243 [0.993; 1.825]
8
0.049
Note: *Probability for a difference between T0 and T28.
Clinical Medicine Insights: Psychiatry 2015:6
9
Girard et al
protein spots for which changes in intensity were observed
during treatment. The nine compounds identified suggested
the involvement of molecular and cellular pathways related
to general homeostasis in MDD. The immunoglobulin
alpha 1 chain is involved in immune function, constituting
the major class of immunoglobulins in bodily secretions. It
is involved in the primary immune response. Haptoglobin
was also identified as a variant spot. Both these spots were
thought to be associated with possible sample contamination.
The function of zinc alpha 2 glycoprotein is not well characterized. This protein has been implicated in cancer mechanisms and lipid mobilization, and it may also be involved in
the immune system.41
Clusterin has been implicated in several physiological
processes, such as the inhibition of plasma protein aggregation,
cellular proteasome-based detoxification, apoptosis, and agerelated processes and diseases42 including neurodegeneration
in Alzheimer’s disease43,44 and Parkinson’s disease.45 It has
been suggested that the role of this molecule depends on the
isoform expressed and that its subcellular distribution may
reflect the level of oxidative stress in the organism.42
Gelsolin is an essential regulator of cell structure and
metabolism involved in immune functions, apoptosis mechanisms, and aging. Blood gelsolin concentrations decrease in
various clinical conditions, including acute respiratory distress syndrome, sepsis, major trauma, prolonged hyperoxia,
malaria, and liver injury.46
The identification of several compounds associated
with the C3 activation pathway provided support for the
involvement of this pathway in the changes occurring between
T0 and T28 in the group displaying clinical improvement.
The direction of change in the concentrations of the various
C3 compounds was consistent with the positions of these molecules on either side of the C3 component cleavage cascade.
Our results suggested a decrease in C3 activation, which plays
a key role in inflammation.47 C3 plays a key role in complement system activation, with C3 activation required for both
the classical and alternative complement activation pathways.
The activation of different complement proteins would induce
the production of C3 convertase, catalyzing the splitting of
C3 into C3a (in the blood) and C3b. This enzyme binds to the
membrane of the cell to be lysed, forming the C5 convertase
complex. Each cleavage in the complement cascade releases
small fragments (C4a, C3a, and C2b) acting on inflammatory cells. The C3 pathway has been associated with MDD in
murine models of depression,48,49 and its activation has already
been reported in subjects with MDD.50,51
The demonstration of changes in the concentrations of
these proteins with clinical improvement in patients on antidepressant treatment is consistent with several hypotheses
concerning the role of the immune system,52,53 apoptosis, and oxidative stress in the pathogenesis of MDD.54–56
Similarly, 2D-DIGE and mass spectrometry analyses of the
hippocampus in the rat model of depression demonstrated
10
Clinical Medicine Insights: Psychiatry 2015:6
changes in the levels of proteins associated with neurogenesis,
cellular localization and transport, cytoskeleton organization,
and apoptosis.57,58 Serum proteomic analysis also led to the
identification of peripheral proteins involved in inflammation and transport proteins as associated with stress and the
response to antidepressants in rats.49,57
A key finding of this study was the association of the
change in protein levels with clinical improvement and symptoms rather than with the type of antidepressant treatment, as
previously reported for the change in cytokine profiles during
electroconvulsive therapy.59
Further validation of our results with ELISA confirmed
the 2D-DIGE observations and the trends observed for the C3a
component. The differences in protein levels between T0 and
T28 were not confirmed with a larger sample of serum samples
from MDD subjects on antidepressant treatment. The 2D-DIGE
analysis detected changes only in the group with clinical improvement: an increase in gelsolin synthesis and a decrease in the synthesis of clusterin. However, this technique is based on the use
of antibodies recognizing specific epitopes, not necessarily corresponding to those carried by the isoform identified in 2D-DIGE
analysis. Indeed, several proteins may have isoforms that cannot
be differentiated in 2D-DIGE analysis and are differentially recognized by the antibodies used in ELISA. This may be a crucial
issue for clusterin, as this protein has several isoforms with very
different roles.60 The use of specific antibodies in ELISA, not
necessarily recognizing the various potential isoforms of the proteins in 2D-DIGE, may account for the discrepant observations
for gelsolin and clusterin. For C3a, we chose to focus on a single
compound from the C3 activation pathway, thereby decreasing
the potential risk of not using the most appropriate antibody for
detection. Furthermore, the range of concentrations of this compound made it easier to detect and follow.
ELISA yielded interesting results for comparisons of
serum samples from MDD patients with those of controls.
Gelsolin levels in MDD patients differed from those in controls before antidepressant treatment, whether or not clinical
improvement subsequently occurred. Differences between
MDD patients and controls after treatment were restricted
to the group of participants displaying no clinical improvement. This suggests that the clinical normalization associated with the improvement in MDD is accompanied by a
similar normalization of gelsolin concentration. The profile
of change in C3a concentration suggests that the C3 pathway is altered during MDD but tends to be influenced by
antidepressant treatment, particularly in cases of clinical
improvement.
The absence of a correlation between the levels of the
biological compounds and MDD intensity, as evaluated with
the HDRS, suggests only that there is no direct quantitative
link between the variables. The relationship between changes
in symptoms and concomitant biological changes may involve
other intermediate molecules, or a shift in time, depending on
the time of data collection.
Serum proteomic analysis in major depression
This study is subject to several limitations. The number of
samples studied was small, but we believe that this limitation
is offset by the similarity of clinical characteristics between
the groups at T0, the similarity of characteristics within
groups at T28, and the difference in clinical characteristics
between groups at T28. This made it possible to identify
changes occurring between T0 and T28, which were common to the patients in one group but not those in the other
group. However, replication of these results is required. The
2D-DIGE results must be interpreted with caution because
they were obtained with only a small number of serum
samples and specific electrophoretic parameters. Proteomic
analyses on serum samples are difficult, because 98% of the
total mass of protein in the serum corresponds to only about
15 proteins. Even after depletion of the most abundant serum
proteins (albumin, immunoglobulins), analysis and identification of the remaining 2% of proteins of interest remain challenging. The electrical and biochemical conditions used here
for protein separation (pH range for isoelectric separation,
molecular weight range for gel electrophoresis, etc) are specific to our study, and the observed changes in serum protein
levels may not be limited to the proteins identified here. The
use of other technical characteristics for protein isolation and
preanalytic serum preparation might well lead to the definition of other protein profiles.
In the study design used here, clinical improvement was
evaluated at 4 weeks, because we aimed to identify early markers of clinical improvement. However, antidepressant treatment
is usually considered effective at 6 weeks, and there is therefore
a risk that our sample evaluation was carried out too early to
detect clinical improvement in some cases. This may have led
to misclassification errors, decreasing the efficacy of marker
identification. It was for this reason that we chose to study
the subjects presenting the most extreme changes in HDRS
score in 2D-DIGE experiment, to maximize the probability
of detecting the presence or absence of clinical improvement
with a high degree of confidence. However, further exploration
at 6 weeks of treatment or after other durations of treatment
would be of interest for further validation and exploration.
MDD subjects and controls should also be compared
on the basis of samples obtained from different groups in the
same conditions. The pattern of intraindividual variation in
gelsolin, clusterin, and C3a concentrations is not well known,
and diurnal variations are therefore possible. The exclusion of
any subject presenting a somatic disorder decreases the likelihood of serum level variations independent of the parameters studied here. However, a comparison with control serum
concentrations over a 28-day period would provide a more
reliable reference.
Despite these limitations and the difficulties involved in
determining the superiority of the results obtained with the
various protein analysis techniques used here, we consider the
results obtained here to implicate gelsolin and the C3 pathway directly or indirectly in MDD. These results therefore
help improve our understanding of the pathophysiological
mechanisms underlying MDD. However, given their lack
of specificity, the reliability of these results and their true
contribution to our understanding of MDD pathology and
treatment outcome remain to be confirmed, and further investigations are required with other technical approaches, larger
samples, and different clinical designs.1,2
Conclusion
This study generated contrasting results, which must therefore be interpreted with caution. However, several key points
can be made. First, our findings suggest that protein levels in
the serum of subjects with MDD change if clinical improvement occurs during antidepressant treatment, suggesting
that peripheral modifications may reflect clinical changes
rather than being associated purely with treatment. Second,
the nature of the proteins displaying a change in abundance
identified here may be relevant, indicating changes to general
pathways worthy of further study and potentially serving as
targets for future therapeutic approaches.
Author Contributions
Conceived and designed the experiments: MG. Analyzed the
data: MG. Wrote the first draft of the manuscript: MG. Contributed to the writing of the manuscript: KV, EP, DM. Agree
with manuscript results and conclusions: MG, DM, KV, EP,
BB, BP. Jointly developed the structure and arguments for the
paper: MG, DM. Made critical revisions and approved final
version: MG, KVD, EP, BB, DM. All authors reviewed and
approved of the final manuscript.
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