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Original article

Proteomic analysis of human saliva: An approach to nd the marker

protein for ovulation
Ganesan Saibabaa , Durairaj Rajesha,b , Subramanian Muthukumara,c ,
Ganesan Sathiyanarayanand , Parasuraman Padmanabhane,** ,
Mohammad Abdulkader Akbarshaf,g , Balzs Gulyse, Govindaraju Archunana,f,*

Center for Pheromone Technology, Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620024, India
Research Institute in Semiochemistry and Applied Ethology, Quartier Salignan, 84 400 Apt, France
Centre for Animal Research, Training and Services (CAReTS), Central Inter-Disciplinary Research Facility (CIDRF), Mahatma Gandhi Medical College and
Research Institute (MGMC-RI) Campus, Puducherry, 607403, India
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 143-701, Republic of Korea
Translational Neuroscience Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore
National Centre for Alternatives to Animal Experiments, Bharathidasan University, Tiruchirappalli, 620024, India
Department of Food Science and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh, Saudi Arabia


Article history:
Received 17 May 2016
Received in revised form 19 October 2016
Accepted 19 October 2016
Available online xxx
Functional annotation


Human saliva contains numerous molecules that play a variety of roles. Among them there are proteins
which serve as biomarkers of various physiological and/or pathological conditions. Compared to other
body uids, saliva is the most convenient material for investigations, and especially for monitoring the
disease conditions. Presently, there is an increasing need to develop a noninvasive method to identify the
time of ovulation in humans to ensure successful fertilization, and for evolving strategies for family
planning. The present investigation has been an attempt to identify one or more proteins in the human
saliva that would be an indicator(s) of ovulation. SDS-PAGE of salivary proteins showed seven prominent
bands during the different phases of the menstrual cycle. Particularly, the 14.5 kDa band was highly
expressed during the ovulatory phase. Eleven proteins were identied in this band of which ten were
highly specic to the ovulatory phase. Among those proteins the intense expression of Cystatin-S was
validated using immunoblot analysis (p < 0.05). The functional annotation of salivary proteins revealed a
high percentage of proteins that engage in binding and regulatory activities. The present results indicate
that salivary proteins, particularly those present during the ovulatory phase, might be used as biomarkers
for impending ovulation.
2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of
Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.

1. Introduction
Human saliva contains major, minor and gingival crevicular
secretions from parotid, submandibular and sub-lingual glands,
which play pivotal roles in digestion of food and maintaining the
oral health [1,2]. Saliva is an excellent biological uid that is useful

* Corresponding author at: Centre for Pheromone Technology, Department of

Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli,
620024, Tamilnadu, India.
** Corresponding author at: The Lee Kong Chian School of Medicine, Nanyang
Technological University, 59 Nanyang Drive 636921 Singapore.
E-mail addresses: (P. Padmanabhan), (G. Archunan).

for noninvasive exploration of the human diseases and physiological conditions [3]. It contains various biomolecules such as
proteins, enzymes and hormones [4,5]. However, the concentration of biomolecules in saliva is generally only one-tenth of that in
the blood [6]. More than two thousand proteins and peptides have
been identied [7] in different secretions of the major salivary
glands [8]. The salivary proteins facilitate bacterial agglutination
[9], digestion of food, antimicrobial activity, lubrication and
cleaning [10].
The salivary gland secretion is regulated by the autonomic
nervous system. The sympathetic and parasympathetic nervous
systems effectively regulate ow rate and composition of saliva.
The parasympathetic system facilitates secretion of a high volume
of saliva containing fewer proteins, whereas the sympathetic
1642-431X/ 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by
Elsevier Sp. z o.o. All rights reserved.

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system has the opposite effect [11]. The enzyme amylase (ptyalin)
and mucins are the major constituents in salivary proteome [12].
Saliva contains immunoglobulins also, and 60% of the total salivary
immunoglobulins is immunoglobulin A (IgA) [13]. Albumin has
also been detected as present in saliva but its concentration varies
from person to person [14]. The human salivary proteins which are
of low molecular weight, such as histatin and proline-rich proteins,
(PRPs) contribute greatly to the oral health [15].
Ovulation is a biological process wherein the mature ovarian
follicle ruptures so as to discharge the ovum, and this happens
under the inuence of luteinizing hormone (LH) surge. The LH
surge triggers a series of proteolytic processes which control
ovulation [16]. Saliva contains a number of hormones. The levels of
estrogen and progesterone in the saliva of premenopausal women
vary in relation to the phases of the menstrual cycle, and the
uctuation correlates with that in blood serum [17]. Similarly, the
salivary testosterone and cortisol levels have been evaluated to
diagnose hypogonadism in males by adopting liquid chromatography-tandem mass spectrometry (LCMS/MS). Compared to other
hormones in human saliva, cortisol exhibits a noticeable diurnal
variation [18].
Over the past decade, proteomics have been considered as one
of the best approaches for identication of biomarkers for various
diseases [19]. Salivary proteins have been identied as biomarkers
for various disease conditions such as Sjogrens syndrome [20],
lung cancer [21], oral cancer [22], a number of systemic diseases
[23], HIV infection [24], dental pellicle development [25], and
hyperglycemia [26]. However, no salivary protein marker for
monitoring the time of ovulation in women has been reported to
date. Although crystallization of saliva is useful in identication of
the fertile period in women [27], this method does not ensure
sufcient sensitivity and specicity. Since no simple and reliable
technique or biomarker is yet available for detection of ovulation in
women, a rapid protein-based detection kit, similar to the
noninvasive pregnancy detection kit that is based on the presence
of hCG (human chorionic gonadotropin) in urine [28], is very much
needed. Perhaps, one or more salivary proteins would serve as a
potential noninvasive biomarker(s) to predict ovulation [29].
Prediction of ovulation in women will be of application in assisted
reproductive technologies (ART), in vitro fertilization (IVF), and
natural family planning. Therefore, an attempt has been made in
this study to investigate the prole of salivary proteins during the
various phases of the menstrual cycle to identify the proteins
which are specically present during the ovulation phase, adopting
high resolution liquid chromatography tandem mass spectrometry
2. Materials and methods
2.1. Chemicals
The chemicals and reagents used in this study were obtained
from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
2.2. Ethics statement
Sample collection from women volunteers and all experimental
protocols were approved by the Institutional Ethical Committee
(IEC) (Approval No: DM/2014/101/38), Bharathidasan University,
Tiruchirappalli, India. All procedures were carried out in accordance with the approved guidelines. Written consent was obtained
from the volunteers who participated in the study.

2.3. Sample collection and process

The saliva was collected between 8.00 and 9.00am from 30
healthy female volunteers (mean age = 24 years, range = 1930
years) who were free from acute or chronic illnesses and ovarian
dysfunction, and did not take medications known to alter the sex
hormone levels. The volunteers brushed the teeth 30 min before
collection of saliva. The saliva was collected by spitting as adopted
in an earlier study [30]. The samples thus collected were
immediately transferred to the laboratory in an ice box. The
samples were centrifuged at 16,000  g for 15 min to remove
insoluble materials and cells, if any, and stored at 80  C until use.
2.4. Hormone analysis
The saliva samples were obtained during the three phases of the
menstrual cycle, namely the preovulatory (days 612), ovulatory
(days 13 and 14) and postovulatory (days 1526) phases, according
to the estradiol concentrations and physical changes of fern pattern
(Fig. S1) [31]. The ovarian follicle status was assessed with
ultrasonography to validate the day of ovulation. Estradiol
concentrations were determined by enzyme immunoassay (EIA)
using commercial kits (Pathozyme Oestradiol OD477 EIA kits,
Omega House, Scotland, UK).
2.5. Protein precipitation
The salivary proteins were precipitated using trichloroacetic
acid (TCA)-acetone method. The saliva samples were mixed with
10% TCA-acetone and 10 mM dithiothreitol (DTT) and incubated for
1 h at 20  C. After incubation, the samples were centrifuged at
5000  g at 4  C for 20 min. The pellets were washed twice with
ice-cold acetone and centrifuged at 5000  g at 4  C for 20 min.
Finally, the pellets were air-dried and re-suspended in 10 mM Tris
buffer. The concentration of protein was determined using the
Bradford method [32].
2.6. Sodium dodecyl sulphate poly-acrylamide gel electrophoresis
To identify the salivary proteins SDS-PAGE was carried out on
12% gel with medium-range molecular weight marker (MerckGeni, Bangalore) used as reference standard. The salivary protein
preparation from each volunteer (30 mg) was thoroughly mixed
with 1  sample buffer [50 mM Tris-Cl (pH 6.8), 2% SDS, 10%
glycerol, 0.1% bromophenol blue, 100 mM b-mercaptoethanol] and
kept for 1 min at 100  C for complete denaturation of proteins, after
which the sample was loaded onto the gel. A constant current of
50 V was applied for the electrophoresis and the entire process was
maintained at room temperature.
2.7. Coomassie brilliant blue (CBB) staining
After the electrophoresis, the gels were immersed in distilled
water for 2 min and subsequently stained with 0.5% CBB solution
(40% methanol, 10% acetic acid and 0.5% CBB R-250) at room
temperature for 2 h. The gels were de-stained using 40% methanol
and 10% acetic acid until their background became clear. Finally,
the gels were rinsed with distilled water and stored in 5% acetic
acid until used for further analysis. The intensity of the protein
bands was measured using gel documentation system (Quantity
One software, Bio Rad, CA, USA), based on the pixel areas occupied
by the protein bands.

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2.8. De-staining and in-gel trypsin digestion

2.12. Antimicrobial activity

Trypsin digestion was conducted according to Muthukumar

et al. [33]. The protein bands were excised using sterile blade and
placed in separate tubes containing 25 mM NH4HCO3 and 50%
acetonitrile (v/v) in 1:1 ratio and incubated at room temperature
until no color was visible. The de-stained gels were sliced into
small pieces and transferred to fresh tubes. The gels were dried in
Speed-Vac and the gel pieces were subjected to reduction
(immersed in 2% b-mercaptoethanol 25 mM NH4HCO3 and
incubated in dark for 20 min at room temperature) followed by
alkylation (in 10% 4-vinylpyridine and 25 mM NH4HCO3 in 50%
acetonitrile). After incubation, the gels were washed with 25 mM
NH4HCO3 for 10 min, dehydrated and digested in 100 ng of trypsin
(Promega, Madison, WI, USA) in 25 mM NH4HCO3 following
overnight incubation. After enzymatic cleavage the trypsin
solution was removed and proteins/peptides were extracted in
0.1% TFA (Triuoroacetic acid) in 50% acetonitrile. The extract was
dried in Speed-Vac and stored at 20  C until further analysis.

Antimicrobial activity of the whole saliva was assessed by welldiffusion method according to the National Committee for Clinical
Laboratory Standards (NCCLS). Mueller-Hinton agar (MHA) plates
were inoculated with 12 h old broth cultures of the test organisms
to create conuent lawns of bacterial growth. The antibacterial
effect of saliva was inferred from the colony-free zone around the
well into which the saliva was introduced. The agar plates were
incubated at 37  C for 24 h and the inhibitory pattern was
determined by measuring the diameter of the zone of inhibition
around the well (in mm). The experiments were repeated thrice
and the average zone of inhibition was calculated.

2.9. Mass spectrometry analysis

Trypsin-digested protein samples were subjected to analysis in
HCT-Ultra ETD II (Bruker Daltonics, Billericia, Bremen, Germany)
using water-saturated acetonitrile containing 0.1% formic acid. A
mass spectrometer coupled with an Agilent 1100 HPLC (High
Performance Liquid Chromatography) system and equipped with
C18 column (Supelco) was used for the analysis. A typical LC linear
gradient was set from 90% H2O to 90% acetonitrile for over a 50 min
runtime at a ow rate of 0.2 mL/min. The LCMS spectra were
averaged by over four scans and 10 L/min were set during the run
for dry gas. Nebulizer pressure was set at 30 psi.

2.13. Functional annotation

The salivary proteins of the ovulatory phase were further
analyzed to retrieve their cellular location, molecular function and
biological process by STRAP 1.5 online database (http://www. [35] and
Gene Ontology (GO) enrichment analysis.
2.14. Statistical analysis
The protein concentrations and relative band intensity values
corresponding to the ovulatory, pre-ovulatory and post-ovulatory
phases are represented as mean  SD, and analyzed using one-way
analysis of variance (ANOVA) with LSD post-hoc comparisons. A p
value  0.05 was considered statistically signicant. All statistical
analyses were performed using Statistical Package for Social
Sciences for Windows, version 16.0 (SPSS Inc., Cary, NC, USA).
3. Results

2.10. Peptide and protein identication

3.1. Determination of the phases of menstrual cycle
The acquired protein masses were processed further for
monoisotopic peptide masses using FLEX analysis software, and
the peptide masses were allocated according to the database
search. SWISS-PROT database entries of MASCOT search engine
( were used for identication of
proteins. The MASCOT search scores were considered as signicant
at p < 0.05. The primary sequences of the detected salivary proteins
were retrieved from NCBI ( All the
mass spectrometry data have been deposited at the ProteomeXchange Consortium [34] ( via PRIDE partner repository with the dataset
identier PXD000921 and DOI 10.6019/PXD000921.

The estradiol concentrations differed to signicant levels

through different phases of the menstrual cycle (p < 0.05), this
being the highest during the ovulatory phase (2.28  0.20 pg/mL)
(Fig. 1). The total protein concentration of saliva was found to be
signicantly higher during the ovulatory phase (2.49  0.39 mg/
mL) than pre-ovulatory (1.14  0.39 mg/mL) and post-ovulatory
(1.50  0.28 mg/mL) phases, respectively (Table 1).

2.11. Western blot analysis

The salivary proteins of the pre-ovulatory, ovulatory and postovulatory phases (n = 10 each) were subjected to SDS-PAGE. After
electrophoresis, the proteins were transferred to PVDF (polyvinylidene diuoride) membrane, which was then incubated with
blocking buffer (5% skimmed milk powder in TBS) for 12 h. After
washing with TBS-T (Tris buffer saline-Tween 20) and TBS buffer,
the membrane was incubated with primary polyclonal anticystatin-S antibody (Santa Cruz Biotechnology, Inc., TX, USA) at a
dilution recommended by the manufacturer. The membrane was
then incubated with alkaline phosphatase (ALP)-conjugated
secondary antibody (anti-rabbit IgG) for 12 h followed by
washing. The reaction was visualized using a chromogenic
substrate BCIP/NBT (Calbiochem, USA). The signal intensities of
the bands were calculated using Image J software (NIH, Bethesda,
MD, USA) and the corresponding b-actin expression was used as

Fig. 1. Salivary estradiol levels during menstrual cycle. The daily pattern of estradiol
level was recorded in the saliva sample of the subject. The estradiol level reached
the peak around day 13 or 14 of menstrual cycle. Data are presented as mean  SD.

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Table 1
Concentrations of salivary proteins during the different phases of menstrual cycle.

Pre-Ovulation Ovulation

Protein Concentration (mg/mL) 1.14  0.39

2.49  0.39*

1.50  0.28**,a

The values are expressed as mean  SD. The mean difference is signicant at
p < 0.05.
p < 0.05, Pre-ovulation v Ovulation.
p < 0.05, Ovulation v Post-ovulation.
p < 0.05, Pre-ovulation v Post-ovulation.

3.2. SDS-PAGE of whole salivary proteins

In 12% SDS-PAGE, the salivary proteins were separated into an
array of protein bands ranging in molecular weight from 70 to
14.5 kDa as guided by standard molecular weight marker proteins
(Fig. 2, Fig. S2). The 14.5 kDa band was highly expressed during the
ovulatory phase as compared with the other phases.
3.3. Total proteome of saliva
The differentially expressed 14.5 kDa bands representing the
three phases were excised and subjected to mass spectrometry
analysis. In total fourteen proteins were identied, combining all
three phases, and listed (Table 2). Importantly, the 14.5 kDa band
during the ovulatory phase represented 11 proteins among which
10 were specically expressed during the ovulatory phase. During
the pre- and post-ovulatory phases there were only ve or less
such proteins. It clearly indicates that the abundance of proteins in
the 14.5 kDa band during the ovulatory phase was high when
compared to the other two phases of menstrual cycle. Cystatin-S
was the predominant protein present in the 14.5 kDa band of the
ovulatory phase. Cystatin-SN, Cystatin-SA, Cystatin-B, Prolactininducible protein, Cystatin-A, Cystatin-C, Thioredoxin, Beta-2microglobulin, Succinate-semialdehyde dehydrogenase, Disintegrin and Metalloproteinase domain-containing protein-7 were the
other proteins that constituted the 14.5 kDa band of the ovulatory
phase. All of these proteins have far-reaching implications as
potential biomarkers of ovulation.
3.4. Western blot analysis
Cystatin-S was identied with a higher number of peptide
matches during the ovulatory phase. Therefore, the intense
expression of Cystatin-S was validated by Western blot analysis
using anti-Cystatin-S antibody with b-actin as the control.
Cystatin-S was highly expressed during the ovulatory phase
compared with other phases (Fig. 3A). The immunoblot results
were consistent with the mass spectrometry results indicating that
the abundance of Cystatin-S during the ovulatory phase has a
functional signicance (Fig. 3B).

Fig. 2. SDS-PAGE of whole salivary proteins. The salivary proteins were separated
by 12% SDS-PAGE. Lane-M, medium range molecular markers. The concentration of
the protein samples in the different lanes was the same. PreO Pre-ovulation phase,
O Ovulation phase, PostO Post-ovulation phase.

Table 2
List of proteins identied in 14.5 kDa band of ovulatory phase.
Swiss-Prot acc. no.a





Lengtha Peptides matched


Prolactin-inducible protein
Lysozyme C





Succinate-semialdehyde dehydrogenase
Disintegrin and metalloproteinase domain-containing
protein 7
Coiled-coil domain-containing protein 171
Uncharacterized protein KIAA0232

Strong inhibitor
Proteinase inhibitor
Thiol proteinase inhibitor
Intracellular thiol proteinase inhibitor
Protein binding
Intracellular thiol proteinase inhibitor
Bacteriolytic function
Inhibitor of cysteine proteinases
Expression of interleukin-2 receptor
Antigen presentation
Role in reproduction

6.07 11.73 119

7.17 52.30
5.92 65.09 754


Nucleotide binding

6.37 152.7 1326

4.71 154.7 1395





Proteins having at least one identied peptides in ovulation period saliva are listed with their Swiss-Prot/TrEmbl accession numbers and length.
Functions were retrieved using the STRAP online database bioinformatics resource [35].
Theoretical pIs (c) and monoisotopic molecular weights (d) were calculated using the Swiss-Prot website [61].

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Fig. 3. Western blot analysis of salivary protein. A) Western blot of Cystain-S in saliva during different phases of menstrual cycle. B) The bar diagram represents relative
abundance of Cystatin-S during the three phases, of the menstrual cycle normalized with the expression of the housekeeping gene b-actin. Equal amount of protein was
loaded to each lane and data are presented as mean  SD. *p < 0.05 as compared with pre- and post- ovulation phases. PreO- Pre-Ovulation, O- Ovulation, PostO- PostOvulation.

activity of saliva, which was assessed by adopting the zone of

inhibition test, increased during the ovulation phase. The results
revealed that K. pneumoniae and E. coli were more sensitive to
saliva (Fig. 4). The high antimicrobial activity during the ovulatory
phase may have signicant role in oral hygiene.
3.6. Functional annotation

Fig. 4. Antimicrobial activity of saliva during the three phases of menstrual cycle.
The antibacterial activity of saliva was tested against test strains by well diffusion
method. The ovulation phase saliva shows highest activity (Inhibition zone- 6 and
5 mm respectively) towards K. pneumoniae and E. coli.

The proteins identied in the saliva during ovulatory phase

were classied on the basis of biological process, cellular location
and molecular function using STRAP database (Table 3). The scatter
plot for ovulatory phase proteins of human saliva was generated,
which was classied by GO. Especially, a few important proteins
appeared as separate clusters in the molecular functions GO term.
A cluster of blue-mixed yellow bubbles represents the Glyco-, Gprotein-, receptor-, protease-, IgA-, IgG- and immunoglobulinbinding proteins. The phospholipid-binding and fatty acid-binding
proteins are denoted by light green bubbles. Metal ion-, iron ion-,
ferric ion-, RNA- and calcium ion- binding proteins are linearly
present in the scatter plot (Fig. S3A and C). Trans-membrane
transporter activity, peptidase activity and amylase activity during
the ovulatory phase are shown as specic co-clusters (Fig. S4A and
4. Discussion

3.5. Antimicrobial activity

The antimicrobial activity of saliva was investigated against ve
test bacterial strains, of which two were gram-positive (Staphylococcus aureus MTCC 98 and Bacillus cereus MTCC 430) and three
were gram-negative (Klebsiella pneumoniae MTCC 432, Escherichia
coli MTCC 78 and Salmonella typhi MTCC 733). The antimicrobial

A typical fern/crystallization pattern has been observed in

cervical mucus due to increased level of NaCl (Sodium chloride)
under the inuence of estrogens [36]. The fern pattern is highly
prominent during the ovulatory phase, which is the best time for
fertilization in women [37]. The present study afrms this status in
respect of both saliva and cervical-vaginal mucus in women. The

Table 3
Functional annotation of salivary proteins during the ovulatory phase.
Biological process

Cellular location

Molecular function







Immune system process
Cellular process
Interaction with cells and organisms
Metabolic process
Response to stimulus
Developmental process


Plasma membrane
Macromolecular complex
Cell surface
Other intracellular organelles


Catalytic activity
Enzyme regulator activity
Structural molecule activity
Antioxidant activity


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salivary electrolytes, hormones and enzyme levels have been

reported to vary among different phases of menstrual cycle [38]. It
has, however, been clearly shown that the salivary fern test to
predict the time of ovulation in regularly cycling women has
specicity only to the extent of about 72% [28]. The major
components of saliva, such as proteins and electrolytes, are
interdependent and, most probably, the changes in hormonal
levels inuence the electrolytes as well as expression of proteins
[36]. The present results suggest that the ovulatory phase of
menstrual cycle has a denite inuence on the protein content of
saliva. Previous reports concerning house rat urine [33], buffalo
salivary proteome [39,40] and human uterine-cervical secretions
[4143] have revealed difference in protein expressions in relation
to the phases of reproductive cycle. It is well known that the
common defensive class of proteins, such as immunoglobulins,
increase to high levels in cervical uid during the ovulatory phase
[42]. The present 1D gel proteome indicates that one or more
proteins may be specically and/or differentially expressed during
the ovulatory phase. The abundance of the 14.5 kDa protein band
during that phase provided the rationale for considering it as the
ovulatory phase-specic protein. The saliva also contains a broad
spectrum of immunologic and non-immunologic proteins having
antibacterial activity [43]. Immunoglobulins play a major role in
inhibition or prevention of microbial growth in oral cavity.
Immunoglobulin-A (IgA) is a major protein of the saliva and it
inhibits microbial growth [44]. The non-immunologic proteins
such as lysozyme, lactoferrin, peroxidase, mucin glycoproteins,
agglutinins, histatins, proline-rich proteins, statherins and cystatins are involved in hydrolysis of bacterial cell wall and inhibition
of bactericidal activity [45]. In the present study, numerous
immunologic and non-immunologic proteins were detected in the
saliva. Specically, the ovulatory phase saliva contained a large
number of defensive proteins that were detected in the antimicrobial activity test. In this regard, this study is in complete
agreement with a previous study [46] and throws a new light on
understanding the defense mechanisms mediated by saliva during
the ovulatory phase of the menstrual cycle.
The functional annotation of the proteins identied in the saliva
was obtained from STRAP database. We found most of the
identied proteins to be associated with binding property and
regulatory function. In addition, these extracellular proteins would
play critical roles during the period of ovulation. It is to be noted
that binding proteins are present along with volatiles in body uids
of mammals and facilitate chemical communication during estrus
[39,47,48]. The binding proteins may also have a role in increasing
the stability of other proteins [39]. Generally, the expression of
HSPs increases during biological stress, inammation, salivary
gland tumors, and microbial invasion, which in turn can activate
protease cascades [4951].
In a previous preliminary report from our group [31] a 48 kDa
protein band was shown as highly expressed during the ovulatory
phase. MALDI-TOF analysis revealed this protein band to match
with UDPN-acetylglucosamine pyrophosphorylase. But, in that
study, we used TCA for protein extraction which is not efcient in
extraction of low molecular weight proteins. Since in the present
study we used TCA in acetone for the protein extraction the low
molecular weight proteins were efciently extracted and so the
14.5 kDa protein expounded to be the dominant protein. Also, in
the present study we adopted the much improved LCMS/MS in
place of MALDI-TOF, and it revealed Cystatin-S to be the dominant
protein of expression during the ovulatory phase. The immunoblot
analysis also revealed a higher expression of Cystatin-S protein
during ovulatory phase compared with other phases of the
menstrual cycle. Therefore, the present study convincingly
demonstrates and validates the 14.5 kDa band, and especially
Cystatin-S, as a much better indicator of ovulation.

The Cystatin gene family has 14 genes, and seven Cystatins

(Cystatin-A, Cystatin-B, Cystatin-C, Cystatin-D, Cystatin-S, Cystatin-SA and Cystatin-SN) are present in the saliva [52]. Cystatins S,
SA, SN, and C are 14 kDa proteins which are essentially
extracellular [53]. The submandibular gland is the major contributor of Cystatins in saliva [54]. Cystatins are strong inhibitors of
cysteine proteases that are responsible for terminal protein
degradation and also have a role in the regulation of salivary
calcium [55]. Human Cystatin-S has high afnity to tooth surface,
which helps in maintaining the surface mineralization [56].
Further, Cystatins are involved in a variety of biological processes
such as antigen presentation, bone resorption, apoptosis and
protein processing. Cystatin has roles during pathological conditions, such as cancer progression, inammation and neurodegeneration [57]. Especially, Cystatin S has been shown to be an
antimicrobial protein which specically inhibits the growth of P.
gingivalis [57]. Other Cystatins, such as Cystatin A, are found in the
basal cells of prostate gland during benign prostatic hyperplasia
[58]. Cystatin C is highly expressed in the male genital tract, and it
has important regulatory role in normal and pathological
proteolysis in the male reproductive system [59]. The high
expression of Cystatin-S in saliva during the ovulatory phase,
found in this study, would probably contribute to the antimicrobial
activity of saliva. It has earlier been suggested that major
electrolytes such as sodium, potassium and calcium are increased
signicantly during the ovulatory phase [60]. Cystatins commonly
participate in mineralization process [57]. Cystatin-S expression
would provide for maintenance of trace elements in the oral cavity.
Cystatin-S as ovulation marker protein has been validated in this
study by immunoblot analysis.
In summary, this is the rst study of salivary proteins as
biomarkers of ovulation in the human. The ovulation-specic
proteins have been listed of which the cystatin-S was identied as
the highly expressed protein. Antibacterial activity was high in
saliva during the ovulatory phase due to the large number of
antimicrobial proteins. Functional annotation of salivary proteins
indicated that they were mostly extracellular proteins participating in regulatory functions. Since contemporarily intensive search
for noninvasive biomarkers for different applications is going on,
the discovery of this study, though preliminary, can go a long way
in bringing up a noninvasive biomarker for ovulation.
Conicts of interest
The authors declare no conict of interest.
Author contributions
G.S. (lead author), P.P. and G.A. designed the study; G.S. (lead
author), D.R., S.M. and G.S. performed research; G.S. (lead author),
S.M., D.R., M.A.A. and B.G. analyzed and interpreted the data; G.S.
(lead author), S.M., D.R., and G.A. wrote the manuscript. M.A.A.
edited the manuscript.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the
institutional and/or national research committee and with the
1964 Helsinki declaration and its later amendments or comparable
ethical standards.
G.S. (lead author) thanks the Indian Council of Medical Research
(ICMR-New Delhi, India; No. 45/5/2014-BIO/BMS) for the Senior

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Research Fellowship. G.A. thanks the INSA (New Delhi)-DFG

(Deutsche Forschungsgemeinschaft, Germany) funding for bilateral visit to University of Jena, Germany. The authors thank Prof.
Dipankar Chatterje and Mr. T Raghu Phaneendra Kumar, Molecular
Biophysics Unit and Proteomics Facility Center, Indian Institute of
Science, Bangalore, for help in the LCMS/MS analysis. The
research facility through DST, DBT, ICAR-National Fund and
UGC-SAP-DRS-II, DST-FIST-Level-I (Stage-II) grants to the University/Department/Centre is acknowledged. The authors thank
Tobias Ternent and Attila Csordas of PRIDE Team for processing
the MS data, which have been deposited with the ProteomeXchange Consortium. PP and BG acknowledge the support from the
The Lee Kong Chian School of Medicine, Nanyang Technological
University, MOE Start-Up Grant, Singapore.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at
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