Proteomic analysis of human saliva: An approach to find the marker protein for ovulation

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Original article

Proteomic analysis of human saliva: An approach to find the marker protein for ovulation Ganesan Saibabaa , Durairaj Rajesha,b , Subramanian Muthukumara,c , Ganesan Sathiyanarayanand , Parasuraman Padmanabhane,** , Mohammad Abdulkader Akbarshaf,g , Balázs Gulyáse, Govindaraju Archunana,f,* a

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 c 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 d Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 143-701, Republic of Korea e Translational Neuroscience Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore f National Centre for Alternatives to Animal Experiments, Bharathidasan University, Tiruchirappalli, 620024, India g Department of Food Science and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh, Saudi Arabia b

A R T I C L E I N F O

Article history: Received 17 May 2016 Received in revised form 19 October 2016 Accepted 19 October 2016 Available online xxx Keywords: Functional annotation Biomarker SDS-PAGE Protein Cystatin-S

A B S T R A C T

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 fluids, 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 identified in this band of which ten were highly specific 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. ã 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 fluid 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: [email protected] (P. Padmanabhan), [email protected] (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 identified [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 flow rate and composition of saliva. The parasympathetic system facilitates secretion of a high volume of saliva containing fewer proteins, whereas the sympathetic

http://dx.doi.org/10.1016/j.repbio.2016.10.005 1642-431X/ã 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.

Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to find the marker protein for ovulation, Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005

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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 influence 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 fluctuation 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 (LC–MS/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 identification of biomarkers for various diseases [19]. Salivary proteins have been identified as biomarkers for various disease conditions such as Sjogren’s 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 identification of the fertile period in women [27], this method does not ensure sufficient sensitivity and specificity. 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 profile of salivary proteins during the various phases of the menstrual cycle to identify the proteins which are specifically present during the ovulation phase, adopting high resolution liquid chromatography tandem mass spectrometry (LC–MS/MS). 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 = 19–30 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 6–12), ovulatory (days 13 and 14) and postovulatory (days 15–26) 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 (SDS-PAGE) 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 (Trifluoroacetic 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 confluent 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 flow rate of 0.2 mL/min. The LC–MS 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. bumc.bu.edu/cardiovascularproteomics/cpctools/strap/) [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 significant. 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 identification 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 (http://www.matrixscience.com) were used for identification of proteins. The MASCOT search scores were considered as significant at p < 0.05. The primary sequences of the detected salivary proteins were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/). All the mass spectrometry data have been deposited at the ProteomeXchange Consortium [34] (http://proteomecentral.proteomexchange.org) via PRIDE partner repository with the dataset identifier PXD000921 and DOI 10.6019/PXD000921.

The estradiol concentrations differed to significant 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 significantly 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 difluoride) membrane, which was then incubated with blocking buffer (5% skimmed milk powder in TBS) for 1–2 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 1–2 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 reference.

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. Details

Pre-Ovulation Ovulation

Protein Concentration (mg/mL) 1.14  0.39

2.49  0.39*

Post-Ovulation 1.50  0.28**,a

The values are expressed as mean  SD. The mean difference is significant at p < 0.05. * p < 0.05, Pre-ovulation v Ovulation. ** p < 0.05, Ovulation v Post-ovulation. a 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 identified, 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 specifically expressed during the ovulatory phase. During the pre- and post-ovulatory phases there were only five 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 identified 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 significance (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 identified in 14.5 kDa band of ovulatory phase. Swiss-Prot acc. no.a

Name

Functionb

pIc

MWc

Lengtha Peptides matched

P01036 P01037 P09228 P04080 P12273 P01040 P61626 P01034 P10599

Cystatin-S Cystatin-SN Cystatin-SA Cystatin-B Prolactin-inducible protein Cystatin-A Lysozyme C Cystatin-C Thioredoxin

4.83 6.92 4.85 6.96 5.40 5.38 9.38 8.75 4.82

14.18 14.31 14.35 11.13 13.52 11.00 14.70 13.34 11.60

141 141 141 98 146 98

P61769 P51649 Q9H2U9

Beta-2-microglobulin 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 TAC Antigen presentation Catalysis Role in reproduction

6.07 11.73 119 7.17 52.30 5.92 65.09 754

1 1 7

Unknown Nucleotide binding

6.37 152.7 1326 4.71 154.7 1395

8 1

Q6TFL3 Q92628 a b c

146

13 11 7 4 2 2 3 3 1

Proteins having at least one identified 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 significant 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 identified in the saliva during ovulatory phase were classified 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 classified 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 specific co-clusters (Fig. S4A and B). 4. Discussion

3.5. Antimicrobial activity The antimicrobial activity of saliva was investigated against five 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 influence 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 affirms 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

Process

Percent

Location

Percent

Function

Percent

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

41.31% 17.13% 16.2%, 15.11% 14.11% 10.8% 7.5% 6.5% 6.5%

Extracellular Plasma membrane Cytoplasm Nucleus Endosome Macromolecular complex Cytoskeleton Mitochondria ER Cell surface Chromosome Other intracellular organelles Others

39.5% 16.14% 10.9% 10.9% 5.4% 3.3% 3.3% 3.3% 3.3% 1.1% 1.1% 10.9% 8.7%

Binding Catalytic activity Enzyme regulator activity Structural molecule activity Antioxidant activity Others

46.60% 17.22% 10.13% 1.1% 1.1% 2.3%

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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 specificity 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 influence the electrolytes as well as expression of proteins [36]. The present results suggest that the ovulatory phase of menstrual cycle has a definite influence on the protein content of saliva. Previous reports concerning house rat urine [33], buffalo salivary proteome [39,40] and human uterine-cervical secretions [41–43] 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 fluid during the ovulatory phase [42]. The present 1D gel proteome indicates that one or more proteins may be specifically 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-specific 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. Specifically, 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 identified in the saliva was obtained from STRAP database. We found most of the identified 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 fluids 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, inflammation, salivary gland tumors, and microbial invasion, which in turn can activate protease cascades [49–51]. 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 efficient 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 efficiently extracted and so the 14.5 kDa protein expounded to be the dominant protein. Also, in the present study we adopted the much improved LC–MS/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 affinity 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, inflammation and neurodegeneration [57]. Especially, Cystatin S has been shown to be an antimicrobial protein which specifically 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 significantly 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 first study of salivary proteins as biomarkers of ovulation in the human. The ovulation-specific proteins have been listed of which the cystatin-S was identified 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. Conflicts of interest The authors declare no conflict 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. Acknowledgements 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 LC–MS/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 http://dx.doi.org/10.1016/j. repbio.2016.10.005. References [1] Defabianis P, Re F. The role of saliva in maintaining oral health. Minerva Stomatol 2003;52:301–8. [2] Edgar WM. Saliva: its secretion, composition and functions. Br Dent J 1992;172:305–12. [3] Wong DT. Towards a simple, saliva-based test for the detection of oral cancer ‘oral fluid (saliva), which is the mirror of the body, is a perfect medium to be explored for health and disease surveillance. Expert Rev Mol Diagn 2006;6:267–72. [4] Haeckel R, Hanecke P. Application of saliva for drug monitoring. An in vivo model for transmembrane transport. Eur J Clin Chem Clin Biochem 1996;34:171–91. [5] Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. J Prosthet Dent 2001;85:162–9. [6] Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011;57:675–87. [7] Bandhakavi S, Stone MD, Onsongo G, Van Riper SK, Griffin TJ. A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva. J Proteome Res 2009;8:5590–600. [8] Castagnola M, Cabras T, Iavarone F, Fanali C, Nemolato S, Peluso G, et al. The human salivary proteome: a critical overview of the results obtained by different proteomic platforms. Expert Rev Proteom 2012;9:33–46. [9] Leito JT, Ligtenberg AJ, Nazmi K, de Blieck-Hogervorst JM, Veerman EC, Nieuw Amerongen AV. A common binding motif for various bacteria of the bacteriabinding peptide SRCRP2 of DMBT1/gp-340/salivary agglutinin. Biol Chem 2008;389:1193–200. [10] Van Nieuw Amerongen A, Bolscher JG, Veerman EC. Salivary proteins: protective and diagnostic value in cariology? Caries Res 2004;38:247–53. [11] Huang AY, Castle AM, Hinton BT, Castle JD. Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells. J Biol Chem 2001;276:22296–306. [12] Meyer-Lueckel H, Hopfenmuller W, von Klinggraff D, Kielbassa AM. Microradiographic study on the effects of mucin-based solutions used as saliva substitutes on demineralised bovine enamel in vitro. Arch Oral Biol 2006;51:541–7. [13] Mestecky J. Saliva as a manifestation of the common mucosal immune system. Ann N Y Acad Sci 1993;694:184–94. [14] Niswander JD, Shreffler DC, Neel JV. Genetic studies of quantitative variation in a component of human saliva. Ann Hum Genet 1964;27:319–28. [15] Perinpanayagam HER, Van Wuyckhuyse BC, Ji ZS, Tabak LA. Characterization of low-molecular-weight peptides in human parotid saliva. J Dent Res 1995;74:345–50. [16] Robker RL, Russell DL, Yoshioka S, Sharma SC, Lydon JP, O’Malley BW, et al. Ovulation: a multi-gene, multi-step process. Steroids 2000;65:559–70. [17] Gann PH, Giovanazzi S, Van Horn L, Branning A, Chatterton Jr. RT. Saliva as a medium for investigating intra- and interindividual differences in sex hormone levels in premenopausal women. Cancer Epidemiol Biomark. Prev 2001;10:59–64. [18] Matsui F. Liquid Chromatography-tandem Mass Spectrometry (LC–MS/MS) assay for simultaneous measurement of salivary testosterone and cortisol in healthy men for utilization in the diagnosis of late-onset hypogonadism in males. Endocr J 2009;56:1083–93. [19] Grus FH, Joachim SC, Pfeiffer N. Proteomics in ocular fluids. Proteom Clin Appl 2007;1:876–88.

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