Salivary proteome profile of women during fertile phase of menstrual cycle as characterized by mass spectrometry

Background Human saliva contains several biomolecules, especially proteins, some of which have been found to serve as biomarkers of different physiological statuses and/or pathological conditions. Saliva is a much superior biological material for investigation over the other body fluids. Several salivary proteins are present in minute quantities whereupon there is possibility to express the hormone receptors, stress proteins or antimicrobial peptides at subtle to major variations which can reveal the physiological and/or pathological statuses. Ovulation is such a critical physiological process that its non-invasive prediction has several advantages. It has been postulated that saliva, with one or more of biomolecules in it such as proteins, amino acids, antioxidants, antimicrobial peptides, and such others would effectively serve as non-invasive predictor(s)/detector(s) of ovulation. Methods Samples were collected from women volunteers and the procedure adopted was approved by the Institutional Ethical Committee (DM/2014/101/38), Bharathidasan University. The saliva samples were collected between 8.00 to 9.00 AM from 30 healthy female volunteers (age, mean = 24, range = 19 - 30), with prior written consent. The protein expression pattern during different phases of menstrual cycle was analyzed using HR-LC-MS/MS and MALD TOF/TOF. Results The functional annotation of salivary proteins identified therein revealed that the proteins get assigned to the class of “extracellular proteins” which are concerned with regulatory functions. As many as 530 proteins showed up in the saliva during ovulation phase whereas there were only 251 proteins during post-ovulation phase. The protein expression was also analyzed using 2D gel electrophoresis and it was found that the salivary proteins focused at pH range 4-7 and molecular weight around 97-14 kDa. The unique/differentially expressed protein spots appeared during ovulation phase was identified by MALDI-TOF analysis. Conclusions According to spot analysis, Cystatin-S, Prolactin-inducible protein, Cystatin-A, Cystatin-SN, BPI fold-containing family A member 2, Alpha-tubulin N-acetyltransferase 1, and Carbonic anhydrase 6 were highly expressed during ovulation phase. These proteins could serve as an indicator(s) for ovulation after extensive validation studies.


Introduction
Saliva is a clear oral fluid composed of 98% water and 2% other compounds, such as mixture of proteins, electrolytes and small molecular weight organic compounds. The term saliva stands for secretions from the major (submandibular, sublingual and parotid) and minor salivary glands, along with crevicular fluid, bacteria and cellular debris. Normally, human salivary glands secrete about 1.0-1.5 L saliva every day, which contains molecules from blood and salivary protein combined [1]. The secretion of saliva is regulated by the autonomic nervous system via signal transduction systems that couple receptor stimulation to ion transport and protein secretory mechanisms [2]. In recent years, saliva has been recognized as a diagnostic tool and general health indicator, and offers many advantages over serum in this regard. Many salivary biomolecules arrive from the blood through passive intracellular diffusion and active transport or extracellular ultrafiltration [3]. As revealed in several, reports the levels of salivary components vary in respect of the spectrum of oral and general health. For example, low levels of lysozymes [4] and presence of lactoferrin [5] were observed in saliva under condition of dental caries.
Likewise, in the beginning of menstruation and during ovulation the protein content of saliva increases considerably, which turns out to be a rich source of food to bacteria. The bacterial count may also increase during menstruation and ovulation [6]. The concentration of analytes in saliva is 100-to 1000-fold lower compared to blood [7]. The major biochemical constituent of saliva is proteins which are influenced by the physiological status of the body. As a diagnostic fluid, saliva and urine offers many advantages over blood, which include (i) simple, cost-effective and non-invasive nature of sample collection, (ii) no need of skilled personal to collect saliva, and (iii) saliva contains lesser protein concentration than plasma. Specifically, the less abundance of proteins in saliva makes it easier to investigate for diagnostic purposes [7].
Menstrual cycle is a complicated physiological process in which different endocrine glands, reproductive organs and brain have roles. Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) are important hormones from pituitary gland, which are concerned with, among other hormones, follicular growth and release of egg, respectively.
The pituitary gland secretes high levels of LH during mid-cycle, which induces the ovary to release the egg which is characterized as ovulation. There is an increasing demand for a biomarker for accurate prediction/detection of fertile phase in women's reproductive cycle [8]. Most of the animals have limited and short fertile period, the estrus. The external indications and attractiveness synchronize with ovulation and maximize the chance for fertilization [9]. Human females do not show any corresponding cyclical changes that would be an indicator of ovulation Therefore, it is felt essential to develop a method to identify the time of ovulation during the reproductive cycle [10].
The time of ovulation is associated with fertile phase of menstrual cycle in human. In the mammals, it is commonly accepted that many reproductive process such asovulation, menstruation, implantation and parturition are linked with inflammation [11]. As consequence of these events there is upregulation of a host of inflammatory mediators, which include cytokines, growth factors and lipid mediators which influence the growth and function of the immune and vascular compartments [12]. Similarly, in response to inflammation in ovary, there are specific pro-or anti-inflammatory cytokine/protein expressions in the body fluids. This can as well be utilized to predict the time of ovulation [13]. However, still there is not yet a reliable non-invasive kit or even method to detect the time of ovulation. Hence, a method for accurate prediction or detection of the fertile phase during menstrual cycle has enormous significance in promoting or controlling fertility. Saliva, for the reasons mentioned above, has long been speculated to possess one or more biomarkers indicting important events in the reproductive cycle. Thus, we adopted a proteome-based approach to detect salivary biomarkers for fertile phase [13].
Initially, polyacrylamide gel electrophoresis (SDS-PAGE), the single dimensional electrophoresis, was carried out to separate out the proteins based on molecular weight and their charges into distinctive protein bands. Thus, complex protein mixtures were analyzed qualitatively by SDS-PAGE [14], which suggested that SDS-PAGE is an adequate technique to assess protein composition of the human saliva [15]. However, in the recent times, 2-D PAGE is used to separate complex protein mixtures of saliva based on the different modifications and isoforms of the same protein. Further, recent advancements in mass spectrometry techniques, i.e., quadrupole/linear ion trap, time-of flight(TOF), quadrupole TOF (QTOF), Fourier transform ion cyclotron resonance (FT-ICR), and OrbiTrap are practiced to improve discovery a protein biomarker in saliva [3]. In our previous study [16], the ovulation-specific proteins in saliva were mapped, and validated adopting immunoblotting. However, the number of proteins and their variants (isoforms) present in the particular region are not known. If what we found is a group of proteins, it would be difficult to work it out for diagnostic purposes. Hence, it was thought that the identification of an ovulation-specific protein in saliva would do well for future application.
Saliva, compared to other body fluids, as already mentioned, is the best biological fluid for exploring the marker to diagnose the reproductive status. Therefore, the present study was directed at identifying the proteins in human saliva in relation to phases in the menstrual cycle by adopting gel electrophoresis followed by mass spectrometry to discern the various proteins, and map the ovulation-specific protein(s)(supplementary Figure S1).

Volunteers' information and ethical statement
Samples were collected from women volunteers and the procedure adopted was approved by the Institutional Ethical Committee (DM/2014/101/38), Bharathidasan University. The saliva samples were collected between 8.00 to 9.00 AM from 30 healthy female volunteers (age, mean = 24, range = 19 -30), with prior written consent [16]. The volunteers were instructed to abstain from smoking and drinking and not to consume food for 10 h before the sample collection. The volunteers were also asked to brush the teeth before collection of saliva so as to prevent microbial contamination.

Sample collection and process
The saliva was collected by spitting into a sterile vial, which was immediately kept in an ice box and brought to the laboratory. The duration of collection of saliva was about 10 min and the saliva secretion over the first minute was discarded. The samples were centrifuged at 16000 x g for 15 min to remove insoluble materials and cells, if any. The samples were stored at -80˚C until further use. The saliva samples were assigned among three phases, viz., preovulatory (days 6 to 12), ovulatory (days 13 and 14) and postovulatory (days 15 to 26) phases according to the pattern of salivary hormones and fern pattern analysis, as was done in our previous study [16].

Protein precipitation and estimation
The salivary proteins were precipitated by trichloroacetic acid (TCA)-acetone precipitation method [17]. The samples were mixed with TCA:acetone (TCA-20% W/V; Acetone-90% V/V) in 1:1 ratio and 20 mMdithiothreitol (DTT) and incubated overnight at -20 °C. After incubation, the samples were centrifuged at 5000 x g at 4 °C for 30 min. The pellets were washed twice with cold acetone and centrifuged at 5000 x g at 4 °C for 30 min. Finally, the pellets were air-dried and re-suspended in UTC (6M Urea, 3M Thiourea, 8% CHAPS) buffer. The protein concentration was determined adopting modified protocol of Bradford (1976) [18].

1D-Gel electrophoresis
The protein concentration in saliva differed slightly between ovulation and post-ovulation phases. The total proteome was fractionated by 12% SDS-PAGE ( Figure 1A). The protein profile of ovulatory phase saliva was compared to that of postovulatory phase. Put together, during the two phases, a total of 12 distinct protein bands appeared in the Coomassie Brilliant blue-stained gels, and their molecular mass ranged from 14 to 97 kDa.

2D-Electrophoresis
The frozen strips were brought to room temperature and subjected to reduction and alkylation. The strips were incubated in SDS-Equilibration buffer I (6 M Urea, 50 mMTris-Cl, 30% Glycerol, 2% SDS, 0.004% Bromophenol blue, and 1% DTT) for 15 min in a gel rocker.

Gel analysis
Digital images of 2D-gels were acquired using ChemiDoc™ XRS imaging system (Bio-Rad) with internal calibration. The acquisition parameters were 300 dpi and epi white illumination. Gel analysis was performed by adopting PDQuest software (Bio-Rad) for spot detection, according to manufacturer's protocol. Spot volume normalization, in the various 2-DE maps, was carried out using the relative spot volumes (% Vol). Initially, automatic spot detection was performed, followed by manual editing for spot splitting and noise removal.The gels containing the largest number of protein spots for each phase were chosen as the reference gels. All other gels were matched with the reference gel by placing user landmarks on approximately 10% of major and minor protein spots, which were visualized to assist in automatic matching. Finally, all matches were checked for errors editied by manually.

Data processing
The acquired mass spectra were processed using DataExplorer® software, and the monoisotopic peptide masses were assigned and used in the database search. The protein identification was analyzed against Homo sapiens protein sequence inMASCOT database search (http//www.matrixscience.com) using SWISS-PROT database entries. Modification of cysteine by carbamidomethylation and methionine by oxidation was allowed. The precursor and product mass tolerance were set as +/-50 and +/-100 ppm, respectively.
Two or more unique peptides for each protein were taken for confirmation of the protein present in the sample.

Functional annotation
The salivary proteins identified during the ovulation and post-ovulation phases were subjected to functional annotation using STRAP online database. The GO terms of proteins were classified based on the biological processes in which they participate, the cellular location and the molecular function. The results revealed that the ovulation phase had more number of GO terms and greater percentage of annotations than during the postovulation phase.

Molecular functional ontology
The GO entries were used to depict the percentage of proteins at molecular functional level through Interproscan analysis in BLAST2GO. The cloud tag image confirmed that the proteins identified in saliva of ovulation phase are essentially those with binding property and catalytic activity. Particularly, among the ovulation phase salivary proteins ( Figure   2A) showed up higher number of binding proteins (41.6%) and metal ion binding proteins (16.1%) compared to other phases ( Figure 2B). Further, the molecular functional network was constructed using GO terms of salivary proteins. The integrated network map revealed the proteins identifiedduring ovulation phase to have glycoprotein binding, ion binding, and immunoglobulin binding properties, also with receptor activity. Additionally, the molecular network analysis revealed the interaction between the identified proteins.

Statistical analysis
The protein concentration and band intensity values corresponding to ovulation, preovulation and post-ovulation phases were represented as mean ± SD and analyzed using one-way analysis of variance (ANOVA) using SPSS 16 software (SPSS Inc., Cary, NC, USA).

Validation of ovulation
It is necessary to assess the ovulation phase after appropriate screening of the menstrual cycle. Not less than 5 cycles were observed to confirm the length of the reproductive cycle of the volunteers prior to sample collection. Those women who exhibited normal 28 day cycle length through the 5 or more cycles were chosen to be the volunteers, and the saliva sample was collected. Ovulatory phase was confirmed from direct fertility marker viz., status of the follicle adopting ultrasonography, and biophysical fertility markers such as basal body temperature and fernpattern in saliva (Supplementary Figure.S1) [23].

HR-LC-MS/MS analysis
In order to identify the salivary proteins, the SDS-PAGE protein profiles of each phase were excised equally into six separate pieces, which were individually subjected to trypsin digestion followed by mass spectrometry analysis. In total, 781 proteins were identified combining ovulation and post-ovulation phases of menstrual cycle. During ovulation phase 530 proteins were found, whereas 251 proteins were found during postovulation phase.
These results clearly indicates that the expression of number of proteins during ovulation phase than post-ovulation phase. Among these proteins, 35 were commonly present during both ovulation and post-ovulation phases. The schematic diagram, Venny, developed by Oliverios (http://bioinfogp.cnb.es/tools/venny) revealed the protein identities in each phase ( Figure 1C).

Ovulation specific proteins
Several functionally important ovulation phase-specific proteins were identified and listed.

Gene ontology
The unknown, uncharacterized and hypothetical proteins were identified and removed.
Further, the repeated proteins in the protein datasets were removed. GO entries were predicted using the GI number of proteins in UNIPROT database. After refinement of datasets the ovulation and post-ovulation phases contained 154 and 117 GO entries, respectively. The gene ontology clearly revealed more UNIPROT entries and greater percentage of annotation during the ovulation phase than the other phases.
Scatter plot for binding proteins Overall, molecular function analysis exposed the proteins thus identified to possess binding property ( Figure 3A). The GO terms were used to analyze the enrichment with log size by adopting REViGO to predict the scatter plot of binding proteins of each phase of menstrual cycle. Scatter plotting was carried out with semantic X and Y axes corresponding to log size value and log10 p-value, respectively. The proteins were segregated and grouped according to their functions, which were displayed with different colors. During ovulation phase, the cluster of yellow bubbles corresponds to the receptor-, protein complex-, peptide-, GABA-binding and MAP kinase activities. The GPCR-, heat shock-, and hyaluronic acid binding and lipid phosphatase-, ligase-, and motor activity proteins shown in linear green bubbles. GABA-A receptor activity and receptor activity proteins are denoted in orange bubbles ( Figure 3B). Toll-like receptor-, fatty acid-, and lipid binding proteins, and protein transporter activity proteins during post ovulation phase are shown as separate clusters ( Figure 3C). These proteins might be having a functional significance during ovulation phase.

2D-Gel electrophoresis
The salivary protein expression profiles were assessed during pre-ovulation, ovulation, and post-ovulation phases of menstrual cycle (Figure 4A-C). In Commassie-stained 2D gels, protein spots were present in the pI range 4-7 and the molecular weight between 14-97 kDa. The spot analyses carried out during different phases using PDQuest software (Bio-rad) showed nearly 50 in each phase. Particularly, there were more number of spots during ovulation phase, and many of them had higher densities compared to the other phases. Additionally, we found that the quantitative differences in the spots were high in 14.5 to 21 kDa spot region of ovulatory phase proteins compared to the other phases. It was noticed that a few low molecular weight protein spots were specifically expressed during the ovulation phase ( Figure 5A)

Differential expression of salivary protein
Based on the visual assessment and multi-channel image analysis from PDQuest (Bio-Rad) protein spot positions during pre-and post-ovulation phases were unchanged. Though ovulation phase had a different protein expression compared to pre-and post-ovulation phases, 16 spots were identified as unique and highly expressed during this phase. These protein spot expressions were also validated in melanie 3D viewer ( Figure 5B).

Identification of proteins from selected spots
Sixteen spots with significant difference in intensities i.e., 2-fold increase, were excised from 2D gel of ovulation phase salivary proteins. The spots were subjected to trypsin digestion and submitted to mass spectrometry. The monoisotopic mass were searched in protein database of human from MASCOT search to identify the full length sequence. In case of spot 1, it closely matched with cystatin-S family protein with 71% sequence coverage and eight matched peptides ( Figure 6A & B, Table 2).. The differentially expressed spots were identified as (Table. 3): Cystatin-S, Prolactin-inducible protein, Cystatin-A,Cystatin-SN, BPI fold-containing family A member 2, Alpha-tubulin N-acetyltransferase 1, Carbonic anhydrase 6, Protein LEG1 homolog, Hemoglobin subunit beta, and Pancreatic alpha-amylase. The spots 1, 3, 4, 7, and 10 were identified as Cystatin-S and these spots might be isoforms/variants of Cystatin-S proteins (Table. 3)..

Discussion
Saliva is an important diagnostic biogical fluid, which reflects the physiological as well as biochemical changes in the body [24]. Human saliva has been subjected to proteomic analysis rather extensively adopting proteomic technique viz., two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and quadrupole time-of-flight mass spectrometry (Q-TOF/MS) [25] as well as comparative proteomic analysis on intra-and inter-person variability of whole saliva using LC/ESI-TOF/MS [26]. Detailed salivary proteomic studies during menstrual cycle adopting the 2D-based mass spectrometry have not been conducted earlier. However, recent studies have been carried out to comprehensively catalog the salivary proteome with regard to cellular localization, biological processes and molecular functions [27]. In our previous study, we have reported that the 14.5 kDa protein band is consistently present and we projected it as the ovulation phase-specific protein. Analysis of this band revealed that the Cystatin-S expression was significantly higher during the ovulation phase, which was further validated adopting immunoblotting, confirming that Cystatin-S is the predominant protein during ovulation [16].
In the present study, a complete proteomic catalogue on human saliva has been mapped with as much as 781 proteins during the major phases of menstrual cycle. Amongst them, 495 proteins are ovulation-specific, and 216 proteins are post-ovulation-specific. Many proteins reported in human saliva have been previously identified in humans under different physiological conditions. The functionally important proteins associated with reproductive mechanisms such as Cystatin-S, Disintegrin and metalloproteinase domain-containing protein 7 (ADAM7),TANK-binding kinase 1, Anoctamin-1,Carbonic anhydrase 6, and so on,appeared during ovulation phase. More specifically, Carbonic anhydrase is present during all phases of animals exhibiting the estrous cycle. Indeed, there is a significant expression of Carbonic anhydrase in cattle, camel and goat [28]. In the human, recent studies have confirmed that secretion of specific peptides/proteins is different in the pediatric age compared to adults [29].
The functional annotation of the identified proteins obtained from STRAP database showed that these proteins are associated with binding property and regulatory function. In addition, these extracellular proteins would play important role during the time of ovulation. It is to be noted that proteins having binding property are widely present along with volatiles in body fluids and facilitate chemical communication during the estrusperiod [30,31]. A more recent report showed that in estrus cycling mammals binding protein are abundant during estrus compared to the other phases [32]. It clearly indicates that there is a specific role for binding proteins during the time of ovulation. The occurrence of large number of binding proteins, during ovulation phase,assigning to molecular function is an aspect of defense response and immunological process. The binding proteins may have a role in increasing the stability of other proteins [32].
The salivary proteins have been shown as biomarkers of various diagnostic contexts [33].
The advantages of salivary proteins as biomarkers include high stability, easy availability with higher half-life and noninvasive collection procedures. The salivary glands are less influenced by unstimulated saliva and are also understood to represent an equilibrated condition. In contrast, in some cases it was suggested that stimulated saliva may provide a more accurate detection of biomarkers [34]. Several salivary biomarkers such as Lactoferrin, Beta-2-microglobulin and Cystatin for Sjögren's syndrome, C-erbB-2, and Epidermal growth factor (EGF) for breast cancer, and Lactoferrin for periodontitis and type 2 diabetes mellitus [35] have been listed using MS-based proteomic techniques. The 2D gel electrophoresis (2-DE) is capable of providing for better biomarker separation (based on both charge and mass) and, therefore, expounds a higher number of biomarkers. In most of the studies, 2DE is used as the first step for protein separation, followed by tandem MS (MS/MS). The whole saliva separated by isoelectric focusing showed most distinctive proteins at different pIs [36]. The outcome of the present study strongly agrees with the previous reports for the IEF separation between the pI 4-7. Recently, the 2DE analysis in periodontitis patients evidenced 15 altered spots out of 128. Further, a relationship that was uncovered between S100A8, S100A9 and S100A6 proteins during the periodontitis confirmed the potential involvement in periodontal inflammation [37].
Likewise, the present study revealed that 16 spots out of 62 were altered during the ovulation phase of menstrual cycle.
Since the composition of salivary proteins is influenced by physiological and environmental factors, they have the potential to access and monitor the diseases [38,24]. Indeed, the differences in protein expression have been well documented in pathological conditions such as cystic fibrosis, dental caries, and periodontitis [39,40]. So far, only limited proteomics studies have been carried out on human saliva with reference to variations in the different phases of menstrual cycle. On the other hand, proteomic analyses of saliva in ruminants are well established with regard to estrous cycle.
Electrophoretic separation revealed many distinct protein bands in sheep and goat parotid secretion [41,42]. A comparative salivary proteomic study was undertaken between goats and sheep [28], and goats and cattle [43]. In the more recent times, a odorant binding protein (OBP), which is around 19 kDa, in buffalo saliva with detailed post-translational modifications and estruslipocalin protein (14.5 kDa) was identified in house rat, which acts as shuttle for chemo-signals during communication with the conspecifics. Likewise, salivary proteomics is a promising tool for the discovery of biomarkers for various diseases. Currently, researchers are interested in developing biochemical-based and/or protein-based marker from saliva for the detection of ovulation time in the human.
In order to make an in-depth salivary proteome analysis the following steps were followed: i) avoid protein loss and get enough protein concentration, ii) a good protein separation to avoid complexity of proteins and to remove abundant proteins, and iii) high mass accuracy peptides sequencing with resolution in scanning [44]. As reported previously, the total concentrations of salivary proteins are low, and do not exceed approximately 2.5 mg/mL [45]. Generally, the concentration of proteins can be assessed by several techniques such as dialysis, lyophilization, reverse-phase separation, ultra-filtration, and enrichment by affinity columns or beads, precipitation with TCA acetone or ammonium sulphate is also advisable [46,47,48]. The salivary proteins were efficiently extracted using TCA-Acetone method followed by the method of Gehrke (2006) [17].Our previous study showed the 14.5 kDa protein specifically expounded to be the dominant protein in saliva [16]. Similarly, the present study also confirms that the proteins are in low molecular weight range highly expressed in ovulation phase compared to other phases.
Recent proteomic platforms have showed about 3000 differentially expressed proteins and peptides in human saliva, many of which are of microbial origin [49]. Similarly, in the present study most of the proteins present in saliva are antimicrobial and defensive proteins. The proteins around 14.5 kDa, such as Cystatin-S, Prolactin-inducible protein, Cystatin-A, Cystatin-SN, BPI fold-containing family A member 2, Alpha-tubulin Nacetyltransferase 1, and Carbonic anhydrase 6 express high during ovulation phase.
Among these proteins, 14.5 kDa Cystatin-S proteins were highly expressed as four isoforms falling in different pI ranges during ovulation phase. Further, the expression of Cystatin-S affirms the previous studies [16]. Likewise, Cystatin-A and cystatin isoforms were reported in gingival crevicular fluid in periodontal patients and chicken egg white during embryogenesis, respectively [50,51]. It is suggested that salivary glands respond to inflammation stimuli to secrete more Cystatin-C into saliva [50] and it has been considered as a biomarker candidate of renal function [52]. Alterations of serum Cystatin C were considered as early marker for hyperthyroidism, cancer, renal function in diabetic patients, and cardiovascular diseases [53][54][55][56]. Cystatin S is a promising tumor biomarker for early cancer diagnosis and treatment evaluation [57]. Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that participate in the regulation of ion, water, and acidbase balance by catalyzing the reversible hydration of carbon dioxide in a reaction: CO2 + H2O ⇔HCO3¯ + H+ [58]. In many body fluids, CA appears to regulate acid-base balance [59], and possible expression of isoenzymes is found in human and animal reproductive organs [60]. Remarkably, carbonic anhydrase is present during all phases of the estrous cycle of animals including cattle, camel and goat [28]. Additionally, the salivary proteins and microbiota as biomarkers for to assess the early childhood caries risk [61]. In the present study, the variations in protein expression indicate that physiological changes influence the protein secretion in saliva. Hence, the specific expression of Cystatin-S in saliva during the ovulation phase would lead to develop an important biomarker for the ovulation detection in human.

Conclusion
The protein expression pattern in human saliva was characterized during different phases of menstrual cycle. The total salivary proteome profile is listed and it was found that as many as 530 proteins appeared during ovulation phase as compared to 251 proteins appearing during post-ovulation phase. Functional annotation of identified salivary proteins revealed them as extracellular proteins associated with regulatory function.The differential protein expression observed following 2D gel electrophoresis revealed that the salivary proteins are focused between pH ranges 4-7 and molecular weight 97-14 kDa.
The unique and differentially expressed protein spots during ovulation phase were identified and confirmed. Of these proteins Cystatin-S, Prolactin-inducible protein, Proteins having at least one identified peptide in ovulation phase saliva are listed with their Swiss-Prot/TrEmbl accession numbers and length.
Functions were retrieved using the STRAP online database bioinformatics resource.
Theoretical pIs and monoisotopic molecular weights were calculated using the Swiss-Prot website. Table 2

Supplementary Files
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