Indian Journal of Dental Research

: 2013  |  Volume : 24  |  Issue : 2  |  Page : 157--163

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS

S Alagendran1, G Saibaba2, S Muthukumar2, R Rajkumar2, RG Guzman3, G Archunan2,  
1 Department of Animal Science, Centre for Pheromone Technology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India; Department of Pharmacology and Physiology, Neurosensorial Physiology Laboratory, UNAM, AV. Universidad, Mexico; Department of Biochemistry, Periyar University, Salem, Tamil Nadu, India
2 Department of Animal Science, Centre for Pheromone Technology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India
3 Department of Pharmacology and Physiology, Neurosensorial Physiology Laboratory, UNAM, AV. Universidad, Mexico

Correspondence Address:
G Archunan
Department of Animal Science, Centre for Pheromone Technology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India


Context: Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. Aims: The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. Settings and Design: Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. Statistical Method Used: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. Materials and Methods: Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. Results: The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. Conclusions: Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.

How to cite this article:
Alagendran S, Saibaba G, Muthukumar S, Rajkumar R, Guzman R G, Archunan G. Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS.Indian J Dent Res 2013;24:157-163

How to cite this URL:
Alagendran S, Saibaba G, Muthukumar S, Rajkumar R, Guzman R G, Archunan G. Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS. Indian J Dent Res [serial online] 2013 [cited 2021 Apr 16 ];24:157-163
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Full Text

Accurate prediction of the time of ovulation is essential for the recovery of a mature oocyte for in vitro fertilization (IVF). The pre-ovulatory luteinizing hormone (LH) surge can be considered the most reliable hormonal change closely related to ovulation. With the use of a rapid LH assay and ovarian ultrasonography, it now appears possible to predict quite accurately the time of ovulation, provided that these technologies are appropriately applied. In recent years, attention has been paid to the biochemical importance of saliva. Hormones that might once have been estimated only through blood analysis are now known to be present in saliva though the quantities are comparatively less. [1] Hence, saliva is considered as the best non-invasive source for chemical and biochemical study including the protein analysis. [2],[3] Reports show that saliva is a very good source of both hormones and enzymes and that their levels change in accordance with the menstrual cycle. [4],[5]

Ovulatory function is commonly monitored by measuring various plasma peptides or steroid hormones. [5] The true fertile period in women only occurs when there is a viable ovum. Ovulation is the crucial event during the menstrual cycle, and it has been calculated that the maximum survival time for the ovum is probably less than 48 h. [6] The mid-cycle LH surge triggers a series of biochemical reactions that lead to follicular rupture and expulsion of the ovum. [7] Other pituitary hormones, prolactin and follicle stimulating hormone (FSH) are also released at this time, [8] but their role in ovulatory events remains uncertain. In addition to well-known patterns of estrogen and progesterone at ovulation, mid-cycle elevations of androstenedione and testosterone have been described; [9] although another report [10] has disagreed with this observation.

Numerous methods based upon the secretion of pituitary and ovarian hormones, or their effects upon responsive tissues, have been described. [11],[12] No single technique, however, is so convenient and effective that it has become a standard procedure in reproductive medicine. A need exists for a non-invasive method for evaluation of luteal function. A single assay of serum progesterone is not sufficient to characterize the duration or adequacy of luteal activity. [13] Attempts have been made to determine the possible effect of hormonal changes during the menstrual cycle on the secretion of saliva and its various components. The analyses have given somewhat contradictory results, probably mainly as a result of variations in the collection and treatment procedures of saliva samples. It should also be noted that some changes found in pure secretions viz., in submandibular saliva, have not been observed in whole mouth saliva.

The activity of alkaline phosphatase and protein increased during ovulation, though could not find any such increase. [14] On the other hand, Cockle and Harkness [15] observed that the activity of human salivary peroxidase increased significantly during ovulation. The amount of calcium and sodium has decreased in submandibular saliva during ovulation and pregnancy, whereas potassium has increased in serum. [16] Ben Aryeh, et al. [17] could not find any such changes in the whole saliva but they observed a significant increase in the concentration of phosphate during mid-cycle. The amount of sialic acid has decreased during ovulation. [18] An increase in the bacterial count in saliva occurs both during menstruation and at ovulation [19] and a decrease in the level of tissue hyaluronidase at menstruation has been found. [20]

Among the salivary components, peroxidase activity is of special interest because peroxidases have been shown to act as marker enzymes in estrogen-responsive tissues. [21] Results supporting this idea have been found in studies conducted on breast cancer, [21] rat uterus, [22] bovine milk, [23] human milk, [24] and yeast. [25] This study was carried out in order to analyze the possible changes in salivary proteins during normal menstrual cycle. The assay includes both products of salivary glands (α-amylase, peroxidase, and sialic acid) and products know to leak from blood (calcium, potassium, and thiocynate). Detailed knowledge of the possible cyclic variations in human saliva is very important because saliva is frequently sed as a diagnostic aid in the treatment of oral diseases. [26] Furthermore, the estimation of some salivary enzymes viz., peroxidase has been suggested to act as an easily applied method of ovulation detection. Currently, there is no single completely reliable parameter for ovulation prediction. Hormonal and clinical parameters that vary during a specific menstrual cycle are troublesome, and there is considerable variation from cycle to cycle even in the same patient. [27]

Saliva protein concentration is dependent on gland production, time of day, diet, age, gender, and presence of disease. [28] In term of protein composition, the main component is α-amylase, which represents 60% of total saliva protein content. Other saliva proteins are heat-shock protein, lactoferrin, immunoglobulins, carbonic anhydrase and albumin, a wide range of peptides that include cystatins, statherin, lysozyme, histatins, and a broad class of typical peptides mainly contributed by prolines labeled s proline-rich proteins. It is also possible to find small peptides due to the salivary proteolytic activity. [29] Components of saliva are secreted from a number of glands including the parotid gland, which in humans secretes a protein-rich fluid of low viscosity and are thus referred to as serous gland, and the submandibular and sublingual glands, which in humans secrete a carbohydrate-rich fluid of higher viscosity and are referred to as mucus glands. Despite these advancements, the primary structure and function of many of the salivary proteins still remain to be determined. A promising approach to the study of saliva is the identification of its protein components in reproductive periods using proteomic techniques. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS/MS) analysis, a mass spectrometer technique specifically designed for use in proteomic studies with the usual sensitivity and accuracy of a standard MALDI-TOF combined with automated MS and MS/MS spectra acquisition of tryptic digest peptides increasing the sensitivity and reliability of protein identification in biological samples. The aim of this study is to gain a better knowledge of saliva protein composition in order to identify possible markers of the ovulatory phase of the menstrual cycle.

From the present investigation, the exact time of ovulation may be predicted by which one can identify the fertile period. The need of prospective diagnosis of ovulation is important in fertility such as assisted reproduction. During the past decade, many indirect tests have been found which make the retrospective diagnosis of ovulation but the prospective diagnosis of ovulation is important for management. [30] A rapid, simple and precise LH assay would appear to be the most appropriate approach to predict ovulation in IVF and artificial insemination programs based on natural ovulation. Thus, prediction of ovulation is necessary; therefore, an attempt was made to detect ovulation through electrophoretic analysis of protein and nature of protein was confirmed through MALDI-TOF/MS analysis and the physical changes of fern pattern in saliva shows the estrogenic changes with salts they form cornified epithelial cells with hair-like structure in ovulatory phase during menstrual cycle in humans in order to predict ovulation. The aim of this study was to find the efficacy of salivary proteins in the prospective diagnosis of ovulation.

 Materials and Methods

Saliva sample collection

Twenty healthy non-smoking women from 20 to 30 years old were studied throughout three menstrual cycles (pre-ovulatory phase: 6-12 days, ovulatory phase: 13-14 days, and post-ovulatory phase: 15-26 days), as determined by history, basal body temperature charting, and saliva ferning test or luteal-phase progesterone measurements. Each one of them was asked to abstain from eating and drinking for 2 h before saliva collection and to rinse their mouth with sterile deionized water before saliva collection. Whole saliva was collected by spitting directly into a pre-chilled sterile 15 ml falcon tube. The samples were kept on ice during collection procedure. The physical appearance of the saliva was recorded and the volume of flow rate was also recorded. A protease inhibitor cocktail (Sigma, St. Louis, MO, USA) was added to the samples to prevent protein degradation during sample preparation. The saliva processing and storage from the time of collection were limited to 2 h.

Saliva protein precipitation method

The experiment was carried out as described above except that acetone and mercaptoethanol were added to the Trichloroacetic acid (TCA) and the washing procedure was modified. A mixture of 500 μl TCA, 20% acetone, and 90% β-mercaptoethanol (0.07%) was taken. The mixture was vortexed to mix thoroughly, then incubated overnight at –20°C, and centrifuged at 11,000 rpm with 4°C for 30 min. The supernatant was decanted and the pellet was washed twice with 200 μl of ice cold acetone containing 0.07% β-mercaptoethanol. The subsequent procedure was similar to Amado, et al.'s (2005) method for protein assay.

Protein assay

The protein pellets obtained were pre-treated with 10 μl of 0.2 M NaOH for 2 min at room temperature prior to the addition of 250 μl of rehydration buffer (7 M urea, 2 M thiourea, and 4% 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). This was because the pellets are fairly insoluble in acidic conditions. The solubilized protein precipitate was then left at room temperature for 1 h and vortexed periodically every 10 min, followed by centrifugation at 11,000 rpm for 10 min at 10°C to remove any insoluble materials. The supernatant collected was then used in the assay of protein content according to the Bradford (1976) [30] method measured by UV-Visible Spectrophotometer (PerkinElmer, 940 Winter street, Waltham, Massachusetts 02451, USA), with bovine serum albumin as a standard.

Single dimension SDS-PAGE analysis

Sample preparation for SDS-PAGE of salivary proteins

Prior to loading on the SDS-PAGE, saliva pellet was mixed with the 2 × sample buffer (1:1 ratio) with equal volume of the sample. [31] Laemmli buffer was (0.0250 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% β-mercaptoethanol) thoroughly mixed with vortex mixer CM 101 (REMI House, Goregaon (East). Mumbai - 400063. India) For few seconds and kept in water bath at 60°C for 60 sec, for the purpose of breaking the polypeptides. SDS-PAGE was performed on a mini-vertical electrophoresis system (Bio-Rad Mini-PROTEAN-3 Cell; Bio-Rad Laboratories, Hercules, CA, USA), in accordance with the discontinuous buffer system. [32] Salivary proteins were separated on a 12.5% separating gel (0.1% SDS, 1.5 M Tris-HCl, and pH 8.8) with a 5% stacking gel on top (0.1% SDS, 0.5 M Tris-HCl, and pH 6.8), under reducing conditions. Typically, 8-10 μl Sample solution containing 30 μg salivary proteins and it was loaded in the lanes. Electrophoresis was performed in electrode buffer (0.1% SDS, 0.25 M glycine, 0.025 M Tris-HCl, and pH 8.3) at 60 V for 10 min, and then switched to 120 V for 120 min at 37°C. Each experiment was repeated in three gels.

The salivary proteins molecular weight were identified by running molecular mass reference standards from Bangalore Genei (Cat No: PMW-M) containing phosphorylase, 97.4 kDa; bovine Serum albumin, 66 kDa; ovalbumin, 43 kDa; carbonic Anhydrase, 29 kDa; soyabean trypsin inhibitor, 20.1 kDa; and lysozyme, 14.3 kDa.

Staining methods

After SDS-PAGE, the gels were stained and destained according to the rapid Coomassie Brilliant Blue (CBB) R-250 staining method following the procedures reported. [33]

In-gel digestion and MALDI TOF/MS

Protein bands from SDS-PAGE were excised and each gel plug was destained using 100 ml of 25 mM NH 4 HCO 3 , 50% (v/v) acetonitrile (1:1) and incubated at 37 ° C for 30 min. This step was repeated until no stain was visible in the gel band. Gel pieces were sliced into small cubes and placed in 1.5 ml Eppendorf tubes. After drying in a Savant Discovery SpeedVac DSR6 (Metavac: 4000 Point Street, Holtsville, NY 11742, USA) the gel was incubated in 100 ml of 2% β-mercaptoethanol/25 mM NH 4 HCO 3 for 20 min at room temperature and in the dark. The same volume of 10% 4-vinylpyridine in 25 mM NH 4 HCO 3/ 50% acetonitrile was added for cysteine alkylation. After 20-min incubation, the gel was soaked in 1 ml of 25 mM NH 4 HCO 3 for 10 min, dried, and then incubated with 25 mM NH 4 HCO 3 containing 100 ng of modified trypsin (Promega) for overnight (18 h). The tryptic digest was removed from the gel, which was extracted with 300 ml of 25 mM NH 4 HCO 3 and 300 ml of 25 mM NH 4 HCO 3 /50% acetonitrile separately. These two fractions were combined together, dried in a Speed-Vac and then kept at −20°C for storage. It was re-suspended in 0.1% formic acid immediately before use. The MALDI-MS data were acquired on an Ultraflex TOF/TOF spectrometer (Bruker Daltonics, Billericia, MA, USA, and Bremen, Germany), equipped with a 50 Hz pulsed nitrogen laser (337 nm), operated in positive ion reflectron mode using a 90-ns time delay, and a 25 kV accelerating voltage. External calibration was done using the peptide I calibration standard supplied by Bruker (peptides include angiotensin II, angiotensin I, substance P, bombesin, Adrenocorticotropic hormone (ACTH) clip 1-17, ACTH clip 18-39, somatostatin 28; with masses ranging from 1000 to 3200 Da). The samples were prepared by mixing an equal amount of peptide (0.5 ml) with matrices, dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid saturated with 0.1% Trifluoroacetic acid (TFA) and acetonitrile (1:1). Masses below 50 m/z are not considered due to interference from the matrix.

Monoisotopic peptide masses were assigned and used in the database search. The protein identification was accomplished utilizing the Mascot search engine 31 (Matrix Science, London, UK). Scores >63 were considered to be significant (P < 0.05) in the Mascot search. Hence, proteins identified with scores less than the significant level were reported as unidentified. Criteria used for protein identification in the Mascot software were: (1) Significant homology scores achieved in Mascot; (2) significant sequence coverage values; and (3) similarity between the protein molecular mass calculated from the gel and the identified protein.


Protein content in saliva across normal menstrual cycle

This study revealed that ferning is due to the formation of NaCl crystals and the presence of mucins, which is associated with estrogen hormonal changes during the period of normal menstrual cycle [Figure 1]. Furthermore, we observed that the total salivary protein concentration is significantly higher in ovulatory phase (16.30 ± 0.95 mg/ml) when compared to the pre-ovulatory and post-ovulatory phases [Figure 2]. Paired t-tests revealed significant differences between ovulatory and pre-ovulatory (P < 0.01) and ovulatory and post-ovulatory phases (P < 0.001). No difference could be detected between pre-ovulatory and post-ovulatory phases (P > 0.05), suggesting a specific effect of ovulation on salivary protein concentration. Variation of protein concentration may be due to the formation of LH surge which mimics with estrogen at the day of pre-ovulatory phase. {Figure 1}{Figure 2}

Single dimension SDS-PAGE

Representative SDS-PAGE salivary protein profiles revealed six distinct proteins with molecular masses of 19, 21.5, 32, 39, 48, 91 kDa [Figure 3]. Further, the gel spots were used for in-gel digestion and protein identification through MALDI-TOF MS analysis followed by Mascot search [Figure 4]. Interestingly, among these proteins, a 48-kDa protein predominated during the ovulatory phase and not in pre-ovulatory and post-ovulatory periods. The results of this study suggest that the protein level and the presence of a 48-kDa band might be considered as indicators of ovulation.{Figure 3}{Figure 4}


The predominant proteins at 19, 48, and 91 kDa were excised and subjected to in-gel tryptic digestion and PMF. High-quality MALDI spectra were obtained for 48-kDa Protein [Figure 3] and [Figure 4]. The bands at 19 and 91 kDa were also processed in the similar manner. It was found that the 91-kDa protein showed nucleotide sequence similarly to a hypothetical protein. The 19-kDa protein was found to show similarity to the salivary lipocalin-1 protein. Finally, the 48-kDa protein was identified as UDP-N-acetylglucosamine pyrophosphorylase of Saccharomyces cerevisiae.

Data base search

The tryptic-digested 48-kDa band was fragmented and 14 peptides were obtained with observed masses viz., 324-331 (996.5 m/z), 379-387 (1104.6 m/z), 392-399 (1006.5 m/z), 484-492 (983.4 m/z), 530-535 (713.3 m/z), 536-544 (923.5 m/z), 593-600 (787.4 m/z), 729-736 (880.4 m/z), 773-780 (1026.5 m/z), 888-893 (722.4 m/z), 937-945 (1003.5 m/z), 977-986 (1140.5 m/z), 1023-1029 (890.4 m/z), and 1024-1031 (977.5 m/z) [Figure 4] and [Table 1]. These peptides were further analyzed with Mascot search and found to show 150 score having the first hit of UDP-N-acetylglucosamine pyrophosphorylase protein in saliva that had the accession number, AB011004-HUMAN. The predicted sequence coverage was ~68%.{Table 1}


This study reveals that the nature of saliva considerably varied depending upon the reproductive period. Saliva samples obtained during the ovulatory period revealed a clear ferning pattern which was not observed in either the pre-ovulatory or the post-ovulatory period [Figure 1]. Consequently, our results suggest a strong relationship between salivary ferning patterns and ovulatory phase of menstrual cycle which is in agreement with earlier studies. [34] However, unlike what was reported for human cervical mucus and bovine vaginal mucus, [35],[36] we did not find the appearance of ferning immediately while observing the saliva under the microscope but only after a slight warm up of about 8-10 min, suggesting that the formation of crystallization in human saliva may take more time. Ferning is caused by equal proportions of sodium and potassium ions, which cyclically increase under the influence of estrogen. [37] The ferning pattern of saliva is also a helpful indicator of the female ovulation period. The ferning is caused by NaCl, which cyclically increases under the influence of estrogen. Unconjugated estrogens are also present in human parotid and submandibular saliva, but at a concentration of only 1-7% of plasma levels. [38] The Rodent submandibular glands, human whole saliva, normal saliva, and inflamed human gingival are known to metabolize estrone to estradiol-17β. [39],[40] In adult rats, the distribution and size of the granular tubules in the submandibular gland are affected by estrogen. [41] Therefore, it is interesting that Cockle and Harkness (1978) have found a mid-cycle peak in human salivary enzymes, which coincided with the ovulatory estrogen peak. The present results show that the protein range was highest at ovulatory phase when compared to pre-ovulatory and post-ovulatory phases [Figure 2], this increase activity is compared with estrogen peak. Therefore, protein assay of saliva seems to be a reliable marker for ovulation prediction. The exact prediction of ovulation is becoming more important in the management of infertile women. There are numerous methods to predict ovulation; however, they often reveal expensive, inaccurate, or invasive. [42] Therefore, an attempt was made to detect ovulation through electrophoretic analysis of protein and fern pattern in saliva during menstrual cycle in human in order to predict ovulation.

In this study, the evaluation of the relationship between UDP-N acetyl glucotransferase and gonadal hormones showed the positive correlations with FSH and LH only when the cycle was ovulatory. Ovulation is a complex process initiated by the LH surge and controlled by the temporal and spatial expression of specific genes. Maturation of the pre-ovulatory follicle by the combined actions of FSH, estradiol, and various growth factors sets the stage for subsequent events that are essential for ovulation and fertilization. Inflammatory mediators and leukocytes play important roles in folliculogenesis and follicular rupture. Elevation of FSH is typically associated with both menstruation and ovulation [43] and changes in this hormone may explain the association with UDP-N acetyl glucotransferase that we found at ovulatory phase. This likely indicates that the ''periovulatory'' samples from many women with ovulatory cycles were not actually collected all that close to ovulation (i.e. the samples were collected on day 10 and the woman could have ovulated on day 14). These observations provide additional evidence in support of the hypothesis that ovulation is an inflammatory-like phenomenon [3]

A large number of lipocalin (low molecular weight protein, sex attractant) and acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin, and plasma amyloid protein, were identified in human follicular fluid [44] and rat urine [45] in relatively high concentrations supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. Our findings confirm the results of earlier investigations with regard to the good correlation between salivary protein patterns and the time of ovulation. These results suggest that in human saliva, the single constituents can be represented in different concentrations, each regulated by a particular hormonal equilibrium. The nature of proteins present in the ovulatory phase was confirmed through MALDI-TOF analysis as biomarkers for the detection of ovulation. The MALDI-TOF/MS of 48-kDa protein showed abundant intense peptides. They start with 324 and end with 1031 m/z, which correspond to the UDP-N-acetylglucosamine pyrophosphorylase sequence - AB011004-HUMAN [Figure 4], [Table 1]. The correlation of humoral anti-sperm antibodies with some cases of unexplained infertility in male and female patients suggests a role for these antibodies in blocking fertilization. In eukaryotes, UDP-GlcNAc is used in the GlcNAc moiety of N-linked glycosylation and the glycosylphosphatidylinositol anchor of cellular proteins. The tryptic-digested peptides have been assigned to the protein sequence of UDP-N-acetylglucosamine pyrophosphorylases. The expression of UDP-N-acetyl glucosamine pyrophosphorylases in ovulatory phase raises the possibility of relationship of this protein mimics with estrogen metabolism. From a clinico-chemical point of view, the reliable cyclic variations in hormones are important as a diagnostic aid in the prediction of ovulation. Identification of the 48-kDa protein may help establishing specific assays to be assessed for clinical use, hopefully leading to the identification of new ovulatory phase markers in saliva. Automated saliva sampling and analysis may be possible as a means of measuring the health status of women, but further work is necessary to identify the optimal techniques of gaining samples. Our results suggest that saliva is a promising and convenient model system for the development of new laboratory tests.


The study was partially supported by a grant from UGC-SAP and DST-FIST and DST-PURSE, New Delhi has kindly acknowledged. Dr. SA was awarded by DGAPA Post-doctoral Research associate in Neurosensorial fisiologia laboratorio, Faculty of Medicine UNAM, Ave. Universidad C.P 04510, Mexico D.F.


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