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Original Research
Characterization of salivary protein during ovulatory phase of
menstrual cycle through MALDITOF/MS
Alagendran S1,2,3, Saibaba G1, Muthukumar S1, Rajkumar R1, Guzman RG2, Archunan G1

Department of Animal
Science, Centre for Pheromone
Technology, Bharathidasan
University, Tiruchirappalli,
Tamil Nadu, India, 2Department
of Pharmacology and Physiology,
Neurosensorial Physiology
Laboratory, UNAM, AV.
Universidad, Mexico, 3Department
of Biochemistry, Periyar
University, Salem, TamilNadu,
India
1

Received
: 240512
Review completed : 121012
Accepted
: 221112

ABSTRACT
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(SDSPAGE) 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 SDSPAGE 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 preovulatory,
ovulatory and postovulatory 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 SDSPAGE.
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 SDSPAGE analysis showed seven different fractions of proteins is
from 1490 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 postovulatory phase. The present study indicates that the protein
level and the specific protein band (48 kDa) through MALDITOF MS analysis might serve as
an indicator for ovulation.
Key words: Human, matrixassisted laser desorption/ionizationmass spectorometry, menstrual
cycle, saliva, single dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis

Accurate prediction of the time of ovulation is essential for

the recovery of a mature oocyte for invitro fertilization(IVF).
The preovulatory 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
Address for correspondence:
Dr.Archunan G
Email:garchu56@yahoo.co.in
Access this article online
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Website:
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PMID:
***
DOI:
10.4103/0970-9290.116669

157

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 noninvasive 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
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Analysis of salivary protein using MALDI-TOF MS

cycle, and it has been calculated that the maximum survival

time for the ovum is probably less than 48h.[6] The midcycle
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 wellknown patterns of estrogen and progesterone at
ovulation, midcycle 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. Aneed
exists for a noninvasive method for evaluation of luteal
function. Asingle 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, etal.[17] could not find any such changes in
the whole saliva but they observed a significant increase
in the concentration of phosphate during midcycle. 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 estrogenresponsive 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 (amylase, peroxidase, and
sialic acid) and products know to leak from blood(calcium,
potassium, and thiocynate). Detailed knowledge of the
Indian Journal of Dental Research, 24(2), 2013

Alagendran, etal.

possible cyclic variations in human saliva is very important

because saliva is frequently used 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 amylase, which represents 60% of total
saliva protein content. Other saliva proteins are heatshock
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 as
prolinerich 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 proteinrich fluid
of low viscosity and are thus referred to as serous gland, and
the submandibular and sublingual glands, which in humans
secrete a carbohydraterich 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. Apromising approach
to the study of saliva is the identification of its protein
components in reproductive periods using proteomic
techniques. Matrixassisted laser desorption/ionizationtime
of flight(MALDITOF) 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 MALDITOF 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
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Alagendran, etal.

Analysis of salivary protein using MALDI-TOF MS

ovulation through electrophoretic analysis of protein and

nature of protein was confirmed through MALDITOF/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 nonsmoking women from 20 to

30 years old were studied throughout three menstrual
cycles (preovulatory phase: 612 days, ovulatory phase:
1314 days, and postovulatory phase: 1526 days), as
determined by history, basal body temperature charting,
and saliva ferning test or lutealphase progesterone
measurements. Each one of them was asked to abstain from
eating and drinking for 2h before saliva collection and to
rinse their mouth with sterile deionized water before saliva
collection. Whole saliva was collected by spitting directly
into a prechilled 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. Aprotease 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 2h.

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. Amixture of 500 l TCA, 20% acetone,
and 90% mercaptoethanol (0.07%) was taken. The
mixture was vortexed to mix thoroughly, then incubated
overnight at -20C, and centrifuged at 11,000 rpm with
4C for 30min. The supernatant was decanted and the
pellet was washed twice with 200 l of ice cold acetone
containing 0.07% mercaptoethanol. The subsequent
procedure was similar to Amado, etal.s(2005) method
for protein assay.

Protein assay

The protein pellets obtained were pretreated with 10 l of

0.2M NaOH for 2min at room temperature prior to the addition
of 250 l of rehydration buffer(7 M urea, 2 M thiourea, and
4% 3[(3Cholamidopropyl)dimethylammonio]1propane
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 11,000rpm for 10min at 10C to remove
any insoluble materials. The supernatant collected was
159

then used in the assay of protein content according to

the Bradford (1976)[30] method measured by UVVisible
Spectrophotometer (PerkinElmer, 940 Winter street,
Waltham, Massachusetts 02451, USA), with bovine serum
albumin as a standard.

Single dimension SDS-PAGE analysis

Sample preparation for SDSPAGE of salivary proteins
Prior to loading on the SDS-PAGE, saliva pellet was mixed
with the 2sample buffer(1:1 ratio) with equal volume
of the sample.[31] Laemmli buffer was(0.0250 M TrisHCl,
pH6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue,
and 10% mercaptoethanol) thoroughly mixed with
vortex mixer CM 101 (REMI House, Goregaon (East).
Mumbai 400063. India) For few seconds and kept
in water bath at 60C for 60 sec, for the purpose of
breaking the polypeptides. SDSPAGE was performed
on a minivertical electrophoresis system(BioRad
MiniPROTEAN3 Cell; BioRad 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 TrisHCl, and pH 8.8)
with a 5% stacking gel on top(0.1% SDS, 0.5 M TrisHCl,
and pH6.8), under reducing conditions. Typically, 810l
Sample solution containing 30 g 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 TrisHCl, and pH 8.3) at 60 V for 10 min, and then
switched to 120 V for 120min at 37C. 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: PMWM) containing phosphorylase,
97.4kDa; bovine Serum albumin, 66 kDa; ovalbumin,
43 kDa; carbonic Anhydrase, 29 kDa; soyabean trypsin
inhibitor, 20.1 kDa; and lysozyme, 14.3kDa.

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 SDSPAGE were excised and each

gel plug was destained using 100ml of 25 mM NH4HCO3,
50% (v/v) acetonitrile (1:1) and incubated at 37oC for
30min. 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 100ml of 2% mercaptoethanol/25 mM NH4HCO3 for
20min at room temperature and in the dark. The same
volume of 10% 4vinylpyridine in 25 mM NH4HCO3/50%
acetonitrile was added for cysteine alkylation. After 20min
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Alagendran, etal.

Analysis of salivary protein using MALDI-TOF MS

incubation, the gel was soaked in 1ml of 25 mM NH4HCO3

for 10min, dried, and then incubated with 25 mM NH4HCO3
containing 100 ng of modified trypsin (Promega) for
overnight(18h). The tryptic digest was removed from the
gel, which was extracted with 300ml of 25 mM NH4HCO3
and 300ml of 25 mM NH4HCO3/50% acetonitrile separately.
These two fractions were combined together, dried in
a SpeedVac and then kept at 20C for storage. It was
resuspended in 0.1% formic acid immediately before use.
The MALDIMS 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(337nm), operated in positive ion reflectron
mode using a 90ns time delay, and a 25kV 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 117, ACTH clip
1839, somatostatin 28; with masses ranging from 1000 to
3200 Da). The samples were prepared by mixing an equal
amount of peptide(0.5ml) with matrices, dihydroxybenzoic
acid and cyano4hydroxycinnamic acid saturated with
0.1% Trifluoroacetic acid (TFA) and acetonitrile (1:1).
Masses below 50m/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.

RESULTS
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[Figure1]. Furthermore,
we observed that the total salivary protein concentration
is significantly higher in ovulatory phase(16.300.95mg/
ml) when compared to the preovulatory and postovulatory
phases[Figure2]. Paired t tests revealed significant
differences between ovulatory and preovulatory(P<0.01)
and ovulatory and postovulatory phases(P < 0.001). No
difference could be detected between preovulatory and
postovulatory phases(P>0.05), suggesting a specific effect
of ovulation on salivary protein concentration. Variation
of protein concentration may be due to the formation
Indian Journal of Dental Research, 24(2), 2013

of LH surge which mimics with estrogen at the day of

preovulatory phase.

Single dimension SDSPAGE

Representative SDSPAGE 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[Figure4]. Interestingly, among these proteins, a
48kDa protein predominated during the ovulatory phase
and not in preovulatory and postovulatory periods. The
results of this study suggest that the protein level and the
presence of a 48kDa band might be considered as indicators
of ovulation.

MALDITOF/MS

The predominant proteins at 19, 48, and 91 kDa were

excised and subjected to ingel tryptic digestion and PMF.
Highquality MALDI spectra were obtained for 48kDa
Protein [Figures 3 and 4]. The bands at 19 and 91 kDa were
also processed in the similar manner. It was found that the
91kDa protein showed nucleotide sequence similarly to a
hypothetical protein. The 19kDa protein was found to show
similarity to the salivary lipocalin1 protein. Finally, the
48-kDa protein was identified as UDP-N-acetylglucosamine
pyrophosphorylase of Saccharomyces cerevisiae.

Data base search

The trypticdigested 48kDa band was fragmented and

14 peptides were obtained with observed masses viz.,
324331 (996.5 m/z), 379387 (1104.6 m/z), 392399
(1006.5 m/z), 484492 (983.4 m/z), 530535 (713.3 m/z),
536544 (923.5m/z), 593600 (787.4 m/z), 729736
(880.4 m/z), 773780 (1026.5 m/z), 888893 (722.4 m/z),
937945 (1003.5 m/z), 977986 (1140.5 m/z), 10231029
(890.4 m/z), and 10241031 (977.5 m/z) [Figure 4 and
Table1]. These peptides were further analyzed with Mascot
search and found to show 150 score having the first hit of
UDPNacetylglucosamine pyrophosphorylase protein in
saliva that had the accession number, AB011004-HUMAN.
The predicted sequence coverage was ~68%.

DISCUSSION
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 preovulatory
or the postovulatory period[Figure1]. 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
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Analysis of salivary protein using MALDI-TOF MS

Figure 1: Salivary ferning pattern across menstrual cycle, (a) Preovulatory phase (6-12 days) fern like appearance was formed due to ESH
stimulation; (b) Ovulatory phase (13-14 days) Estrogen fluctuation stimulates with salts like sodium ions. Fern like crystals was formed; (c)
Postovulatory phase (15-26 days), Fern like formation was decline due to formation of Luteinising hormone

Figure 2: Salivary protein concentration across the menstrual cycle.

Results represent the Mean SEM of three experiments in all phases. *P
< 0.05 significantly high at ovulatory phase when compare to other phases

Figure 3: Onedimensional electrophoresis of salivary protein during

menstrual cycle. M, marker; L1, preovulatory phase (612 days); L2,
ovulatory phase (1314 days); and L3, postovulatory phase (1526 days)

Figure 4: Mass spectrum of 48kDa protein (UDPNacetylglucosamine pyrophosphorylase). The sequence stretches that are covered by the
peptide ion signals (68% sequence coverage) in the mass spectrum are underlined in red color

but only after a slight warm up of about 810min, suggesting

that the formation of crystallization in human saliva may
take more time. Ferning is caused by equal proportions of
161

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
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Analysis of salivary protein using MALDI-TOF MS

Table1: The observed and molecular expected masses(M+H)
of 48kDa UDPNacetylglucosamine pyrophosphorylase
present in saliva during ovulatory phase(1314days) of
menstrual cycle
Start
324
379
392
484
530
536
593
729
773
888
937
977
1023
1024

End
331
387
399
492
535
544
600
736
780
893
945
986
1029
1031

Observed
996.5
1104.6
1006.5
983.4
713.3
1074.9
787.4
880.4
1026.5
722.4
1003.5
1140.5
890.4
977.5

Expected
994.25
1103.57
1004.25
982.45
712.69
1073.4
786.94
879.48
1025.48
721.56
1002.3
1139.57
889.35
976.24

Sequence
MNINDLK.L*
M.NINDLKLTLSK.A*
K.AGQEHLLR.F
K.NVDARMEPVPR.E
R.MEPVPREVLGSATR.D
K.VAVLLLAGGQGTR.L*
K.GMYDVGLPSR.K
K.GMYDVGLPSRK.T
K.TLFQIQAER.I
K.CIIPWYIMTSGR.T
R.TMESTK.E
K.YFGLKK.E*
VADKEFHAPL*
IIDENGVHEL.VKNGI

*Matched peptides

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 17% of plasma levels.[38] The Rodent
submandibular glands, human whole saliva, normal saliva,
and inflamed human gingival are known to metabolize
estrone to estradiol17.[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 midcycle 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 preovulatory and postovulatory phases[Figure2], 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
UDPN 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 preovulatory 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
Indian Journal of Dental Research, 24(2), 2013

hormone may explain the association with UDPN 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 inflammatorylike
phenomenon[3]
A large number of lipocalin(low molecular weight protein,
sex attractant) and acutephase 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 MALDITOF analysis as biomarkers
for the detection of ovulation. The MALDITOF/MS of
48kDa protein showed abundant intense peptides. They
start with 324 and end with 1031 m/z, which correspond
to the UDPNacetylglucosamine pyrophosphorylase
sequence AB011004HUMAN [Figure 4, Table 1]. The
correlation of humoral antisperm antibodies with some cases
of unexplained infertility in male and female patients suggests a
role for these antibodies in blocking fertilization. In eukaryotes,
UDPGlcNAc is used in the GlcNAc moiety of Nlinked
glycosylation and the glycosylphosphatidylinositol anchor
of cellular proteins. The trypticdigested peptides have been
assigned to the protein sequence of UDPNacetylglucosamine
pyrophosphorylases. The expression of UDPNacetyl
glucosamine pyrophosphorylases in ovulatory phase raises
the possibility of relationship of this protein mimics with
estrogen metabolism. From a clinicochemical point of view,
the reliable cyclic variations in hormones are important as a
diagnostic aid in the prediction of ovulation. Identification of
the 48kDa 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.

ACKNOWLEDGMENTS
The study was partially supported by a grant from UGCSAP and
DSTFIST and DST-PURSE, New Delhi has kindly acknowledged.
Dr. SA was awarded by DGAPA Postdoctoral Research associate in
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Analysis of salivary protein using MALDI-TOF MS

Neurosensorial fisiologia laboratorio, Faculty of Medicine UNAM,

Ave. Universidad C.P 04510, Mexico D.F.

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How to cite this article: Alagendran S, Saibaba G, Muthukumar S, Rajkumar
R, Guzman RG, Archunan G. Characterization of salivary protein during
ovulatory phase of menstrual cycle through MALDI-TOF/MS. Indian J Dent
Res 2013;24:157-63.
Source of Support: Nil, Conflict of Interest: None declared.

Indian Journal of Dental Research, 24(2), 2013