Buffalo Cervico-Vaginal Fluid Proteomics with Special Reference to Estrous

BOR Papers in Press. Published on March 12, 2014 as DOI:10.1095/biolreprod.113.113852
Buffalo Cervico-Vaginal Fluid Proteomics with Special Reference to Estrous
Cycle: Heat Shock Protein (Hsp)-70 Appears to Be an Estrus Indicator1
Running title: BUFFALO CERVICO-VAGINAL FLUID PROTEOME
Subramanian Muthukumar3, Ramalingam Rajkumar4, Kandasamy Karthikeyan3, Chen-Chung
Liao5, Dheer Singh6, Mohammad Abdulkader Akbarsha7, Govindaraju Archunan2,3
3
Center for Pheromone Technology, Department of Animal Science, Bharathidasan University,
Tiruchirappalli, India
4
Proteomics Core Facility, Department of Biology and Chemistry, City University of Hong Kong, Hong
Kong, SAR
5
Proteomics Research Center, National Yang-Ming University, Taipei, Taiwan
6
Animal Biochemistry Division, National Dairy Research Institute, Karnal, India
7
Mahatma Gandhi-Doerenkamp Centre, Department of Animal Science, Bharathidasan University,
Tiruchirappalli, India
1
Supported by the Department of Biotechnology (Order No. BT/PR/10247/AAQ/1/364/2007), the Indian
Council of Agricultural Research (ICAR), and the instrumentation facility from UGC-SAP and DSTPURSE, Government of India. S.M. was supported by a Senior Research Fellowship from the Council of
Scientific and Industrial Research (CSIR), New Delhi. The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the
PRIDE partner repository with the dataset identifier PXD000620.
2
Correspondence: Govindaraju Archunan, Centre for Pheromone Technology, Department of Animal
Science, Bharathidasan University, Tiruchirappalli-24, Tamilnadu, India. Phone numbers : Tel: 91-4312407040; Fax: 91-431-2407045; E-mail address : [email protected]
ABSTRACT
Cervico-vaginal fluid (CVF) plays significant roles in coitus, sperm transport and implantation. It is
believed to be a good non-invasive biomarker for various diagnostic purposes. In this study, a
comprehensive proteomic analysis of buffalo CVF was performed during the estrous cycle in order to
document the protein expressions, utilizing SDS-PAGE, mass spectrometry, and immunoblot. The main
objective was to screen CVF of buffalo for one or more estrus-specific proteins. A total of 416 proteins
were identified in the CVF of both estrus and diestrus phases put together. Out of these proteins, 68 were
estrus-specific. The major physiological reflections of estrus CVF proteins appeared to be stress response,
immune response and metabolism. Eventually, the expression level of the heat shock protein-70 (HSP-70)
in the CVF during the estrus phase, as revealed in SDS-PAGE analysis, was higher than during diestrus.
That the protein we dealt with here was HSP-70 was confirmed in the immunoblot analysis. The findings
provide a potential lead for the evaluation of these proteins for estrus detection in buffalo since CVF
biomarker detection is a non-invasive technique. The mass spectrometry data of identified proteins have
been deposited at the ProteomeXchange with the identifier PXD000620.
Key words: Buffalo; cervico-vaginal fluid; estrous cycle; proteome analysis; Heat shock protein-70
1
Copyright 2014 by The Society for the Study of Reproduction.
INTRODUCTION
Interaction between sperm and the luminal microenvironment of the female genital tract plays a
sizeable role in mammalian sperm transport and fertilization. The molecular mechanisms of sperm
transport through the female genital tract have not yet been fully understood. During this transit, the
spermatozoa have to traverse different kinds of mucus secretions. One of these secretions, called the
cervico-vaginal fluid, is a complex biological fluid derived mainly from cervical secretion [1]. It plays an
essential role in mammalian reproduction [2] viz., immune surveillance against pathogens [3]. The
physical and chemical properties of bovine CVF clearly indicate that changes in composition of CVF
during estrous cycle are under hormonal control [4], since hormonal fluctuations influence the
biochemical properties of cervix [5].
In buffalo estrous cycle, estrus is a short period of time, and ovulation occurs at 11 hrs following
its onset [6]. It is important to note that estrus detection is a major problem in buffalo reproduction since
estrus signs are not well established. When compared to cow and other mammals the visual signs of
estrus are not prominent in the buffalo. Therefore, the buffalo estrus is denoted as a “silent heat” [7]. To
overcome this problem, presently several attempts have been made to devise methods to detect estrus in
buffaloes, but till date none of these methods is efficient or reliable [8]. Hence, there is a pertinent and
urgent need to find a modality to identify the precise time of estrus in buffalo for enhancing the success of
fertilization through artificial insemination. We have identified estrus-specific volatile compounds in
buffalo urine [9] and feces [8]. Development of a volatile-based kit, availing the knowledge thus gained,
to enable estrus detection in buffalo is in progress in our lab. Though the volatile compounds are
reportedly one of the reliable indicators to discern estrus [10], estrus-specific protein expression can be
considered as a better marker for estrus detection and which discerns the physicochemical changes in the
body fluids; these criteria are most accepted in diagnosis of cancer and various other diseases [11].
Considering the problem in buffalo estrus detection, it is important to identify specific proteins in body
fluids during estrus to develop a protein-based kit.
Presently, CVF analyses facilitate the identification of several human disease-related changes in
their bacterial flora [12, 13, 14] and also offers a potential diagnostic tool to predict maternal and fetal
health. Thus, a comprehensive catalogue of protein expression in CVF during estrous cycle would reveal
the proteins which are expressed specifically or highly at the time of estrus. Therefore, it will be of
advantage if the protein profiles of CVF and their expression during the estrous cycle, with special
emphasis to estrus-specific or estrus-indicating protein, is perceived in order to locate a marker protein to
detect estrus to successfully inseminate the buffalo. Thus far, there has been no direct report adopting
proteomics analysis with reference to differentially expressed proteins during estrus. On the other hand,
there is one report relating to the study of oviduct proteins in buffalo [15], but the study did not dissect the
phase-specific proteins. Therefore, we embarked on investigation of proteins in buffalo CVF during estrus
and diestrus. The current proteomic approaches enable the effective identification of proteins in complex
biological/body fluids [16, 17]. In the present study, for the first time, we show the proteomic profile of
buffalo CVF collected during estrus and diestrus. The SDS-PAGE was employed to fractionate the total
proteome and, subsequently, high resolution LC-MS/MS was used to decipher the total proteome.
MATERIALS AND METHODS
Experimental Animals
Buffaloes maintained at Veterinary College and Research Institute (VCRI), Namakkal
(11°9'44"N latitude and 78°9'37"E longitudes), Tamil Nadu, India, were used. Twelve heifer murrah
buffaloes, free from anatomical and/or reproductive disabilities and diseases, were chosen for the
investigation. The animals were maintained in separate 100 m2 paddock housing system. The animals
were tagged with a number in the ear for individual identification. The buffaloes were fed ad libitum with
green fodder @ 1.5 to 2 kg per day. All the experimental procedures were approved by the Institutional
Animal Ethics Committee of VCRI.
2
Estrus Observations
The animals were observed for six consecutive estrous cycles by trained personnel. The onset of
estrus was recorded based on the behavioral signs such as restlessness, frequent urination, sniffing of
vagina and flehmen behaviour by the male. The onset of estrus was further confirmed by cardinal visual
signs such as swelling and reddening of vulva, mucus discharge, mounting and also by trans-rectal
examination. The typical fern crystallization analysis of CVF was done and onset of estrus was noted as
day ‘0’ [18]. The mucus was collected by inserting a sterile swab into the cervical region and smeared
onto a glass slide. The smear was allowed to dry at room temperature and then directly observed by
microscope at 150X.
Sample Collection
CVF was collected for six consecutive estrous cycles of individual females during estrus and
diestrus [8, 18]. A portion of the samples was utilized for the fern crystallization analysis and the
remaining samples were immediately transferred to vials and maintained at -80°C for further analysis.
Sample Preparation and Protein Estimation
Frozen CVF was brought back to room temperature and homogenized using a glass homogenizer
to obtain a clear liquefied sample and, then, centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant
was collected and used for the protein estimation according to the modified protocol of Bradford [19].
SDS-PAGE Analysis
The 12% SDS-PAGE, as modified after Laemmli [20], was adopted. The protein profiles were
identified by running molecular mass reference standards (Bangalore Genie; cat. No. PMW-M) that
comprised of Phosphorylase 97.7, Bovine serum albumin 66, Ovalbumin 43, Carbonic anhydrase 29,
Soybean trypsin inhibitor 21.1 and Lysozyme 14.1 kDa. The supernatant of CVF equivalent of 40 µg
protein sample from each phase was loaded onto the gel, and electrophoresis was carried out with a
constant voltage current (50 V) at room temperature for 6 hr. The gel was rinsed with distilled water for 2
min and stained with 0.5 % Coomassie Brilliant Blue R-250, prepared in 40% methanol and 10% acetic
acid, at room temperature for 2 hr. The gel was then destained in a solution containing 40% methanol and
10 % acetic acid, until the appropriate background was obtained.
Trypsin in-Gel Digestion
Seven separate portions were excised from each of estrus and diestrus gel lanes and destained
using 100µL of 25mM NH4HCO3/50% (v/v) acetonitrile (1:1) by incubation at 37°C for 30 min. The
procedure was repeated until no stain was visible in the protein band and then dried in a Speed-Vac
(Savant, Germany). The gel slices were incubated in 100 mL of 2% β-mercaptoethanol/25mM NH4HCO3
for 20 min at room temperature in dark. For cysteine alkylation an equal volume of 10% 4- vinylpyridine
in 25mM NH4HCO3/50% acetonitrile was added. After 20 min incubation, the slices were soaked with
1mL of 25mM NH4HCO3 for 10 min, dried and then incubated overnight (~18 h) in 25mM NH4HCO3
containing 100 ng of modified trypsin (Promega, Germany). The digested samples were separated and
dried in a Speed-Vac. The preparation was resuspended in 0.1% formic acid immediately before mass
spec-analysis.
Mass Spectrometric Analysis
LTQ-Orbitrap (Discovery) hybrid mass spectrometer with a nano-electrospray ionization source
(ThermoElectron, San Jose, CA, USA) and coupled with nano-flow HPLC (Agilent Technologies 1200
series, Germany) was used for the analysis of all excised bands from the gels. The entire spectrometric
analysis was carried out with an Agilent C18 column (100 x 0.75 mm. 3.5µm particle diameters). The
mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The
flow rate of pump was optimized at 0.5 µL/min. The conventional MS spectrum (Scurvey Scan) was
3
acquired at high resolution (M/∆M, 60,000 full width half maximum) over the acquisition range m/z 2002000. The series of precursor ions were selected for the MS/MS scan. Further, the spectrum (CID
spectrum or MS/MS spectrum) was acquired for the fragment ions generated by collision-induced
dissociation.
Data Analysis
Xcalibur software (version 2.0. SR1) was used for analyzing the mass spectrometry data. Product
ion scans gathered from tandem mass spectrometry were involved in the database search software
SEQUEST (TURBO)
(http://sjsupport.thermofinnigan.com/project/product_support/xcal_layered_apps_turbosequest.htm) [21].
Peak lists were created from the products in the scan data (threshold set to 10,000) and these were
searched against the mammalian protein sequence database from NCBI (http://www.ncbi.nlm.nih.gov/).
Modification of cysteine by carboxymethylation and methionine by oxidation modifications was allowed.
The mass tolerance for the precursor peptide ions was set to 3.5 and the fragment ion tolerance was set to
1. Singly charged, doubly charged and triply charged peptides have higher cross-correlation score viz.,
1.9, 2.2 and 3.75, respectively, which give high confidence in terms of protein identification. More than
two unique peptides for each protein were taken for confirmation of the protein present in the sample.
Western Blotting
CVF from the two phases were loaded on 12% SDS-polyacrylamide gels. After SDS-PAGE, the
proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by semidry Western blotting
system (Bio-Rad). The following procedure was conducted using ECL Western blotting protein detection
kit (GE Amersham, product code: RPN2135). A mouse monoclonal HSP-70 (Santa Cruz Biotechnology)
antibody at 1:2000 dilution was used as primary blotting, and anti-rat IgG at 1:2000 dilution was used as
secondary antibody. The images were developed and fixed on X-ray film. The band intensity of HSP-70
expression in estrus and diestrus were analyzed using one-way analysis of variance (ANOVA) by SPSS
version 16 (SPSS Inc., Cary, NC, USA).
RESULTS
Estrus Validation by Individual Estrus Cardinal Signs
A recent study has proved that identification of the estrus phase is mandatory to fix the ovulation
period [6]. Thus far the available reports demonstrate the presence of volatile molecules that are
specifically expressed during the estrus phase, and these volatiles may play a significant role in the
buffalo reproduction [8, 9]. To accomplish this, totally 72 estrous cycles were observed in the present
study including the two major phases, estrus and diestrus. The percentage of overall observation of estrus
signs is denoted in the figure 1A. The majority of animals had well-pronounced CVF crystallization
(86.11%), intense vulva swelling (84.72%) and vulva reddening (77.77%). The mucus discharge and
mounting were observed as weak symptoms (36.11% & 6.14%). The CVF crystallization was found well
pronounced during estrus, which can be considered as main validation for estrus detection (Fig. 1B).
1D Gel Proteome Expression in CVF
After validation of estrus and diestrus phases, the CVF was collected. The total proteome was
fractionated by SDS-PAGE. The protein profile of estrus phase CVF was compared with that of diestrus.
In the two phases put together, totally 9 bands were revealed in Coomassie brilliant blue stained gel; their
molecular mass ranged between 15 and 133 kDa (Fig. 2A). The staining intensity of all bands was found
to be similar in the high molecular range, where as bands in the low molecular range viz., 42, 27, 22 and
15 exhibited a significant difference in the intensity (band area).
4
Mass Spectrometric Analysis and Functional Annotation
The CVF proteome representing the two phases were sliced into fourteen fractions and subjected
to mass spectrometric analysis (biological replicates). The results revealed totally 416 proteins from both
phases. Of these, as much as 127 proteins were specific to estrus phase and 63 proteins were specific to
diestrus phase and 113 proteins were present in both phases (Fig. 3). The identified proteins from estrus
phase CVF were utilized to retrieve their molecular function, and cellular component using STRAP
online database (http://www.geneontology.org/GO.tools) [22] with the input of proteins Uniprot ID. The
results of the ontology analysis of estrus proteins expounded the following Molecular functions: binding
activity 52%, catalytic activity 29%, enzyme regulatory activity 8%, structural molecule activity 5%,
others 5%, antioxidant 1% and Molecular transducer activity 1%(Fig. 4B). As regards Cellular
component, the classification revealed that 21% cytoplasmic proteins, 14% other components, 13%
extracellular region proteins, 12% cytoskeletol proteins, 10% nuclear proteins, 9% other intracellular
organelle and plasma membrane proteins, 5% endoplasmic reticulum proteins, 3% macromolecular
complex proteins, 2% chromosome proteins, 1% cell surface and 1% endosome protein (Fig. 4A).
Estrus-Specific Proteins
The results obtained in the mass spectrometry analysis helped us to map the phase-specific
proteins. From the 127 estrus-specific proteins identified, only 68 candidates were most reviewed in
Protein Database (PDB). Their biological functions were retrieved from DAVID database
(http://david.abcc.ncifcrf.gov/) [23], and the theoretical pIs and monoisotopic molecular weight were
calculated from Swiss-Prot database (http://web.expasy.org/compute_pi/) [24] and these are listed in
Table 1 as estrus-specific proteins. Among the identified proteins, heat shock proteins were expressed in
five different places in the SDS-PAGE (Table 1).
Western Blotting
We were set to examine further the 70 kDa protein present on the SDS-PAGE protein profile and
confirm it adopting mass spectrometry approach. To further understand the intrinsic properties of the
band, we ran the SDS-PAGE and transferred the proteins onto PVDF membrane, and then probed using
anti-HSP monoclonal antibody detection by ECL kit. The immunoblot revealed that the CBB-stained 70
kDa protein detected in the SDS-PAGE could be reliably identified as HSP-70. Interestingly, the Western
blot analysis showed that a significantly high level expression of HSP-70 was obvious in estrus CVF,
compared to diestrus CVF (Fig. 2B & 2C).
DISCUSSION
CVF plays a significant role in the process of reproduction [1]. Thus far, identification of buffalo
CVF proteins with special emphasis on estrous cycle has been sketchy. In the present investigation, we
collected the CVF of buffaloes representing estrus and diestrus phases, fractionated the proteome by SDSPAGE, and identified the proteins utilizing mass spectrometry which was further confirmed by
immunoblot. The buffalo cardinal estrus signs provided for identification of the estrus phase. We also
observed a higher percentage of vulva swelling and vulva reddening which are in line with a previous
report on cow [25]. On the other hand, mucus discharge and mounting were considerably less. In the
overall manifestation of estrus, the vulva swelling and the CVF crystallization were well pronounced,
which confirm the report of Deo and Roy [26]. Trans-rectal examinations were carried out for the
validation of estrus phase. It is interesting to note that the amount of mucus was high during estrus, and
the mucus was dilute and looked clear, which would offer lesser resistance for sperm to swim [27],
whereas during diestrus the mucus was scanty but dense. The crystallization patterning of the CVF took a
typical white colour when dried. SDS-PAGE revealed a much more significant difference in the
expression of the low molecular weight proteins between estrus and diestrus CVF. Recently, an estrusspecific urinary low molecular weight lipocalin protein has been identified in a rodent [28]. In order to
5
make a combinatorial and comprehensive protein study, we took the entire gel from each phase in two
replicates, made each into seven pieces each of estrus and diestrus CVF and subjected to tryptic digestion.
Further, each fraction was subjected to mass spectrometry analysis.
In this study, each protein was identified based on more than two unique peptide matches and
listed of the phases in estrous cycle. A total of 240 proteins were identified during estrus phase, whereas
only 176 proteins were identified during diestrus. In order to screen the specific proteins during the estrus
phase, the identified proteins from both phases were executed in Venny online tool. Interestingly, 127
proteins were found to be exclusive for the estrus phase. Thus, the results expounded that estrus phase
CVF has more proteins than the diestrus phase. Expression of more number of proteins during the estrus
phase substantiates a functional significance during the period in order to make condition for sperm
movement comfortable so as to facilitate fertilization. Further, we have classified the identified proteins
based on the intrinsic properties such as molecular function and cellular component of the identified
proteins.
The comparative proteomic analysis of buffalo CVF during the estrus phase revealed a large
number of proteins. The major functional groups identified in estrus CVF proteins are metabolic
molecules: ranging from proteases such as Glycogen phosphorylase, Pyruvate kinase, Alpha-enolase, Llactate dehydrogenase, Hypoxanthine-guanine phosphoribosyltransferase; protein- and ion-binding
proteins such as Plasminogen activator inhibitor type 1, ACTN1 protein, EF-hand domain-containing
protein D2, Resistin, HEAT repeat-containing protein 5B; immunologically responsive proteins:
Neurogenic locus notch homolog protein 1, Alpha-1 acid glycoprotein; and stress responsive proteins:
Heat shock 70 kDa protein 1B, Heat shock protein 70 cognate, Alpha-actinin-2, and Heat shock protein
beta-1. As far as immune response proteins are concerned, anti-inflammatory and antimicrobial molecules
appeared in the estrus CVF.
The appearance of a histone H2B in the estrus CVF of buffalo is unique. Basically, histones are
primarily involved in chromatin remodeling and are mostly limited to intra-cellular localization. Recent
reports indicate that extracellular neutrophils also contain histone molecules [29, 30]. The histone
molecules have further been reported to involve in antimicrobial activities [31, 32]. Therefore, the
presence of histone specifically in estrus CVF provides strong circumstantial evidence that this protein
would exert an antimicrobial effect during coitus.
The heat shock proteins, which are stress-response molecules, were expressed highly during
estrus in five different regions of the SDS-PAGE. This protein group, said to be a part of the molecular
chaperones, is involved in “house keeping” of the cells, and facilitates other proteins in transport, folding,
unfolding, assembly and disassembly of multi-structured units, and degradation of misfolded or
aggregated proteins [33]. For instance, the presence of HSP-70 in the cervical region during the estrus
phase of animals complicated with an infection induces the expression of interleukins [34]. This whole
comparative proteomic study provided a lead to discovery of a high level of expression of heat shock
proteins exclusively during estrus, and we have further proved the higher expression of HSP-70 in estrus
CVF using anti-mouse HSP-70 in the immnoblot analysis. Thus, we confirmed an intense expression of
heat shock protein 70 molecule during the estrus phase. It is well documented that HSP-70 is involved in
steroidogenesis and also in the assembly and traffiking of steroid receptors [35, 36]. HSP-70 acts as a
coactivator for nuclear estrogen receptor, thus regulating estrogen receptor-α activity in breast cancer
cells [37]. The expression of HSP-70 and heat shock transcription factor are under regulation by estrogen.
A significantly higher level of expression of HSP-70 was observed in epithelial cells during diestrus in
rat, when the maximum expression of estrogen receptor-α occurred in oviduct [38]. However, our study
showed that the extracelluar fluid CVF has higher expression of HSP-70 during estrus phase. Also,
hormonal changes in infundibulum and ampullar regions of rat ovidut were influenced by HSP-70, i.e., it
was abundant in the oviduct during preganancy, indicating that HSP-70 may modulate the estrogen- and
other pregnancy-related hormones [38].
Investigation on HSP-70, until now, has documented on inseparable relationship of HSP-70 with
reproduction process, and our present report also proves that HSP-70 expression is truly depended on the
phases of the estrous cycle. The very high expression of HSP-70 in estrus phase of buffalo CVF would
6
suggest a significant role to HSP-70 in the maintanance of healthy cervical region, to facilitate effective
fertilization. During estrus, the animal is amenable to coitus with the male partner. The cervix may have
microbial infection and/or the pro-inflammatory process during the ovulation, which may also be
considered as stresses, which would facilitate the influx of stress-response proteins to maintain a healthy
cervical condition to facilitate the fertilization purposes. Up-regulation of HSPs has been elaborately
studied in various stress issues such as high temperature [39], low temperature [40], radiation [41],
bacterial and viral infection [42], heavy metal exposure [43], oxidative stress [44], and physical activity
[45]. Thus, this is the first ever report showing very high expression of HSP-70 in CVF during the estrus
phase of buffalo. It is concluded that the molecules identified in the estrus CVF have many functions
including anti-microbial and anti-inflammatory, which are eventually important during the mating
scenario and in the medium for the sperm transportation.
CONCLUSION
This is the first report on buffalo CVF proteome centering around the estrous cycle. We report the
occurrence of a total of 416 proteins that have many biological functions with a comprehensive difference
between estrus and diestrus phases. The heat shock protein at 70 kDa was highly expressed during the
estrus phase when compared to diestrus, which is the most crucial time in the reproductive cycle to ensure
successful insemination. Now, we have found a new landmark avenue for estrus detection in buffalo,
making use of data on set of proteins during the estrus phase. The proteins HSPs and histone could be
considered as the marker proteins for estrus. Further studies to confirm these proteins to represent of
estrus phase in buffalo, are in progress.
ACKNOWLEDGMENT
We thank PRIDE Team for the processing and deposition of our mass spectrometry data into the
ProteomeXchange database. S.M. thanks Council of Scientific and Industrial Research (CSIR), New
Delhi, for the award of Senior Research Fellowship.
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9
FIGURE LEGENDS
Figure 1: Estrus cardinal signs of buffalo. (A) Histograms show the percentage of estrus signs around
seventy two estrous cycles. (B) Typical CVF crystallization of buffalo (air dried, X 150). (a) Compact and
continuous leafy arrangement of crystal during estrus phase. (b) Discontinuous and improper
crystallization during diestrus.
Figure 2: SDS-PAGE and Western blot of buffalo CVF. (A) Electrophoretic separation of CVF collected
during estrus and diestrus buffalo. M- Protein molecular markers, E- Estrus, D- Diestrus; each lane
contains 40 µg protein. (B) Shows the representative Western blot analysis of HSP-70 expression in CVF
of estrus (E) and diestrus (D). (C) Shows the representative western blot of HSP-70 band intensity of
CVF. The band intensity significantly high during estrus (E) compared to diestrus (D) using Fisher’s least
significant difference post-hoc comparisons (*p<0.05). Values are mean ± SE from six individuals.
Figure 3: Heat map profiling of buffalo CVF proteins. Shows the proteins identified during estrus and
diestrus phases, representing through its Uniprot ID (presence- deep black, absence- light gray).
Figure 4: Functional annotation of estrus proteins using STRAP database [22]. (A) Cellular component.
(B) Molecular function (all functions and component are represented as percentage of total proteins
identified).
10
TABLE 1: Estrus-specific CVF proteins of buffalo.
Swiss-Prot
acc.no.a
Protein description
Functionb
pIc
MWd
Total
peptides
identified
Q0VCM4
Glycogen phosphorylase, liver form
Metabolic process
6.7
97.32
5
Q5IST7
Heat-shock 70-kDa protein 5
(Fragment)
Plasminogen activator inhibitor type
1 (Fragment)
Phosphorylase
Nucleotide binding
-
-
6
Protein binding
-
-
3
Metabolic process
6.71
93
6
P35609
Alpha-actinin-2
Regulation of apoptosis
5.31
103.85
4
O97896
Oxidation reduction
-
-
2
A1L0V1
Xanthine:oxygen oxidoreductase
(Fragment)
ACTN1 protein (Fragment)
Ion binding
-
-
2
P04157
Leukocyte common antigen
B cell proliferation
5.6
140.7
5
P17879
Heat shock 70 kDa protein 1B
Response to stress
5.52
70.17
7
A5A8V7
Heat shock 70 kDa protein 1-like
Response to stress
6
70.34
8
Q07439
Heat shock 70 kDa protein 1A/1B
Catabolic process
5.6
70.05
3
B7Z6M1
cDNA FLJ50683, highly similar to
Plastin-3
WD repeat-containing protein 1
Protein binding
5.92
65.63
5
Sensory perception of sound
6.18
66.06
2
Q9NZM3
Intersectin-2
Endocytosis
8.32
193.46
5
A1YKV7
Response to stress
-
-
2
Peptidyl-serine
Phosphorylation
Unknown
6.35
286.1
2
9.76
22.42
3
P17066
Heat shock protein 70 cognate
(Fragment)
Leucine-rich repeat serine/threonineprotein kinase 2
cDNA FLJ57081, moderately similar
to WD repeat protein 1
Heat shock 70 kDa protein 6
Unknown
5.81
71.02
5
B6D983
Alpha-1 acid glycoprotein
Immune response
5.2
23.14
4
Q2HJ86
Tubulin alpha-1D chain
Microtubule
4.91
50.28
3
A6QPT4
MPO protein
Oxidation reduction
9.9
81.89
6
A6MK23
Cell redox homeostasis
-
-
5
Q3ZBD7
Disulfide-isomerase A3-like protein
(Fragment)
Glucose-6-phosphate isomerase
Gluconeogenesis
7.45
62.72
8
A5D984
Pyruvate kinase
Glycolysis
7.96
57.94
9
Q1KLB8
Protein disulfide isomerase
(Fragment)
Glucose-6-phosphate isomerase
(Fragment)
Putative tubulin-like protein alpha4B
Cytosol aminopeptidase
Cell redox homeostasis
-
-
2
Glycolysis
-
-
2
Microtubule
7.71
27.55
4
Proteolysis
6.07
56.28
6
Protocadherin alpha-7
Homophilic cell adhesion
5.05
97.74
7
Merlin
Cell adhesion
6.11
69.69
4
Q6R745
B4DUB7
O75083
Q5S007
B4DS71
Q95M65
Q9H853
P00727
Q9UN72
P35240
11
Protein description
Functionb
pIc
MWd
Total
peptides
identified
P05164-2
Isoform H14 of Myeloperoxidase
Oxidation reduction
9.11
67.07
4
Q5THJ4
Protein localization
6.15
491.84
8
Immune response
5.69
89.32
9
Phosphorylation
6.72
78.49
4
Q15084
Vacuolar protein sorting-associated
protein 13D
Neurogenic locus notch homolog
protein 1
Receptor-type tyrosine-protein
phosphatase epsilon
Protein disulfide-isomerase A6
Cell redox homeostasis
4.95
46.17
2
Q9XSJ4
Alpha-enolase
Glycolysis
6.37
47.19
2
A5A6I5
Fructose-bisphosphate aldolase A
Glycolysis
8.39
39.3
3
A6ZI47
Fructose-bisphosphate aldolase
Glycolysis
7.1
39.37
5
P11974
Pyruvate kinase isozymes M1/M2
Glycolysis
7.6
57.91
2
O70371
Lipocortin V (Fragment)
-
-
2
P31947
14-3-3 protein sigma
Negative regulation of
coagulation
Signal transduction
4.68
27.77
3
A0FH34
L-lactate dehydrogenase
Glycolysis
6.02
36.74
3
Q63610
Tropomyosin alpha-3 chain
Brain development
4.75
28.87
4
Q8QPC7
Nucleoprotein (Fragment)
Nucleic acid binding
-
-
2
P98106
P-selectin
5.54
79.08
5
A0A1F3
L-lactate dehydrogenase A chain
Positive regulation of cell
adhesion
Glycolysis
8.21
36.55
2
Q9BV35
Calcium-binding mitochondrial
carrier protein SCaMC-3
Transaldolase
Transmembrane transport
6.85
52.37
2
Pentose shunt
6.36
37.54
2
Fructose-bisphosphate aldolase
(Fragment)
Isoform 2 of 14-3-3 protein sigma
Glycolysis
-
-
2
Signal transduction
4.68
27.77
2
Unknown
-
-
2
Ubl conjugation pathway
6.25
148.65
8
Response to nutrient
5.34
40.31
4
Intermediate filament-based
process
Transport
-
-
2
6.85
52.37
4
Unknown
4.77
27.17
2
Metabolic process
6.59
24.4
2
Metabolic process
-
-
2
Glycolysis
7.95
57.8
5
Swiss-Prot
acc.no.a
P46531
B2GV87
P37837
A3FKT6
P31947-2
Q29219
Q5XPI3
P04899
A4IF59
Q9BV35-4
Q5VU59
P47959
Q64531
P14618-2
Tropomysin, cytoskeletal type
(Fragment)
E3 ubiquitin-protein ligase RNF123
Guanine nucleotide-binding protein
G(i) subunit alpha-2
Vim protein (Fragment)
Isoform SCaMC-3d of Calciumbinding mitochondrial carrier protein
Tropomyosin 3
Hypoxanthine-guanine
phosphoribosyltransferase
Hypoxanthine-guanine
phosphoribosyltransferase
(Fragment)
Isoform M1 of Pyruvate kinase
isozymes M1/M2
12
Protein description
Functionb
pIc
MWd
Total
peptides
identified
P47756
F-actin-capping protein subunit beta
Actin filament capping
5.36
31.21
3
Q3T149
Heat shock protein beta-1
Response to heat
5.98
22.39
2
P28783
Protein S100-A9
Chemotaxis
6.29
17.11
2
P17439
Glucosylceramidase
Metabolic process
7.32
55.49
3
Protein binding
5.01
26.62
2
P06899
EF-hand domain-containing protein
D2
Histone H2B type 1-J
Nucleosome assembly
10.32
13.77
2
Q762I5
Resistin
Protein binding
7.64
95.66
6
C9J4S4
Putative uncharacterized protein
RAB7A
HEAT repeat-containing protein 5B
Nucleotide binding
8.91
10.96
2
Binding
6.77
224.3
8
Swiss-Prot
acc.no.a
Q4FZY0
Q9P2D3
a
Proteins having at least two peptide identification in estrus buffalo CVF are listed with Swiss-pro/TrEmbl
accession number.
b
Functional annotation was retrieved using the DAVID database a bioinformatics resource [23].
c,d
Theoretical pIs (c) and monoisotopic molecular weight (d) were calculated using the Swiss-prot website [24].
13
FIGURE 1
FIGURE 2
14
FIGURE 3
15
FIGURE 4
16