Sample Collection
This study was approved by ethical review committee of Institute of Zoology, University of the Punjab. Pregnant females (n=260) in their second and third trimester were registered from public hospitals of Lahore (Sheikh Zaid Hospital, Jinnah Hospital and Lady Wellington Hospital). The purpose of the study was explained and informed consent clearly stating the study purpose was signed by the patients or their attendants. As the research involved human participants, it was performed in accordance with the relevant guidelines and regulations mentioned in the ‘Declaration of Helsinki’.
The risk factors for preterm birth were examined by the medical history of the patients. Before sample collection each patient was asked about smoking status, age, previous medical history, parity, gravidity and complete history of miscarriages and abortion (table: 1). Systolic and diastolic blood pressure was recorded before sample collection. The subjects of age less than 18 and more than 40 were excluded from the study. Subjects with complications during pregnancy like extensive bleeding, multiple miscarriages, fetal congenital irregularities, ectopic pregnancy and any other confounding pathology were also excluded.
At first, 350 pregnant females were registered in the study, fifty five females did not show any interest and withdrawn from the study. Thirty five females were not sure about their last menstrual period (LMP) date. Overall 260 females were followed till the end of their pregnancy. At their first visit, blood sample was taken and after delivery their total gestational age was noted. Out of 260, 150 females delivered preterm. First we separated them into second and third trimester samples based on time of sample collection. Further, we divided them into very preterm group (gestational age ≤ 32weeks) and extremely preterm group (gestational age ≤ 28 weeks). 100 females delivered at term with gestational age ≥ 37 weeks. Out of 150, 70 females were in the second trimester at the time of sampling and were considered as control I and 80 were in the third trimester at the time of sampling and considered as control II.
Demographic features
|
2nd trimester samples
|
|
|
3rd trimester samples
|
|
|
Controls (≥37 weeks)
|
very preterm (≤
32weeks)
|
Extremely preterm (≤ 28 weeks)
|
P-
VALUE
|
Controls (≥37 weeks)
|
Very preterm (≤ 28 weeks)
|
extremely
preterm (≤ 28 weeks)
|
P-
Value
|
Age (years)
|
26.5±5.468
|
26.67±6.623
|
28.5±2.074
|
0.7568
|
28.71±6.448
|
24.14±5.757
|
28.43±3.309
|
0.2278
|
Parity
|
0 =50 %
>0 =50 %
|
0 =0 %
>0 =100 %
|
0 = 25%
>0 = 75%
|
-
|
0 =36 %
>0 =54 %
|
0 =26 %
>0 =74 %
|
0 =0 %
>0 =100 %
|
-
|
Gravidity
|
1.250±0.50
|
2.429±1.512
|
2.500±1.00
|
0.2692
|
2.571±2.070
|
2.429±1.813
|
3.143±2.340
|
0.7969
|
Smoking
|
No
|
No
|
No
|
-
|
No
|
No
|
No
|
-
|
Baby sex
|
50% Male
50%female
|
28%Male
72%female
|
25%Male
75%female
|
NA
|
38%Male
62%female
|
42%Male
58%female
|
50%Male
50%female
|
NA
|
Table 1: The demographic features of the study group.
Serum Separation
Within 3 hours of phlebotomy the serum was separated. For serum separation, centrifugation of each blood sample was done for 10 minutes at 2500 rpm. Autoclaved cryovials were used for serum storage and labeled clearly with date and sample ID. For further processing, samples were stored at -80 ˚C.
Protein Estimation
For protein estimation, Bradford assay [9] was used. For quantification of protein, Bradford method is the highly specific and most accurate one. It is based on binding of coomassie blue
G250 with protein molecules and gives maximum absorption at 595nm.
Electrophoretic Analysis of Proteins
After protein quantification, 2-dimensional gel electrophoresis was done that involves first dimensional analysis by Iso-Electric Focusing (IEF) and second dimensional analysis by SDS PAGE [10].
For iso-electric focusing, IPG strips of 17cm, linear, 3-10pH with catalog No. 163-2007 of BioRad Company were used. By using rehydration buffer samples of 250 µg protein/ 300 µL were prepared. To avoid evaporation, mineral oil was overlaid after loading the sample during rehydration. For 12hrs at 50V, rehydration was done and then focusing was performed at 20°C with 10000V. After the completion of iso-electric focusing, the IPG strip was equilibrated in SDS equilibration buffers 1 and 2, respectively in shaker.
For second dimensional analysis, PROTEAN II XL cell (Bio-Rad) was used. On the inner plates of electrophoresis cell, 12% polyacrylamide gel was mounted. After shifting strip to the gel, it was over laid with 0.5% low melting point agarose. After that on the +ve end of the IPG strip, protein marker (15µL) was loaded. The electrophoretic cell was filled with running buffer. The gel was run firstly at 16mA and then at 35mA. After completion, the gel was transferred in fixative solution for overnight. Later on, staining of the gel was done in coomassie brilliant blue G250 as described by [11]. Finally, gel was destained in destaining solution.
Protein Identification
The proteins were identified by using peptide sequence databases through Mascot software search engine. Each sample was digested by trypsin to obtain peptides by set protocol [12]. After that Phase Nano-LC-MS/MS analysis was done and the peptides were separated by Shimadzu LC-20AD Nano nanoliter liquid chromatograph. Mass Spectrometry Detection was performed by ESI tandem mass spectrometer: TripleTOF 5600 (SCIEX, Framingham, MA, USA). Finally, the spectra obtained were analyzed to identify protein of interest using Mascot 2.3.02 (Fig. 1).
2D gel analysis by SameSpots
After identification by LC-MS/MS and Mascot, Samespots software was used to determine the volume and area covered by each protein. Statistical analysis of data was done by using ANOVA Tukey’s Post Hoc analysis.