**2.6. 2D gel electrophoresis**

The 2D-placenta proteome of the fetal and maternal sides of sPTL-IM was compared to their term controls, respectively. The purified tissue lysates from both case and control placentas (maternal and fetal sides) were diluted in a rehydration buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% biolyte 3–10, 20 mM DTT, 40 mM Tris, 0.002% (w/v) bromophenol blue to a final concentration of 266 μg, and a total volume of 250 μL. The protein samples were passively rehydrated at room temperature onto the 13 cm, pH 3–10 nonlinear IPG strips (GE Healthcare) for 16 h. Strips were overlaid with a dry strip cover fluid and focused using IPGphor II IEF system (Ettan IPGphor 3 IEF, GE Healthcare, USA) at 20°C and 50 μA/strip of maximum current limit using the following program: step and hold at 500 V for 500 Vh, gradient at 1000 V for 750 Vh, gradient at 8000 V for 11,250 Vh, and step and hold at 8000 V for 7333 Vh.

After completion of the first dimension IEF, IPG strips were equilibrated for 15 min at room temperature in a buffer containing 6 M urea, 50 mM Tris/HCl pH 8.8, 40% (v/v) glycerol, 2% (w/v) SDS, 0.02% bromophenol blue, and 65 mM DTT, followed by a 15-min incubation at room temperature in the same buffer containing 135 mM IAA in place of DTT. After equilibration, IPG strips were placed and sealed on top of a 1.0-mm thick 10% wide-range PAGE with 0.5% agarose containing 0.02% bromophenol blue dye for second dimension wide-range PAGE analysis. The gels were run using the SE 600 Ruby electrophoresis tank (GE Healthcare). Each gel was run at a constant current of 10 mA for 15 min at 15°C followed by 25 mA for the remainder of the run until the tracking dye migrated to within 1 cm of the bottom of the gel.

alkylated in 150 μL of 55 mM iodoacetamide in 100 mM ammonium bicarbonate at room temperature for 20 min in the dark. The gel plugs were dehydrated twice in 150 μL of 50% acetonitrile (20 min each time), then 50 μL of 50% acetonitrile (15 min); following acetonitrile

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers…

http://dx.doi.org/10.5772/intechopen.74925

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Each protein spot was "in-gel" digested in 25 μL of 7 ng/μL trypsin (Promega, cat# V5111) in 200 mM ammonium hydrogen citrate for overnight at 30°C; 25 μL of 50% acetonitrile was added to each protein spot, incubated for 15 min (room temperature), quick spin, trypsin digestion solutions were collected, and dried by vacuum centrifugation. The pellet was recon-

The resulting tryptic peptides obtained after in-gel digestion were analyzed by using a Bruker Ultraflexreme MALDI-TOF/TOF MS (Ultraflexreme; Bruker Daltonics GmbH, Breman, Germany) to give a peptide mass fingerprint and peptide sequence information. A solution of α-cyano-4-hydroxycinnamic acid (0.7 mg/mL) in 85% acetonitrile, 15% water, and 0.1% TFA was used as the matrix. Equal volumes of the matrix solution were mixed with tryptic digest peptides in 1:1 and spotted onto the MALDI plate (Achorchip plate). By routine, a standard peptide calibration mix in the mass range of 800–3200 Dalton (Bruker Daltonics, Leipzig,

MALDI-TOF/TOF mass spectra were acquired with smartbeam laser at 355 nm and operated in a positive and reflectron mode with 25-kV accelerating voltage; TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 5000 laser shots per fragmentation spectrum on each of the seven to 10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). The collision energy was set to

The resulting peptide mass fingerprints were submitted to a computer equipped with the MASCOT search program (Matrix Science Ltd., UK) (www.matrixcsience.com) for identification of the protein present in the gel spot. Searches were performed without constraining to the protein molecular weight or isoelectric point. MASCOT search parameters were set as follows: carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as a variable modification, trypsin as a proteolytic enzyme, residues up to one missed cleavage site with trypsin, and mass tolerance as 200 ppm. MS/MS spectra were searched as above using a peptide mass tolerance of 100 ppm and a fragment mass tolerance of ±0.2 Dalton. The protein identification database used was the National Center for Biotechnology Information (NCBInr), and the species selected for analysis was *Homo sapiens*. Results were scored using probability-based MOWSE scores (protein score is −10 × Log10 (*P*), where *P* is the probability that the observed match is a random event). Protein scores greater than 67 were considered significant (*P* < 0.05).

Student's *t*-test (SPSS version 19.0) was applied on 2D quantification data to compare and evaluate the statistical significance of targeted protein among the study groups. *P*-value less

removal, the gel plugs were completely dried by vacuum centrifugation.

stituted with 10 μL of 0.1% TFA for their mass spectrometric analysis.

Germany) was analyzed for external calibration of the mass spectrometer.

1 kV, and nitrogen was used as the collision gas.

than 0.05 was considered to be significant in both cases.

**2.9. Mass spectrometry**

**2.10. Data analysis**

To minimize gel-to-gel variation, placenta tissue lysates from both sPTL-IM and control placentas were ran simultaneously in a quart gel electrophoresis system (Ruby, GE Healthcare) for the second dimension and stained.
