**2.7. Protein visualization, statistical analysis, and spot detection**

Case and control 2D gels (maternal and fetal sides) were visualized by silver nitrate [14] or colloidal Coomassie blue staining [15]. After staining, silver-stained 2D gels were scanned using the Image Scanner III imager (Image scanner III, GE Healthcare, USA). Gels stained with colloidal Coomassie blue were kept in 1% (v/v) acetic acid at 4°C until spot picking for protein identification.

All gel images were imported, and the protein spots were quantified by using ImageMaster 2D platinum software, version 7 (GE Healthcare Amersham Biosciences) program for comparative analysis. Protein spots were quantified in terms of their relative volume where the individual spot volume is divided by the total volume of the whole set of gel spots.

2D-gel images of sPTL-IM (maternal and fetal sides) were compared with term controls (maternal and fetal sides), respectively. Automatic gel image alignment was applied first and adjusted manually. Every single spot detected by the software was reviewed, and artifacts were excluded. To avoid variation in analysis, the same parameters were used for each gel. Besides, a single master gel image with the best quality and containing all the spots was prepared in each group as the reference gel. Normalization of the spot intensities was conducted according to the total optical density of the gel. After determining the percentage of intensity for each spot, the mean intensity of each spot between the cases and controls was analyzed by Student's *t*-test. Only protein spots with complete reproducibility across the sample set, expression intensity more than twofold changes (increase or decrease) as well as the *P*-value less than 0.05 were considered statistically significant for protein identification.

### **2.8. In-gel tryptic digestion**

The protein spots from colloidal Coomassie blue-stained wide-range PAGE selected for further protein identification were excised manually using a sterile surgical blade. These gel plugs were cut into smaller pieces and transferred to a microcentrifuge tube filled with gel plaque storage buffer (7% (v/v) acetic acid and 10% (v/v) methanol) and stored at 4°C until in-gel digestion.

Gel plugs were washed with 150 μL of 100 mM ammonium bicarbonate for 10 min, de-stained and dehydrated in 150 μL of 50% acetonitrile in 50 mM ammonium bicarbonate for five times (15 min each time), and rehydrated in 150 μL of 10 mM DTT in 100 mM ammonium bicarbonate at room temperature for 30 min. DTT solution was removed and the gel plugs were 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 removal, the gel plugs were completely dried by vacuum centrifugation.

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

### **2.9. Mass spectrometry**

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.

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)

Case and control 2D gels (maternal and fetal sides) were visualized by silver nitrate [14] or colloidal Coomassie blue staining [15]. After staining, silver-stained 2D gels were scanned using the Image Scanner III imager (Image scanner III, GE Healthcare, USA). Gels stained with colloidal Coomassie blue were kept in 1% (v/v) acetic acid at 4°C until spot picking for

All gel images were imported, and the protein spots were quantified by using ImageMaster 2D platinum software, version 7 (GE Healthcare Amersham Biosciences) program for comparative analysis. Protein spots were quantified in terms of their relative volume where the

2D-gel images of sPTL-IM (maternal and fetal sides) were compared with term controls (maternal and fetal sides), respectively. Automatic gel image alignment was applied first and adjusted manually. Every single spot detected by the software was reviewed, and artifacts were excluded. To avoid variation in analysis, the same parameters were used for each gel. Besides, a single master gel image with the best quality and containing all the spots was prepared in each group as the reference gel. Normalization of the spot intensities was conducted according to the total optical density of the gel. After determining the percentage of intensity for each spot, the mean intensity of each spot between the cases and controls was analyzed by Student's *t*-test. Only protein spots with complete reproducibility across the sample set, expression intensity more than twofold changes (increase or decrease) as well as the *P*-value

The protein spots from colloidal Coomassie blue-stained wide-range PAGE selected for further protein identification were excised manually using a sterile surgical blade. These gel plugs were cut into smaller pieces and transferred to a microcentrifuge tube filled with gel plaque storage buffer (7% (v/v) acetic acid and 10% (v/v) methanol) and stored at 4°C until in-gel digestion.

Gel plugs were washed with 150 μL of 100 mM ammonium bicarbonate for 10 min, de-stained and dehydrated in 150 μL of 50% acetonitrile in 50 mM ammonium bicarbonate for five times (15 min each time), and rehydrated in 150 μL of 10 mM DTT in 100 mM ammonium bicarbonate at room temperature for 30 min. DTT solution was removed and the gel plugs were

individual spot volume is divided by the total volume of the whole set of gel spots.

less than 0.05 were considered statistically significant for protein identification.

for the second dimension and stained.

6 Electrophoresis - Life Sciences Practical Applications

protein identification.

**2.8. In-gel tryptic digestion**

**2.7. Protein visualization, statistical analysis, and spot detection**

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

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

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).

### **2.10. Data analysis**

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 than 0.05 was considered to be significant in both cases.
