Rhod-2 was excited at 543 nm and fluorescence emission was measured from 560 nm to 600 nm

with gestational diabetes mellitus were recruited for the GDM group. The diagnosis of GDM was based on American Diabetes Association criteria. To prevent potential interference from insulin or metformin, only diet controlled GDM women were recruited for the study. The control group consisted of twenty-five agematched healthy pregnant women with normal glucose tolerance during pregnancy. 6. Protein Visualization and Computer Analysis of Protein Spots 2-D gels were fixed in 50% methanol for 2 h, visualized by Coomassie Blue Colloidal Staining, and scanned using the ImageScanner system combined with LabScan software. All gel images were analyzed using ImageMaster 2D Platinum software. Gel images from each group were edited, and spots were matched manually. A unique identification number was assigned to matching spots on different gels. Normalization of the spot intensities was conducted according to the total optical density of the gel. Spots which had more than 2 fold change in relative spot volume were identified as significantly differential spots between gels. 3. Sample Collection To improve homogeneity and comparability, placentas selected for the study were from pregnant women that received Cesarean sections. Placenta villi of approximately 3 cm3 were obtained from five different intact cotyledons immediately following delivery. After the maternal decidual layer was removed, the tissue was quickly washed with ice-cold PBS, frozen in liquid nitrogen, and stored at 280uC for further protein or mRNA extraction. Samples for immunohistochemistry were fixed in 4% paraformaldehyde for three days and embedded in paraffin before sectioning. 7. In-gel Tryptic Digestion Spots from 2-DE gels selected for further analysis were excised using a blade and gel pieces were transferred to microfuge tubes. After rinsing with distilled water and destaining with potassium ferricyanide and sodium thiosulfate, the gel pieces were dehydrated in 100% acetonitrile. 2 mL of modified porcine trypsin in 25 mM ammonium bicarbonate, pH 8, was added to each sample then incubated at 37uC overnight. The trypsindigested solutions were collected and the remaining peptides were extracted from the gel pieces by incubating in 0.1% Trifluoroacetic acid/60% order 71939-50-9 acetonitrile for 15 min prior to drying in a vacuum centrifuge. 4. Protein Preparation for Two-dimensional Electrophoresis Eight placenta villi samples from the GDM group and eight from the NGT group were randomly chosen for two-dimensional electrophoresis. For each sample, a total of 300 mg placental tissue was disrupted in liquid nitrogen and solubilized in 1000 mL lysis buffer consisting of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v IPG buffer, 1% v/v PMSF and 1% v/v protease inhibitor cocktail. After incubating on ice for 2 hours, the sample was centrifuged at 16000 g for 30 min at 4uC to remove debris. The supernatant was collected and further purified using a ReadyPrep 2D Clean-up kit to improve 2DE gel quality by removing contaminating substances in the sample. Next, the concentrations of the purified protein samples were determined by Bradford protein assay according to the manufacturer’s instructions, using bovine serum albumin as standard. The purified protein samples were stored at 280uC until 2DE was performed. 8. MALDI-MS and MS/MS Analysis MALDI-TOF/TOF mass spectrometry measurements were performed using a Bruker Ultraflex III MALDI-TOF/TOF MS operating in reflectron mode with 20 kV acceleratin