Protein Extraction from Placental Tissues

Proteins from placental tissues were extracted as described in a previous study.22 (link) Briefly, approximately 50 mg of each sectioned tissue was washed twice with PBS to remove the serum. Then, tissues were denatured in 8 M urea/1 M NH₄HCO₃ buffer and homogenized with a tissue homogenizer (60 Hz, Shanghai Jing Xin, China). Lysates were precleared by centrifugation at 13,000 ×g for 15 min at 4°C, and protein concentrations were determined by BCA assay (Pierce, Wisconsin, USA). Proteins were reduced using 5 mM DTT at 37°C for 1 h, alkylated using 15 mM iodoacetamide at room temperature in the dark for 30 min, and then another 2.5 mM DTT was added to quench the excess iodoacetamide at room temperature for 10 min. Then, sequencing-grade trypsin (Promega, Madison, WI; protein: enzyme, 100:1, w/w) was added to the samples and incubated at 37°C overnight in less than 2 M urea/0.25M NH₄HCO₃ buffer. Samples were centrifuged at 13,000 rpm for 15 min to remove any particulate matter and purified with a HLB column (Waters, Milford, MA, USA). Peptides were eluted from the HLB column in 60% ACN/0.1% TFA, and the peptide concentrations were measured using Nanodrop^TM one spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).