The phosphoprotein isolation from EC and healthy endometrium tissue samples was performed by Phosphoprotein Enrichment Kit (Pierce, Appleton, USA) [50 (link)]. About 5 mg of tissue from the EC or the healthy endometrium was homogenized in lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), NaCl 150 mM) with Phosphatase Inhibitor Cocktail Set II 1× (Millipore, Burlington, VT, USA) and 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine. Protein concentration was determined by Bradford assay. According to the manufacturer’s instructions, 3mg of tissue lysate was used for the phosphoprotein purification by immobilized metal affinity chromatography (IMAC).
3 EC was used to isolate phosphopeptides. Tissues were lysed, and proteins were digested with EasyPep Maxi MS Sample Prep Kit (Thermo Fisher, Boston, MA, USA). After protein digestion, peptides were transferred in dioxide (TiO2) spin tips (Thermo Fisher) for phosphopeptide enrichment, according to the manufacturer’s instructions.
3 EC was used to isolate phosphopeptides. Tissues were lysed, and proteins were digested with EasyPep Maxi MS Sample Prep Kit (Thermo Fisher, Boston, MA, USA). After protein digestion, peptides were transferred in dioxide (TiO2) spin tips (Thermo Fisher) for phosphopeptide enrichment, according to the manufacturer’s instructions.
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