Phosphoproteomics Analysis of Embryos

To perform phosphoproteomics, 30 embryos from the same group were randomly pooled together as one sample. The samples were lysed in SDT lysis buffer containing 4% (w/v) SDS, 100 mM Tris -HCl (pH 7.6) and 0.1M DTT. The samples were then centrifuged for 15 minutes at 16,000 × g at 4°C, and the protein concentration was determined by the BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA). A total of 200 mg of protein was digested using a filter-aided proteome preparation assay [57 (link)]. Digested samples were desalted by running through the C18 cartridge (Empore™ SPE Cartridges C18, bed I.D.: 7 mm, volume: 3 ml; Sigma, St. Louis, MI, USA) and dried by a SpeedVac (Thermo Fisher Scientific). A total of 100 μg of peptides in each sample were labeled using TMT reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Labeled peptides were fractionated by strong cation exchange (SCX) chromatography using the AKTA purifier system (GE Healthcare, Piscataway, NJ, USA). The enrichment of phosphopeptides was carried out using the sequential IMAC method by the High-SelectTM Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). After lyophilization, the phosphopeptides were resuspended in 20 μL of loading buffer (0.1% formic acid).