2.9 Preparation of protein samples for LC-MS/MS

For LC-MS/MS sample preparation, 50 µg seminal EV protein from each biological replicate was pooled to produce 150 µg protein from each fertility group. Out of 150 µg stock solution, 50 µg of protein sample was digested and reduced with 5 mM tris(2-carboxyethyl) phosphine (TCEP). The protein sample was further alkylated with 50 mM iodoacetamide and digested with trypsin (1:50, Trypsin/lysate ratio), and then incubated for 16 h at 37°C. The dried pellet was resuspended in buffer A (2% acetonitrile, 0.1% formic acid) for downstream LC-MS/MS analysis.