Fresh ejaculate was collected from a healthy, 27-year-old Caucasian male and immediately spun down at 13,000 g for 5 minutes at 4°C to separate seminal fluid from spermatozoa. Phenylmethylsulphonylfluoride (PMSF, 0.2 mM), benzamidine (0.1 mM), and 1 μg/ml each of aprotinin, leupeptin, and pepstatin (Sigma, St. Louis, USA) were added to the sample to avoid digestion by powerful proteases present in seminal fluid. To ensure complete separation of cell debris or occasional spermatozoa from seminal plasma, the sample was centrifuged at 100,000 g for 30 minutes at 4°C. Protein concentration was assessed by Coomassie Plus assay (Pierce, Rockford, USA) and 1 mg protein was resolved on 10% NuPAGE Novex Bis-Tris gel (Invitrogen, Carlsbad, USA). The gel was cut into 14 pieces and subjected to standard in-gel trypsin digestion protocol [39 (link)]. Briefly, the pieces were washed twice with 25 mM ammonium bicarbonate/50% ethanol, dehydrated with absolute ethanol, reduced for 1 hour at 56°C with 10 mM dithiothreitol (DTT), alkylated for 45 minutes in the dark with 55 mM iodoacetamide. After extensive washing with ammonium bicarbonate and dehydratation, the 12.5 ng/μl trypsin solution (modified sequencing grade; Promega, Madison, USA) was added and the enzyme was allowed to function overnight at 37°C. The peptides were extracted with 30% acetonitrile, 3% trifluoroacetic acid (TFA) and the organic solvent was evaporated in a vacuum centrifuge. TFA was added to the final concentration of 2% and stop-and-go extraction tip purification was performed as previously described [40 (link)].
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