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"Centrifuge 10 ml of original semen at 1,500× g for 5 minutes. Wash the resulting sperm pellet with PBS solution three times using the same method, and then preserve the sperm pellet by storing it at -80 ℃. Thaw the sperm pellet on ice when needed. The sperm pellet is lysed using SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6) to extract proteins. Quantify the protein content using the BCA Protein Assay Kit (Bio-Rad, USA) and analyze the protein extract using 12.5% SDS-PAGE gel (constant current 14 mA, 90 min) and Coomassie Blue R-250 staining for quality control. Protein digestion is performed using trypsin according to the filter-aided sample preparation (FASP) protocol. In brief, each sample is mixed with 200 μg of protein and 30 μl of SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCI pH 8.0). The mixture is then filtered using Microcon units (10 kD) with UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) to remove low molecular weight components. Next, 100 μl of iodoacetamide (100 mM IAA in UA buffer) is added to block the reduced cysteine residues and incubated for 30 minutes in the dark. The filter is washed three times with UA buffer and twice with 25 mM NH4HCO3 buffer. Finally, the protein suspension is digested overnight at 37 ℃ using 4 μg of trypsin (Promega) in 40 μl of 25 mM NH4HCO3 buffer. The resulting peptides are collected as filtrate and then desalted using C18 Cartridges (Empore™ SPE Cartridges C18, bed I.D. 7 mm, volume 3 ml, Sigma), followed by vacuum concentration and reconstitution with 40 μl of 0.1% (v/v) formic acid for peptide quantification (OD280)."