Sperm Sample Preparation and Protein Digestion for Mass Spectrometry Analysis

Centrifuge 10 ml of original semen at 1,500× g for 5 minutes. Wash the resulting sperm pellet thrice with PBS solution using the same centrifugation method, and preserve the pellet by storing it at -80 ℃. Thaw the sperm pellet on ice when required. Lyse the sperm pellet 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 assess the protein extract using 12.5% SDS-PAGE gel (constant current 14 mA, 90 min) and Coomassie Blue R-250 staining for quality control. Employ trypsin for protein digestion adhering to the filter-aided sample preparation (FASP) protocol. Briefly, combine each sample with 200 μg of protein and 30 μl of SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCI pH 8.0). Filter the mixture using Microcon units (10 kD) with UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) to eliminate low molecular weight components. Subsequently, add 100 μl of iodoacetamide (100 mM IAA in UA buffer) to block the reduced cysteine residues and incubate for 30 minutes in the dark. Wash the filter thrice with UA buffer and twice with 25 mM NH4HCO3 buffer. Finally, digest the protein suspension overnight at 37 ℃ using 4 μg of trypsin (Promega) in 40 μl of 25 mM NH4HCO3 buffer. Collect the resulting peptides as filtrate, desalt 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).