Protein Extraction and Digestion for Sperm Sample Analysis: A Detailed Protocol

Centrifuge 10 mL of original sperm at 1,500 x g for 5 minutes. The precipitated sperm is then subjected to three washes with PBS solution using the same centrifugation method. The sperm precipitate is preserved by storing it at -80 ℃. For subsequent use, the sperm precipitate is thawed by placing it on an ice box. Protein extraction and lysis are performed using SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6). Subsequently, protein content is quantified using the BCA Protein Assay Kit (Bio-Rad, USA). A 20 μg protein extract is run on a 12.5% SDS-PAGE gel (constant current 14 mA, 90 min) and stained with Coomassie Blue R-250 for quality control testing. Protein digestion is carried out using trypsin following the filter-aided sample preparation (FASP) protocol. Briefly, 200 μg of protein is mixed with 30 μL of SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCI pH 8.0) for each sample. The mixture is then filtered through Microcon units (10 kD) using UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) to remove low molecular weight components. Reduced cysteine residues are blocked by adding 100 μL of iodoacetamide (100 mM IAA in UA buffer) and incubating the mixture in the dark for 30 minutes. The filter is washed thrice with UA buffer and twice with 25 mM NH4HCO3 buffer. The protein suspension is finally digested with 4 μg of trypsin (Promega) at 37 ℃ overnight in 40 μL of 25 mM NH4HCO3 buffer. The resulting peptides are collected as filtrate, desalted using C18 Cartridges (Empore™ SPE Cartridges C18, bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 μL of 0.1% (v/v) formic acid for peptide quantification (OD280).