Protein Extraction and Digestion: A Refined Protocol for Sperm Sample Preparation

Ten milliliters of original essence were subjected to centrifugation at 1,500g for five minutes. The precipitated sperm were washed thrice with phosphate-buffered saline (PBS) solution and centrifuged using the same method. The resulting sperm precipitate was preserved and stored at -80°C. Upon thawing on an icebox, the sperm precipitate was lysed, and proteins were extracted using a solution of sodium dodecyl sulfate (SDS) and Tris-HCl buffer (4% SDS, 100 mM Tris-HCl, pH 7.6). Protein content was quantified using a BCA protein assay kit (Bio-Rad, USA). Subsequently, 20 μg of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Blue R-250 staining for quality control testing.

Protein digestion was performed using trypsin following the filter-aided sample preparation (FASP) protocol. Briefly, 200 μg of protein were mixed with 30 μl of SDT buffer (4% SDS, 100 mM dithiothreitol [DTT], 150 mM Tris-HCl, pH 8.0). This mixture was filtered through a Microcon unit (10 kD) using a solution of 8 M urea and 150 mM Tris-HCl (pH 8.0) to remove low molecular weight components. Subsequently, 100 μl of 100 mM iodoacetamide (IAA) were added to block reduced cysteine residues, and the mixture was incubated in the dark for 30 minutes. The filter was washed thrice with UA buffer and twice with 25 mM ammonium bicarbonate (NH4HCO3) buffer. Finally, the protein suspension was digested overnight at 37°C with 4 μg of trypsin (Promega) in 40 μl of 25 mM NH4HCO3 buffer. The resulting peptides were collected as filtrate, desalted using a C18 filter (Empore SPE filter C18), vacuum concentrated, and reconstituted with 40 μl of 0.1% (v/v) formic acid for peptide quantification (OD280).