Updated FTP location. Sperm isolated from WT mice and PITHD1-deficient mice in triplicate were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants containing 100 µg protein each were reduced in 5 mM DTT at room temperature for 30 min and alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate buffer. After repeated sonication and vortexing, the proteins were digested with 1 μg of trypsin/Lys-C mix (Promega) for 16 h at 37 °C. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT6plex reagents for 1 h at room temperature. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using a Pierce High pH Reversed-phase Peptide Fractionation Kit (Thermo Fisher). Nine fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (ACN) in 0.1% triethylamine. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA.