HPLC-MS was carried out using a protocol as described previously by Su et al. (54). Briefly, testes from wild-type and Zcwpw1−/− mice at PD14 were collected (three replicates). Testes were lysed in protein extraction buffer consisting of 75 mM NaCl, 50 mM tris (pH 8.2), 8 M urea, 1 mM NaF, 1% (v/v) EDTA-free protease inhibitor cocktail, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and 1 mM phenylmethylsulfonyl fluoride. The samples were then subjected to tandem mass tags labeling. Aliquots of the same samples were combined, lyophilized, resuspended, and then loaded onto an XBridge BEH130 C18 column (2.1 mm by 150 mm, 3.5 μm; Waters) with an UltiMate 3000 HPLC system at a flow rate of 200 μl/min. For MS evaluation, 30 fractions were sequentially resuspended in 0.1% formic acid, and an LTQ Orbitrap Velos mass spectrometer (Thermo Finnigan, San Jose, CA) coupled on-line to a Proxeon EASY-nLC 1000 was used for analysis.

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