Sperm Proteome Maturation in the Mouse Epididymis

Samples consisted of intact epididymides that were harvested from five euthanized adult male mice (Mus musculus). Each epididymis was then defatted, separated from the testis and vas deferens and cut into three segments, corresponding to the caput, corpus, and cauda epididymis, based on morphological analysis. The sperm from each tissue segment were then purified using repeated rounds of centrifugation and pellet resuspension, solubilized in SDS, and ran on a 1-dimensional SDS-PAGE for in-gel digestion. Extracted peptides were analyzed by nanoflow reverse phase liquid chromatography using a nanoAcquity LC system (Waters) coupled in-line with the linear trap quadrupole (LTQ) Orbitrap Velos instrument (Thermo Fisher Scientific). Mass spectrometry data were collected with the instrument operating in data dependent MS/MS mode and analysed using X!Tandem (The GPM; version 2009.10.01.1) searched against the NCBI Mus musculus protein fasta file (dated 3/4/2011, with 29617 entries). X!Tandem was searched with a fragment ion mass tolerance of 0.50Da and a parent ion tolerance of 2.0Da. Iodoacetamide derivative of cysteine was specified as a fixed modification, while oxidation of methionine was specified as a variable modification.