Sperm Proteome Maturation in the Mouse Epididymis

Mass Spectra Data Files: 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.

质谱数据文件：本实验样本取自5只经安乐死处理的成年雄性小家鼠（Mus musculus）的完整附睾。随后对每一例附睾开展脱脂处理，将其与睾丸、输精管分离，并基于形态学分析将其切割为附睾头（caput）、附睾体（corpus）与附睾尾（cauda）三个区段。随后对每一段组织中的精子进行纯化：通过多轮离心与沉淀重悬操作完成纯化，将精子溶解于十二烷基硫酸钠（SDS）中，随后进行一维SDS-聚丙烯酰胺凝胶电泳（1-dimensional SDS-PAGE）以进行胶内酶解。提取得到的肽段采用纳流反相液相色谱进行分析，实验使用沃特世（Waters）公司的nanoAcquity液相色谱系统，与赛默飞世尔科技（Thermo Fisher Scientific）的线性离子阱四极杆-轨道阱（LTQ）Orbitrap Velos质谱仪在线联用。质谱数据采集时，仪器以数据依赖性MS/MS模式运行；数据分析采用X!Tandem（The GPM，版本2009.10.01.1）完成，检索数据库为NCBI小家鼠蛋白质FASTA文件（发布于2011年3月4日，共包含29617条条目）。X!Tandem检索参数设置如下：碎片离子质量容忍度为0.50Da，母离子质量容忍度为2.0Da；半胱氨酸的碘乙酰胺衍生物修饰被设为固定修饰，甲硫氨酸的氧化修饰被设为可变修饰。