目的 探索分离猪附睾头部精子释放蛋白的新方法，确定释放蛋白的功能特点及信号通路。方法 利用Transwell小室独特的共培养系统，充分利用0.4 μm聚碳酸酯膜分离精子释放的蛋白。使用同位素标记相对和绝对定量（iTRAQ）技术分析释放的蛋白，对鉴定到的差异蛋白，使用GO数据库描述蛋白功能，运用KEGG数据库Pathway分析，确定差异蛋白最主要的生化代谢途径和信号转导途径，同时用IPR数据库对差异蛋白的结构域进行非冗余分析。结果 Transwell小室上、下室共鉴定到542种蛋白，其中，464种蛋白表达量显著上调，78种蛋白表达量显著下调。差异蛋白主要参与跨膜转运、离子运输、ATP合成等生物过程。差异蛋白主要参与的信号通路为醛固酮调节的钠盐吸收、囊泡转运、EGFR酪氨酸激酶抑制等。硫氧还蛋白、半乳糖、糖苷水解酶等构成差异蛋白的主要结构域，为蛋白发挥作用提供结构基础。结论 Transwell小室分离蛋白的方法切实有效，iTRAQ技术在附睾头部精子释放蛋白中成功鉴定出差异蛋白，差异性分析及功能注释为探究差异蛋白参与的生命活动及功能奠定了基础。
Objective To explore a new method for isolating proteins released by spermatozoa of pig epididymis, and determine the functional characteristics and signaling pathways of released proteins.Method The unique co-culture system of Transwell chamber was used to separate proteins secreted by sperms through 0.4 μm polycarbonate membrane. The isotopically labeled relative and absolute quantitation (iTRAQ) technique was used to analyze the released proteins and identify the differential proteins.The most important biochemical metabolic pathway and signal transduction pathway of differential proteins were confirmed by GO database describing their functions and KEGG database analyzing their pathways. Meanwhile, IPR database was used to conduct non-redundancy analysis for the domains of differential proteins.Result A total of 542 proteins were identified in the upper and lower rooms of Transwell chamber, of which 464 proteins were significantly up-regulated and 78 proteins were significantly down-regulated. The differential proteins were mainly involved in biological processes such as transmembrane transport, ion transport and ATP synthesis. The main involved signaling pathways of differential proteins were aldosterone-mediated sodium salt uptake, vesicle transport, and EGFR tyrosine kinase inhibition. Sulfur-oxidized protein, galactose, glycoside hydrolase and other major domains that constituted differential proteins provided structural bases for protein functions.Conclusion The method of separating proteins using Transwell chamber is effective. iTRAQ technique successfully identifies the differential proteins in proteins released by spermatozoa in epididymal head. The difference analysis and function annotation lay the foundation for exploring life activities and functional roles that differential proteins involved.