Abstract: In order to explore the protein dynamic changes of buffalo sperm before and after capacitation，the total protein of buffalo sperm before and after capacitation was quantitatively analyzed by tandem mass tag（TMT） combined with liquid chromatography‑mass spectrometry（LC‑MS/MS） analysis technology.The results showed that there were 93 differentially expressed proteins before and after buffalo sperm capacitation，52 up‑regulated proteins and 41 down‑regulated proteins were expressed after capacitation. Differentially expressed proteins were involved in biological processes such as sexual reproduction，cytoskeleton，transport and spermatogenesis，and were also involved in peroxisome proliferators‑activated receptors（PPARs） and soluble NSF attachment protein receptors（SNARE）signaling pathways.The hub proteins（Hub）of the regulatory pathway screened by protein interaction network analysis included topoisomerase 2（TOP2A），enolase 3（ENO3）and so on.Western blot results showed that the expression levels of vesicle associated membrane protein 4（VAMP4）and apolipoprotein C‑3（APOC3）increased after capacitation of buffalo sperm.

Key words: Buffalo, Sperm capacitation, Tandem mass tag, LC?MS/MS, Proteomics

摘要： 为了探索获能前后水牛精子的蛋白质动态变化，通过串联质谱标签（TMT）结合液质联用（LCMS/MS）分析技术，对获能前后水牛精子的总蛋白质进行定量分析。结果表明，获能前后水牛精子的差异表达蛋白质共有93个，获能后表达上调蛋白质52个，表达下调蛋白质41个。差异表达蛋白质参与了有性生殖、细胞骨架、转运和精子发生等生物学进程，也参与过氧化物酶体增殖物激活受体（PPARs）和可溶性NSF附着蛋白受体（SNARE）等信号通路。经蛋白质互作网络分析，筛选出调节通路的中心蛋白（Hub）有拓扑异构酶2（TOP2A）、烯醇化酶3（ENO3）等。Western blot 结果表明，囊泡相关膜蛋白4（VAMP4）和载脂蛋白C 3（APOC3）在水牛精子获能后表达量升高。

关键词: 水牛, 精子获能, 串联质谱标签, 液质联用, 蛋白质组学