非梗阻性无精子症和健康生育男性精浆差异蛋白质的“shotgun”鉴定

Identification of differential proteins in the seminal plasma of healthy fertile and non-obstructive azoospermia men by shotgun proteomic strategy

目的:利用"shotgun"蛋白质组学策略鉴定正常生育男性和非梗阻性无精子症男性精浆的差异蛋白质。方法:正常生育男性和非梗阻性无精子症男性各3例自愿捐献的精液标本,通过Percoll分离获取精浆,等量混合后在SDS-PAGE上进行分离,切胶取条带进行胶内酶解,抽提肽段后利用"shotgun"蛋白质组学策略鉴定蛋白质。然后,将两组鉴定到的惟一肽段数≥2和惟一肽段数=1,但肽段总数≥4的蛋白质对比分析。结果:正常生育男性和非梗阻性无精子症男性精浆中共得到213个差异蛋白质,其中133个蛋白质存在于非梗阻性无精子症者,80个蛋白质存在于正常生育者。这些差异蛋白质集中在信号转导、细胞骨架和酶催化活性类,其中酶催化活性类中尤以氧化还原酶类为多。通过进一步分析,有18个差异蛋白质尤其值得关注,这18个蛋白质中包括:胞质动力蛋白重链、脂肪酸合成酶、微管蛋白α6等。结论:本研究中鉴定到的二者的差异蛋白质数量多、功能分布广,不仅再次证明"shotgun"蛋白质组学策略是鉴定蛋白质的一个较好的方法,也提醒我们:非梗阻性无精子症的发生原因复杂,类型多样,选择标本必须从组织学和基因水平严格细化,方能得到蛋白质研究的可信结果。本研究选取的无精子症男性精浆标本从蛋白质水平上表明其病因与有丝分裂M期有关,具体机制尚不清楚。

Objective：To identify differential proteins in the seminal plasma of healthy fertile men and non-obstructive azoospermia patients by the shotgun proteomic strategy. Methods：Six seminal plasma samples from 3 healthy fertile and 3 non-obstructive azoospermia volunteers were collected by Percoll isolation,balanced-mixed,and followed by separation of the mixture by SDS-PAGE. The proteins were subjected to in-gel enzymolysis and isolation of peptide fragments,and then identified by the shotgun proteomic strategy. Then comparative analyses were made between the two groups on the identified proteins with the unique peptide count ≥2 and =1 but with the peptide count ≥4. Results：A total of 213 differential proteins were identified,133 in the non-obstructive azoospermia patients and 80 in the healthy fertile men. According to the molecular function,these differential proteins mainly fell into the types of signal transduction,cytoskeleton and catalytic activity,especially oxidoreductase activity in the latter type. Eighteen of the differential proteins were found to be of particular significance,including dynein heavy chain,fatty acid synthase,and tubulin alpha-6 chain. Conclusion：The differential proteins identified in this study were many in number and various in function,which not only demonstrated the value of the shotgun proteomic strategy in protein identification,but also suggested the complicated pathogenesis and varied types of non-obstructive azoospermia. The samples must be selected strictly based on their gene and histological types. Non-obstructive azoospermia was shown to be related with the M phase of the mitotic cell cycle at the protein level,but its specific mechanism remains unknown.