我国利什曼原虫伽师株有毒力和无毒力前鞭毛体定量蛋白质组学比较分析

Comparative proteomics analysis of the virulent promastigotes and avirulent promastigotes of a Leishmania strain from Jiashi, Xinjiang, China

目的应用定量蛋白质组学方法比较我国利什曼原虫伽师株有毒力和无毒力前鞭毛体蛋白表达的差异。方法将分离于我国新疆伽师县黑热病患者婴儿利什曼原虫JS5有毒力株前鞭毛体和无毒力株前鞭毛体经酶解后进行液相色谱串联质谱(Liquid chromatography-tandem mass spectrometry, LC-MS/MS)的非标记(Label-free)定量分析,利用MaxQuant软件查库,进行Label-free非标定量(LFQ)分析;每个样品重复3次质谱分析,只有1次有LFQ值的蛋白舍弃。结果本研究共鉴定蛋白3 994个,有毒力和无毒力株间差异蛋白为298个(差异倍数>1.2或差异倍数<0.83,P<0.05),其中利什曼原虫有毒力株前鞭毛体特有表达蛋白15个,无毒力株前鞭毛体特有表达蛋白23个,二者间表达丰度有显著差异蛋白260个,其中有毒力前鞭毛体表达上调蛋白(高表达)78个,表达下调蛋白(低表达)182个。这些差异表达蛋白中已鉴定功能的蛋白分别涉及糖与脂质代谢、核酸代谢、压力反应、细胞骨架形成、细胞周期与增殖等生物功能。结论利什曼原虫伽师株有毒力株和无毒力株前鞭毛体蛋白质组的表达存在差异,为筛选和鉴定与利什曼原虫感染相关关键分子奠定了基础。

We analyzed the protein abundance differences between the virulent promastigotes and avirulent promastigotes in the same Leishmania strain by comparative proteomics method.Tryptic digests of total proteins were analyzed using liquid chromatography-tandem mass spectrometry(LC-MS/MS),followed by Label-free quantitative differential expression analysis.The MS data were analyzed with MaxQuant software(ver 1.3.0.5)against database.The mass spectrometric analysis of each sample was repeated for three times.Only the proteins that were present in all three replicates(LFQ intensity)for at least one condition were included in the data set.This study resulted in the identification of 3 994 proteins across 2 samples.Of these,298 differentially expressed proteins(ratio >1.2 or ratio<0.83,P﹤0.05)were identified in at least two of the three technical replicates with two or more LFQ intensity number considered.Among these differentially expressed proteins,15 proteins were uniquely in the virulent promastigotes,23 proteins in the avirulent promastigotes.Of 260 differentially expressed proteins were the same in the two groups,among which 162 proteins were dow n-regulated and 78 proteins were up-regulated based on protein annotation.Some of the identified differentially expressed proteins were demonstrated and they involved in biological functions such as glucose and lipid metabolism,nucleotide acid metabolism,stress response,cytoskeleton,cell cycle and proliferation.It is evident that different protein expression profile of the virulent promastigotes and avirulent promastigotes in the same Leishmania strain and this finding may lay the foundation for screening and identification of the key leishmanial molecules for infecting mammal host cells.