Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment.
Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment.
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DOI：
10.1371/journal.pone.0004098
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发表时间：
2008
期刊：
影响因子：
3.7
通讯作者：
Becker K
中科院分区：
文献类型：
--
作者：
Prieto JH;Koncarevic S;Park SK;Yates J 3rd;Becker K
蛋白质组学研究对我们理解寄生虫生物学、宿主-寄生虫相互作用和药物作用机制有显著贡献。对于大多数抗疟药物而言，其作用方式和耐药性发展机制都没有得到充分阐明，尽管这将是成功开发迫切需要的新型抗疟药物的重要先决条件。在这里，我们建立了一个大规模的定量蛋白质组学的方法来检查氯喹和青蒿素治疗后的恶性疟原虫滋养体阶段的蛋白质表达变化。为此目的，使用14 N-异亮氨酸和13 C6，15 N1-异亮氨酸的SIL（稳定同位素标记）被优化以获得99%原子百分比富集。使用阴离子交换层析进行蛋白质组分级分离以降低样品复杂性并增加蛋白质表达的定量覆盖。对亚组分的胰蛋白酶肽进行SCX/RP分离，通过LC-MS/MS测量，并使用新型软件工具Census进行定量。在药物处理的寄生虫中，我们鉴定了总共1，253种蛋白质，从而使滋养体阶段鉴定的蛋白质总数增加了30%。获得了800多种蛋白质的相对定量。在青蒿素和氯喹处理下，分别有41和38种蛋白质上调（>1.5），而14和8种蛋白质下调（<0.5）。除了特异性调节的蛋白质外，我们还鉴定了作为对药物治疗的一般反应而被调节的蛋白质组。蛋白质组学数据通过蛋白质印迹法证实。这里描述的方法允许有效的大规模差异蛋白质组分析恶性疟原虫研究药物治疗或环境变化的反应。分析仅需要100 µg蛋白质，这表明该方法也可以转移到其他顶复门寄生虫。
Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using 14N-isoleucine and 13C6,15N1-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (<0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 µg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites.
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