Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition
Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition
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DOI：
10.1111/j.1365-2958.2004.04049.x
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发表时间：
2004-05-01
影响因子：
3.6
通讯作者：
Hyde, JE
中科院分区：
文献类型：
--
作者：
Nirmalan, N;Sims, PFG;Hyde, JE
准确测量药物攻击、代谢应激、发育编程或其他干扰后蛋白质表达的比较水平的能力代表了后基因组学疟疾研究中最重要的目标之一。我们在这里描述了一个简单而强大的定量方法，非常适合在体外实验，旨在研究最重要的人类寄生虫，致命的恶性疟原虫的蛋白质组的变化。我们已经开发的代谢标记技术使用寄生虫摄取重同位素的异亮氨酸在正常生长过程中，然后通过二维分离的个别蛋白质和质谱。该方法基本上适用于从完整的基因组序列预测的约5300种恶性疟原虫蛋白质中的每一种，从而允许从经受不同刺激的体外培养物合成的这些蛋白质中的任何一种中容易地鉴定和准确地比较定量标记的肽。我们证明了它的应用程序的细胞周期变化的研究，在那里我们观察到不同的蛋白质和报告的转录水平指示在翻译水平上的调制模式。我们的数据还提供了证据，在寄生虫的翻译后修饰的显着水平，我们测量的磷酸乙醇胺N-甲基转移酶和肌动蛋白-I在整个细胞周期的变体之间的差异。我们还监测了寄生虫对等效剂量的临床抗疟抑制剂乙胺嘧啶和四环素的反应，并观察到与这些药物的可能靶点无关的一些蛋白质的差异效应。
The ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post-genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope-containing isoleucine during normal growth followed by two-dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the approximate to 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post-translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N-methyltransferase and actin-I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.