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A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation.
PLOS Neglected Tropical Diseases ( IF 3.8 ) Pub Date : 2019-12-19 , DOI: 10.1371/journal.pntd.0007651 Karunakaran Kalesh 1 , Paul W Denny 2
PLOS Neglected Tropical Diseases ( IF 3.8 ) Pub Date : 2019-12-19 , DOI: 10.1371/journal.pntd.0007651 Karunakaran Kalesh 1 , Paul W Denny 2
Affiliation
Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo. The identification of parasite proteins synthesised during starvation is key to unravelling the underlying molecular mechanisms facilitating adaptation to these conditions. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesised Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified over 250 starvation-responsive NSPs in the parasite. Our results show a starvation-specific increased relative abundance of several translation regulating and stress-responsive proteins in the parasite. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 2.47e-35) and peptide biosynthetic process (enrichment P value 4.84e-35) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find widespread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.
中文翻译:
BONCAT-iTRAQ方法可在饥饿过程中暂时解析墨西哥利什曼原虫中新合成蛋白质的定量分析。
适应饥饿是利什曼原虫生命周期不可或缺的一部分。在体外和体内,该寄生虫都能在营养缺乏的情况下幸存下来。鉴定饥饿期间合成的寄生虫蛋白是揭示有助于适应这些条件的潜在分子机制的关键。另外,由于利什曼原虫中的压力适应机制与毒力和传染性相关,因此在饥饿状态下对新合成蛋白质(NSP)完整库的概况分析对于药物靶标发现很重要。但是,事实证明,从现有的寄生虫蛋白质组的较大背景下,鉴定和定量低丰度,饥饿特定的NSP困难,因为这需要高度选择性和灵敏的方法。本文中,我们介绍了墨西哥乳杆菌前体中的一种综合化学蛋白质组学方法,该方法涉及BONCAT技术和iTRAQ定量蛋白质组学质谱(MS)的强大组合,该方法可在饥饿的寄生虫中从头开始进行蛋白质合成的时间分辨定量分析。独特的是,这种方法将BONCAT技术对NSP的高特异性与iTRAQ蛋白质组学MS的高灵敏度和多重定量能力相结合。概念验证实验在寄生虫中鉴定出250多个饥饿反应性NSP。我们的结果表明,该寄生虫中几种翻译调节蛋白和应激反应蛋白的饥饿特异性增加了相对丰度。对已鉴定的用于生物过程的NSP的GO分析显示了翻译(富集P值2。47e-35)和肽的生物合成过程(富集P值4.84e-35)作为极为显着富集的术语,表明NSP对调节蛋白质合成具有高度特异性。我们相信,这种方法将在研究利什曼原虫物种的发育阶段以及更广泛的原生动物生物学领域中得到广泛使用。
更新日期:2019-12-20
中文翻译:
BONCAT-iTRAQ方法可在饥饿过程中暂时解析墨西哥利什曼原虫中新合成蛋白质的定量分析。
适应饥饿是利什曼原虫生命周期不可或缺的一部分。在体外和体内,该寄生虫都能在营养缺乏的情况下幸存下来。鉴定饥饿期间合成的寄生虫蛋白是揭示有助于适应这些条件的潜在分子机制的关键。另外,由于利什曼原虫中的压力适应机制与毒力和传染性相关,因此在饥饿状态下对新合成蛋白质(NSP)完整库的概况分析对于药物靶标发现很重要。但是,事实证明,从现有的寄生虫蛋白质组的较大背景下,鉴定和定量低丰度,饥饿特定的NSP困难,因为这需要高度选择性和灵敏的方法。本文中,我们介绍了墨西哥乳杆菌前体中的一种综合化学蛋白质组学方法,该方法涉及BONCAT技术和iTRAQ定量蛋白质组学质谱(MS)的强大组合,该方法可在饥饿的寄生虫中从头开始进行蛋白质合成的时间分辨定量分析。独特的是,这种方法将BONCAT技术对NSP的高特异性与iTRAQ蛋白质组学MS的高灵敏度和多重定量能力相结合。概念验证实验在寄生虫中鉴定出250多个饥饿反应性NSP。我们的结果表明,该寄生虫中几种翻译调节蛋白和应激反应蛋白的饥饿特异性增加了相对丰度。对已鉴定的用于生物过程的NSP的GO分析显示了翻译(富集P值2。47e-35)和肽的生物合成过程(富集P值4.84e-35)作为极为显着富集的术语,表明NSP对调节蛋白质合成具有高度特异性。我们相信,这种方法将在研究利什曼原虫物种的发育阶段以及更广泛的原生动物生物学领域中得到广泛使用。