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Development of Chemical Proteomics for the Folateome and Analysis of the Kinetoplastid Folateome.
ACS Infectious Diseases ( IF 5.3 ) Pub Date : 2018-09-28 , DOI: 10.1021/acsinfecdis.8b00097
Lauren A Webster 1 , Michael Thomas 1 , Michael Urbaniak 1 , Susan Wyllie 1 , Han Ong 1 , Michele Tinti 1 , Alan H Fairlamb 1 , Markus Boesche 2 , Sonja Ghidelli-Disse 2 , Gerard Drewes 2 , Ian H Gilbert 1
Affiliation  

The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited by a number of drugs. However, there has been little success in developing drugs that target folate metabolism in the kinetoplastids. Despite compounds being identified which show significant inhibition of the parasite enzymes, this activity does not translate well into cellular and animal models of disease. Understanding to which enzymes antifolates bind under physiological conditions and how this corresponds to the phenotypic response could provide insight on how to target the folate pathway in these organisms. To facilitate this, we have adopted a chemical proteomics approach to study binding of compounds to enzymes of folate metabolism. Clinical and literature antifolate compounds were immobilized onto resins to allow for "pull down" of the proteins in the "folateome". Using competition studies, proteins, which bind the beads specifically and nonspecifically, were identified in parasite lysate ( Trypanosoma brucei and Leishmania major) for each antifolate compound. Proteins were identified through tryptic digest, tandem mass tag (TMT) labeling of peptides followed by LC-MS/MS. This approach was further exploited by creating a combined folate resin (folate beads). The resin could pull down up to 9 proteins from the folateome. This information could be exploited in gaining a better understanding of folate metabolism in kinetoplastids and other organisms.

中文翻译:

叶组化学蛋白质组学的发展和动质体叶组分析。

叶酸途径已在许多生物体中进行了广泛研究,其重要性已被许多药物所利用。然而,在开发针对动质体中叶酸代谢的药物方面几乎没有成功。尽管已鉴定出对寄生虫酶有显着抑制作用的化合物,但这种活性并不能很好地转化为疾病的细胞和动物模型。了解抗叶酸在生理条件下与哪些酶结合以及这与表型反应的对应关系可以提供有关如何靶向这些生物体中的叶酸途径的见解。为了促进这一点,我们采用了化学蛋白质组学方法来研究化合物与叶酸代谢酶的结合。临床和文献中的抗叶酸化合物被固定在树脂上以允许“ 在每种抗叶酸化合物的寄生虫裂解物(布氏锥虫和大利什曼原虫)中鉴定出。通过胰蛋白酶消化、肽的串联质量标签 (TMT) 标记以及随后的 LC-MS/MS 来鉴定蛋白质。通过创建组合叶酸树脂(叶酸珠)进一步利用了这种方法。该树脂可以从叶组中提取多达 9 种蛋白质。该信息可用于更好地了解动质体和其他生物中的叶酸代谢。在每种抗叶酸化合物的寄生虫裂解物(布氏锥虫和大利什曼原虫)中鉴定出。通过胰蛋白酶消化、肽的串联质量标签 (TMT) 标记以及随后的 LC-MS/MS 来鉴定蛋白质。通过创建组合叶酸树脂(叶酸珠)进一步利用了这种方法。该树脂可以从叶组中提取多达 9 种蛋白质。该信息可用于更好地了解动质体和其他生物中的叶酸代谢。
更新日期:2018-09-28
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