Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects
Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects
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DOI：
10.1021/acs.jproteome.8b00972
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发表时间：
2019-05-01
影响因子：
4.4
通讯作者：
Bai, Gang
中科院分区：
文献类型：
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作者：
Wang, Zhihua;Kim, Unchol;Bai, Gang
此前已证明人参皂苷可有效抑制动物模型和细胞系中癌细胞的生长和存活。然而，作为通过与靶蛋白相互作用来治疗癌症的活性成分的特定人参皂苷成分仍然未知。通过亲和质谱（MS）技术的集成定量蛋白质组学方法，我们破译了源自人参皂苷粗提物的人参皂苷活性成分的核心结构，并在鉴定介导其抗癌活性的靶蛋白方面取得了进展。串联质量标签 (TMT) 标记定量蛋白质组学技术获得了 55620 个 MS/MS 谱图，鉴定了 5499 个蛋白质和 3045 个修饰蛋白质。在这些已鉴定的蛋白质中，224 个差异表达蛋白质和修饰蛋白质在非小细胞肺癌细胞系中发生显着改变。生物信息学工具综合分析表明Ras蛋白在多种功能通路中发挥普遍调节作用，可能是人参皂苷化合物的直接靶蛋白。然后，基于Ras蛋白的亲和力MS筛选鉴定出20(s)-原人参二醇、20(s)-人参皂苷Rh2和20(s)-人参皂苷Rg3在不同条件下与Ras蛋白具有亲和力。特别是20(s)-原人参二醇，其衍生物是已报道的抗肿瘤化合物20(s)-人参皂苷Rh2和20(s)-人参皂苷Rg3，通过1.22 μM的低KD和G12和G60的突变位点对Ras具有更高的亲和力，被证明在这些相互作用中发挥着核心作用。此外，分子机制和生物活性评估结果证实了化学配体的身份，该化学配体直接作用于Ras的GTP结合袋，并显示在癌细胞生物活性谱中有效。
Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 mu M and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.