Background

Natural products are an important source for discovering novel drugs due to their various pharmacological activities. Salvia miltiorrhiza Burge (Danshen) has been shown to have promising therapeutic potential in the management of heart diseases, making it a candidate for cardiovascular drug discovery. Currently, there is limited quantitative analysis of the phosphorylation levels of Danshen-derived natural products on a proteome-wide, which may bias the study of their mechanisms of action.

Purpose

This study aimed to evaluate the global signaling perturbation induced by Danshen-derived bioactive compounds and their potential relationship with myocardial ischemia/reperfusion (IR) injury therapy.

Study design

We employed quantitative proteome and phosphoproteome analysis to identify dysregulated signaling in IR injury hearts from mice. We compared changes induced by Danshen-derived compounds based on IR-associated phospho-events, using an integrative approach that maps relative abundance of proteins and phosphorylation sites.

Methods

Isobaric chemical tandem mass tags (TMT) labeled multiplexing strategy was used to generate unbiased quantitative proteomics and phosphoproteomics data. Highly accurate and precise TMT quantitation was performed using the Orbitrap Fusion Tribrid Mass Spectrometer with synchronous precursor selection MS3 detection mode. Mass spectrometric raw files were analyzed with MaxQuant (2.0.1.0) and statistical and bioinformatics analysis was conducted with Perseus (1.6.15).

Results

We quantified 3661 proteins and over 11,000 phosphosites in impaired heart tissue of the IR mice model, expanding our knowledge of signaling pathways and other biological processes disrupted in IR injury. Next, 1548 and 5545 differently expressed proteins and phosphosites were identified by quantifying the proteome and phosphoproteome of H9c2 cells treated by five Danshen bioactive compounds respectively. Results revealed the vast differences in abilities of five Danshen-derived bioactive compounds to regulate phosphorylation modifications in cardiomyocytes, with dihydrotanshinone I (DHT) showing potential for protecting against IR injury by modulating the AMPK/mTOR signaling pathway.

Conclusions

This study provides a new strategy for analyzing drug/natural product-regulated phosphorylation modification levels on a proteome-wide scale, leading to a better understanding of cell signaling pathways and downstream phenotypic responses.

中文翻译：

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综合蛋白质组学和磷酸蛋白质组学分析揭示了丹参生物活性化合物的心脏保护机制

背景

天然产物因其多种药理活性而成为发现新药的重要来源。丹参（丹参）已被证明在治疗心脏病方面具有良好的治疗潜力，使其成为心血管药物发现的候选者。目前，对丹参衍生天然产物的蛋白质组磷酸化水平的定量分析有限，这可能会影响其作用机制的研究。

目的

本研究旨在评估丹参衍生的生物活性化合物引起的整体信号扰动及其与心肌缺血/再灌注（IR）损伤治疗的潜在关系。

学习规划

我们采用定量蛋白质组和磷酸蛋白质组分析来识别小鼠 IR 损伤心脏中失调的信号传导。我们使用绘制蛋白质和磷酸化位点相对丰度图的综合方法，比较了基于红外相关磷酸化事件的丹参衍生化合物引起的变化。

方法

使用同量化学串联质量标签 (TMT) 标记的多重策略来生成无偏差的定量蛋白质组学和磷酸化蛋白质组学数据。使用具有同步母体选择 MS3 检测模式的 Orbitrap Fusion Tribrid 质谱仪进行高精度 TMT 定量。使用 MaxQuant (2.0.1.0) 分析质谱原始文件，并使用 Perseus (1.6.15) 进行统计和生物信息学分析。

结果

我们对 IR 小鼠模型受损心脏组织中的 3661 个蛋白质和超过 11,000 个磷酸位点进行了定量，扩大了我们对 IR 损伤中受损的信号通路和其他生物过程的了解。接下来，通过对五种丹参生物活性化合物处理的 H9c2 细胞的蛋白质组和磷酸蛋白质组进行定量，分别鉴定了 1548 和 5545 个不同表达的蛋白质和磷酸位点。结果显示，五种丹参衍生的生物活性化合物调节心肌细胞磷酸化修饰的能力存在巨大差异，其中二氢丹参酮 I (DHT) 显示出通过调节 AMPK/mTOR 信号通路来预防 IR 损伤的潜力。

结论

这项研究提供了一种在蛋白质组范围内分析药物/天然产物调节的磷酸化修饰水平的新策略，从而更好地了解细胞信号传导途径和下游表型反应。