Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
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DOI：
10.3791/61489
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发表时间：
2020-06-01
影响因子：
1.2
通讯作者：
Wang, Pengcheng
中科院分区：
文献类型：
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作者：
Hsu, Chuan-Chih;Tsai, Chia-Feng;Wang, Pengcheng
蛋白质磷酸化对于渗透条件下酶活性和基因表达的调节至关重要。基于质谱 (MS) 的磷酸蛋白质组学改变了植物信号转导的研究方式。然而，需要大量起始材料和延长 MS 测量时间来实现覆盖深度一直是植物整体磷酸化蛋白质组变化高通量研究的限制因素。为了提高植物磷酸化蛋白质组学的灵敏度和通量，我们开发了一种基于停止和继续提取（阶段）尖端的磷酸化蛋白质组学方法，结合串联质量标签（TMT）标记，用于快速、全面地分析植物响应渗透胁迫的磷酸化扰动。利用阶段吸头技术的简单性和高通量，整个过程使用两个吸头完成磷酸肽富集、分级分离和样品清洁步骤大约需要一小时，表明该方法易于使用且效率高。该方法不仅提供深入的植物磷酸化蛋白质组学分析（> 11,000 个磷酸肽鉴定），而且还证明了相邻组分之间卓越的分离效率（< 5% 重叠）。此外，还使用 TMT 标记实现了多重分析，以量化野生型和 snrk2 十倍突变体植物的磷酸化蛋白质组变化。该方法已成功用于揭示 Raf 样激酶响应渗透胁迫的磷酸化事件，这有助于理解陆地植物早期渗透信号传导。
Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (> 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (< 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.