Takemori Ayako, Nakashima Taiken, Ômura Hisashi, Tanaka Yuki, Nakata Keisuke, Nonami Hiroshi, Takemori Nobuaki
1Department of Bioresource Production Science, The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, 790-8566 Japan.
2Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
Plant Methods. 2019 Apr 24;15:40. doi: 10.1186/s13007-019-0427-7. eCollection 2019.
Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins.
Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins.
We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes.
维管植物中的腺毛被称为天然细胞工厂，因为它们在腺细胞中合成和储存次生代谢产物。为了系统地了解腺细胞中的代谢过程，分析细胞蛋白质组动力学是必不可少的。基于质谱的传统蛋白质组学方法能够进行大规模蛋白质分析，但需要大量的毛状体样本进行深入分析，且不适用于对目标蛋白质进行快速灵敏的定量分析。
在此，我们提出了一种高通量策略，用于使用选择反应监测（SRM）分析法定量特定毛状体腺细胞中的目标蛋白质。以番茄VI型毛状体腺细胞中的蛋白质为模型系统的SRM分析平台，证明了其在从有限量样本中定量多种蛋白质方面的有效性。大规模SRM分析使用与纳流液相色谱仪在线连接的三重四极杆质谱仪，在120分钟内准确测量了从100个腺毛状体中回收的腺细胞样本中221种目标蛋白质的表达水平。使用SRM分析对不同器官（叶片、绿色果实和花萼）的VI型毛状体腺细胞进行比较定量蛋白质组学，揭示了特定器官富集的蛋白质。
我们提出了一种使用已建立的SRM分析的靶向蛋白质组学方法，该方法能够以最少的采样量对感兴趣的蛋白质进行定量。SRM分析的显著成功及其简单的实验工作流程将增加腺毛状体中的蛋白质组学研究。
