Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.
Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.
复制标题
整个植物的水培同位素标记和用于定量植物蛋白质组学的高性能质谱分析。
DOI：
10.1007/978-1-61779-885-6_12
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Bindschedler LV
中科院分区：
文献类型：
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作者：
Bindschedler LV
水培同位素标记整株植物（HILEP）结合水培植物培养和代谢标记与稳定同位素使用含15 N的无机盐标记整株和成熟植物。采用15 N盐作为HILEP的唯一氮源导致产生外观健康的植物，其含有近100%标记的15 N蛋白。因此，HILEP适用于定量植物蛋白质组分析，其中植物生长在14 N或15 N水培培养基中，并在收集生物样品进行相对蛋白质组定量时合并。汇集的14 N-/15 N-蛋白质提取物可以以任何合适的方式分级分离，并用蛋白酶消化用于鸟枪蛋白质组学，通常使用反相液相色谱纳米电喷雾电离串联质谱法（RPLC-nESI-MS/MS）。使用混合离子阱/FT-MS质谱仪获得了最佳结果，将MS数据采集的高质量准确度和灵敏度与速度和高通量MS/MS数据采集相结合，增加了鉴定和定量的蛋白质数量，并改善了蛋白质定量。使用集成MS数据分析软件以高度自动化的方式进行峰处理和从原始MS数据文件中挑选、蛋白质鉴定和定量，人工干预最少，从而简化了分析工作流程。在这篇方法学论文中，我们描述了如何使用15 N盐水培拟南芥植物进行同位素标记，以及如何使用不需要广泛的生物信息学技能的方便工作流程来定量所得蛋白质组。
Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using15N-containing inorganic salts to label whole and mature plants. Employing15N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain15N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either14N- or15N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled14N-/15N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using15N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.