Parker Jennifer, Zhu Ning, Zhu Mengmeng, Chen Sixue
Plant Molecular and Cellular Biology Program, University of Florida, Florida, USA.
J Vis Exp. 2012 Mar 24(61):3766. doi: 10.3791/3766.
Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant(1,2). The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato(3). Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas(3). Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)(4). DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue(4). ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins(5). Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes(6,7). Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry(8,9). The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter(10). After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.
丁香假单胞菌番茄致病变种DC3000不仅会在番茄上引发细菌性斑点病，还会在芸苔属植物以及模式植物拟南芥上引发该病(1,2)。接种DC3000的子叶中活性氧(ROS)的积累表明ROS在番茄细菌性斑点病坏死细胞死亡的调控中发挥作用(3)。过氧化氢作为ROS的组成成分，在番茄植株接种假单胞菌后产生(3)。过氧化氢可以用组织化学染色剂3'-3'二氨基联苯胺(DAB)进行检测(4)。DAB染色与过氧化氢反应，在叶片组织上产生棕色染色(4)。ROS对细胞氧化还原环境具有调节作用，可改变某些蛋白质的氧化还原状态(5)。半胱氨酸是一种对氧化还原变化敏感的重要氨基酸。在轻度氧化条件下，半胱氨酸巯基的可逆氧化作为氧化还原传感器和信号转导器，调节多种生理过程(6,7)。串联质量标签(TMT)试剂能够使用串联质谱同时鉴定和多重定量不同样品中的蛋白质(8,9)。半胱氨酸反应性TMT (cysTMT)试剂能够对多达六个生物样品中含半胱氨酸的肽段进行选择性标记和相对定量。每个等压cysTMT标签具有相同的标称母离子质量，由一个巯基反应基团、一个质谱中性间隔臂和一个串联质谱报告基团组成(10)。标记后，样品进行蛋白酶消化。使用含有抗TMT抗体的树脂富集半胱氨酸标记的肽段。在串联质谱分析过程中，一系列报告离子(即126 - 131 Da)出现在低质量区域，提供相对定量信息。该工作流程对于降低样品复杂性、提高动态范围和研究半胱氨酸修饰是有效的。在此，我们展示了使用cysTMT技术对经Pst DC3000处理的番茄(里奥格兰德)叶片进行氧化还原蛋白质组学分析。这种高通量方法有潜力应用于研究其他氧化还原调节的生理过程。
