Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.
Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.
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DOI：
10.1016/j.molp.2016.10.011
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发表时间：
2017-01
期刊：
影响因子：
27.5
通讯作者：
P. Lundquist;O. Mantegazza;A. Stefanski;K. Stühler;A. Weber
中科院分区：
文献类型：
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作者：
P. Lundquist;O. Mantegazza;A. Stefanski;K. Stühler;A. Weber
蓝色非变性PAGE（BN-PAGE）可将蛋白质复合物解析为非变性状态。当与免疫印迹结合时，它可以用于在给定合适抗体的情况下以高分辨率鉴定任何蛋白质的高分子量复合物的存在。为了大规模地鉴定高分子量复合物中的蛋白质，并绕过对特异性抗体的要求，我们将串联质谱（MS/MS）方法应用于BN-PAGE分辨的叶绿体。将凝胶分成6个条带，可以用天然分子量分离法对1000种叶绿体蛋白进行鉴定和无标记定量。值得注意的是，这种方法实现了与传统的叶绿体鸟枪蛋白质组学分析相媲美的识别深度，表明许多已知的叶绿体蛋白质组是适合MS/MS鉴定根据我们的分馏方案。通过限制分馏带的数量为6个，我们方便放大比较分析，因为我们证明与reticulatachloroplast突变体显示网状叶表型。我们的比较蛋白质组学方法确定了一个候选的相互作用蛋白的网以及对脂质重塑蛋白，氨基酸代谢酶和质体分裂机制的影响。我们还强调了从叶绿体的每个亚室中选择的蛋白质，这些蛋白质提供了对已知或假设的蛋白质复合物的新见解，以进一步说明这种方法的实用性。我们的研究结果表明，该技术的高灵敏度和重现性，预计将广泛适用于其他亚细胞区室。
Blue native-PAGE (BN–PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN–PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with thereticulatachloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.