Illuminating Biological Interactions with in Vivo Protein Footprinting
Illuminating Biological Interactions with in Vivo Protein Footprinting
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DOI：
10.1021/acs.analchem.9b00244
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发表时间：
2019-05-21
影响因子：
7.4
通讯作者：
Jones, Lisa M.
中科院分区：
文献类型：
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作者：
Espino, Jessica A.;Jones, Lisa M.
蛋白质足迹法与质谱联用越来越多地用于蛋白质相互作用和构象的研究。羟基自由基足迹法，蛋白质的快速光化学氧化（FPOP），利用羟基自由基氧化修饰溶剂可及的氨基酸。在这里，我们描述了FPOP的进一步发展，在体内蛋白质结构分析（IV-FPOP）与秀丽隐杆线虫。C.线虫是线虫科的一部分，被用作许多人类疾病的模型系统。在这些蠕虫中进行结构研究的能力将提供对结构在疾病发病机制中的作用的深入了解。优化了蠕虫内标记的许多参数，包括微流控流动系统和过氧化氢浓度。IV-FPOP能够改变蠕虫体内各种器官中的数百种蛋白质。该方法成功地探测溶剂的可及性类似于在体外FPOP，证明其作为一种结构技术在多器官系统中使用的潜力。该方法与质谱法的耦合允许氨基酸残基水平的结构信息，比目前可用的体内方法更高的分辨率。
Protein footprinting coupled with mass spectrometry is being increasingly used for the study of protein interactions and conformations. The hydroxyl radical footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids. Here, we describe the further development of FPOP for protein structural analysis in vivo (IV-FPOP) with Caenorhabditis elegans. C. elegans, part of the nematode family, are used as model systems for many human diseases. The ability to perform structural studies in these worms would provide insight into the role of structure in disease pathogenesis. Many parameters were optimized for labeling within the worms including the microfluidic flow system and hydrogen peroxide concentration. IV-FPOP was able to modify several hundred proteins in various organs within the worms. The method successfully probed solvent accessibility similarily to in vitro FPOP, demonstrating its potential for use as a structural technique in a multiorgan system. The coupling of the method with mass spectrometry allows for amino-acid-residue-level structural information, a higher resolution than currently available in vivo methods.