Zhu Yi, Serra Aida, Guo Tiannan, Park Jung Eun, Zhong Qing, Sze Siu Kwan
School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive 637551, Singapore.
Department of Pathology and Molecular Pathology, University Hospital Zürich , Zürich, Switzerland.
J Proteome Res. 2017 Jun 2;16(6):2282-2293. doi: 10.1021/acs.jproteome.7b00154. Epub 2017 May 8.
Mass spectrometry-based protein footprinting emerged as a useful technology to understand protein ligand interactions in vitro. We have previously demonstrated the application of footprinting in live E. coli cells. Here, we further optimized an ultrafast laser photolysis hydroxyl radical footprinting method and applied it to study the interaction of EGF and EGFR in live mammalian cells. This method used a nanosecond laser to photochemically generate a burst of hydroxyl radicals in situ in-cell suspension to oxidize the amino acids on the protein surface. Mass spectrometric analysis of the thus modified peptides was interpreted to probe the solvent-accessible surface areas of the protein in its native biological state with and without EGF activation. Our footprinting data agreed with the two relevant EGFR crystal structures, indicating that this in-cell laser photolysis footprinting technique is a valid approach to study the structural properties of integral membrane proteins directly in the native environment.
基于质谱的蛋白质足迹分析成为一种了解体外蛋白质-配体相互作用的有用技术。我们之前已经证明了足迹分析在活的大肠杆菌细胞中的应用。在此，我们进一步优化了一种超快激光光解羟基自由基足迹分析方法，并将其应用于研究活的哺乳动物细胞中表皮生长因子（EGF）与表皮生长因子受体（EGFR）的相互作用。该方法使用纳秒激光在细胞内悬浮液中原位光化学产生一阵羟基自由基，以氧化蛋白质表面的氨基酸。对如此修饰后的肽段进行质谱分析，以探测在有和没有EGF激活的情况下，处于天然生物学状态的蛋白质的溶剂可及表面积。我们的足迹分析数据与两个相关的EGFR晶体结构一致，表明这种细胞内激光光解足迹分析技术是直接在天然环境中研究整合膜蛋白结构特性的有效方法。
