根据同步加速器 X 射线束线评估台式闪氧化系统的基于质谱的羟基自由基蛋白足迹,Journal of the American Society for Mass Spectrometry

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根据同步加速器 X 射线束线评估台式闪氧化系统的基于质谱的羟基自由基蛋白足迹
Journal of the American Society for Mass Spectrometry ( IF 3.2 ) Pub Date : 2024-02-09 , DOI: 10.1021/jasms.3c00368
Rohit Jain _{1,

2,

3} , Nanak S. Dhillon _{2,

3} , Vijaya Lakshmi Kanchustambham _{1,

3} , David T. Lodowski _{2,

3} , Erik R. Farquhar _{1,

3} , Janna Kiselar _{2,

3} , Mark R. Chance _{1,

2,

3}

Affiliation

使用同步加速器 X 射线辐射 (XFP) 和质谱法的羟基自由基蛋白足迹 (HRPF) 是一种经过充分验证的结构生物学方法，可为大分子结构动力学提供重要的见解，例如确定结合位点、测量亲和力和绘制表位。产生 HRPF 所需的羟基自由基 (·OH) 的多种替代来源，例如激光光解和等离子体照射、基于同步加速器的 HRPF 的补充，以及最近开发的基于闪光灯光解的商用仪器（FOX 系统），可以获取实验室台式 HRPF。在这里，我们评估了使用台式 FOX 仪器进行内部 HRPF 实验与 NSLS-II XFP 光束线上基于同步加速器的 X 射线足迹实验。使用乳酸氧化酶 (LOx) 作为模型系统，我们使用两台仪器进行了·OH 标记实验，然后进行 nanoLC-MS/MS 自下而上的肽质量图谱。实验在高葡萄糖浓度下进行，以模拟生物缓冲液和人类临床样本中存在的高清除条件，其中可用于与感兴趣的生物分子反应的·OH 较少。对 FOX 和 XFP HRPF 方法的性能进行了比较，我们发现调整·OH 剂量可以在生理相关的高清除条件下实现两种设置的最佳标记覆盖范围。我们的研究证明了 FOX 和 XFP 标记方法的互补性，证明 FOX 光解系统等台式仪器可以提高 HRPF 技术的通量和可及性。

"点击查看英文标题和摘要"

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

更新日期：2024-02-09

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