Sonoma State University, Rohnert Park, Sonoma, CA, 94928, US.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, US.
Commun Biol. 2022 Aug 25;5(1):866. doi: 10.1038/s42003-022-03775-1.
X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.
X 射线辐射标记使用宽带 X 射线对原位羟基自由基进行标记，以绘制蛋白质相互作用和构象图。高通量密度束对于克服自由基清除剂至关重要。然而，传统的样品输送环境（如毛细管流动）限制了充分利用完全未衰减的聚焦宽带束的使用。一种替代方法是使用液体射流，我们之前已经证明，在非聚焦 X 射线源下，使用这种形式的样品输送可以将标记增加十倍。在这里，我们报告了首次在高通量微聚焦同步加速器光束线上使用液体射流进行自动在线定量荧光剂量学特性和样品暴露的情况。我们的方法可以在单个微秒的曝光时间内实现，同时保持高水平的侧链标记。这种发展极大地提高了该方法的整体效果和效率，生成高质量的数据，并为高通量和超快时间分辨原位羟基自由基标记开辟了领域。
