Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures. Graphical Abstract ᅟ.

中文翻译：

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比较蛋白质的氢氘交换和快速光化学氧化：野生型和 ΔN6 β2-微球蛋白的结构表征。

氢氘交换 (HDX) 与质谱 (MS) 耦合是一种成熟的技术，用于结构 MS 领域，用于探测蛋白质中骨架酰胺的溶剂可及性、动力学和氢键。相比之下，蛋白质的快速光化学氧化 (FPOP) 使用从过氧化氢的光解中释放出来的羟基自由基，在微秒毫秒的时间尺度上共价标记溶剂可接近的氨基酸侧链。在这里，我们使用这两种技术来研究蛋白质 β2-微球蛋白 (β2m) 与其淀粉样截断变体 ΔN6 之间的结构和动力学差异。我们表明 HDX 和 FPOP 突出了蛋白质区域的结构/动力学差异，定位于 N 末端截断周围的区域。此外，我们证明，通过精心优化的 LC-MS 条件，FPOP 数据可以探测亚氨基酸水平的溶剂可及性，并且可以有意义地解释这些数据，以更详细地了解蛋白质结构中的局部环境和侧链方向。图形摘要ᅟ。