Photocleavable crosslinker design, synthesis and analysis for enhanced crosslinking mass spectrometry methods

交联质谱是一种多尺度的蛋白质行为测定技术。它现在是一个建立和强大的工具，用于阐明单个蛋白质和复合物的结构。该方法目前正在积极开发中，用于蛋白质构象变化和蛋白质-蛋白质相互作用的研究。至关重要的是，交联质谱法有能力在蛋白质存在的天然环境（活细胞）中实现上述所有目标。该方法还可以在细胞裂解物、细胞器和完整细胞上进一步扩展到系统水平和蛋白质组范围的分析。然而，更高水平的蛋白质混合物复杂性给分析带来了新的挑战。交联质谱技术的基础是用于引入共价键的化学反应，这保留了关键的空间信息。因此，交联剂开发是交联质谱法开发的关键驱动力。新型交联剂可以通过它们在数据采集和数据分析方法之间的相互作用来增强分析过程。紫外光解离（UVPD）的最新进展在这里打开了一个新的角度，我们现在想利用交联质谱。我们将开发具有增强功能性（反应性和交联剂UV裂解性）的化学交联剂，以及新的分析和计算方法，以更好地利用所得数据。我们希望通过改善信噪比，在交联质谱分析中实现高复杂性蛋白质分析的更大深度，并将与标准交联剂直接比较进行评估。

Crosslinking mass spectrometry is a multi-scale, protein behaviour determination technology. It is now an established and robust tool for elucidating structures of individual proteins and complexes. The method is currently under active development for use in the study of protein conformational changes and protein-protein interactions. Crucially, crosslinking mass spectrometry has the capacity to achieve all of the above, in the native environment in which proteins exist, the living cell. The method can also scale further, to systems level and proteome-wide analysis, on cell lysates, organelles and intact cells. However, higher levels of protein mixture complexity create new challenges for analysis.Crosslinking mass spectrometry technology is underpinned by the chemistry used to introduce covalent links, which preserve the critical spatial information. Crosslinker development is therefore a key driver of crosslinking mass spectrometry method development. Novel crosslinkers can enhance the analytical process with their interplay between data acquisition and data analysis approaches. Recent progress in ultraviolet photodissociation (UVPD) opens a new angle here, which we now want to exploit for crosslinking mass spectrometry. We will develop chemical crosslinkers with enhanced functionality (reactivity and crosslinker UV-cleavability), as well as new analytical and computational methods for better utilisation of the resulting data. We expect to achieve significantly greater depth for high-complexity protein analysis in crosslinking mass spectrometry analysis by improving signal-to-noise and will assess this in direct comparison to standard crosslinkers.