Huntington’s disease (HD) is one of the most devastating neurodegenerative disorders (NDs) that currently lacks effective therapies. Caused by toxic aggregation of mutant Huntingtin (Htt) proteins carrying abnormally long polyglutamine (polyQ) repeats, their timely removal through proteasomal degradation is critical for delaying onset of the disease. Given its crucial role as the central machine responsible for degradation of damaged and misfolded proteins such as Htt and other aggregation-prone proteins, 26S proteasome impairment has been recognized as one of the hallmarks of NDs associated with neuropathology. While it has been suggested that protein aggregates can induce conformational changes in the 26S to reduce its function, activation of proteasomes through phosphorylation appears to enhance the removal of Htt mutants. However, the molecular details underlying proteasome inhibition and activation in HD remain unclear. To address these unknowns, it is essential to quantitatively assess Htt aggregation and phosphorylation-dependent conformations of the 26S in cells to obtain a mechanistic understanding of the structure-function relationship of HD-associated proteasomes. Such investigations have remained previously unexplored due to lack of proper strategies. During the current funding cycle, we have demonstrated that cross-linking mass spectrometry (XL- MS) is effective for studying in vivo structural dynamics of proteasome complexes. While the development of specific residue-targeting MS-cleavable cross-linkers has further improved our capability to map protein-protein interactions (PPIs), interactions at hydrophobic regions remain difficult to characterize due to lack of targetable residues. Therefore, it is necessary to explore alternative chemistries for capturing structural details in those regions in order to comprehensively dissect proteasome conformational dynamics in cells. Here, we aim to develop photochemistry-based XL-MS platforms to enable their application for complex PPI mapping in vivo and in vitro. In addition, we intend to develop integrated QXL-MS platforms to define the temporal dynamics of the 26S proteasome upon Htt aggregation and phosphorylation, yielding molecular details to delineate the structure-function relationship of HD-impaired proteasomes. This project not only represents a great leap in XL-MS technology, but also helps address important yet unresolved biological questions associated with HD that have great potential for future therapeutic exploitation.