Structural\nAnalysis of the 20S Proteasome Using Native\nMass Spectrometry and Ultraviolet Photodissociation

Owing to the role of the 20S proteasome in a wide spectrum\nof pathologies,\nincluding neurodegenerative disorders, proteasome-associated autoinflammatory\nsyndromes (PRAAS), and cardiovascular diseases, understanding how\nits structure and composition contribute to dysfunction is crucial.\nAs a 735 kDa protein assembly, the 20S proteasome facilitates normal\ncellular proteostasis by degrading oxidized and misfolded proteins.\nDeclined proteasomal activity, which can be attributed to perturbations\nin the structural integrity of the 20S proteasome, is considered one\nof the main contributors to multiple proteasome-related diseases.\nDevising methods to characterize the structures of 20S proteasomes\nprovides necessary insight for the development of drugs and inhibitors\nthat restore proper proteasomal function. Here, native mass spectrometry\nwas combined with multiple dissociation techniques, including ultraviolet\nphotodissociation (UVPD), to identify the protein subunits comprising\nthe 20S proteasome. UVPD, demonstrating an ability to uncover structural\nfeatures of large (>300 kDa) macromolecular complexes, provided\ncomplementary\ninformation to conventional collision-based methods. Additionally,\nvariable-temperature electrospray ionization was combined with UV\nphotoactivation to study the influence of solution temperature on\nthe stability of the 20S proteasome.