Time-Resolved In-Cell Protein Interactions and Structural Dynamics via Rapid Buffer Online Exchange-Ion Mobility-Mass...

Observing protein structural dynamics directly within the complex cellular milieu is crucial for understanding biology but is hampered by severe matrix interference in mass spectrometry. Here, we introduce the RapiBOX-IM-MS platform, which enables time-resolved analysis of proteins directly in crude lysates and living cells. The device integrates rapid online buffer exchange with pulsed electrophoretic separation at a nano-electrospray emitter, eliminating matrix suppression and providing sub-second temporal resolution. Its power is demonstrated by capturing transient, asymmetric intermediates in hemoglobin refolding and visualizing the lobe-specific calcium binding dynamics of in-cell calmodulin. Most significantly, we applied the platform to the neurodegenerative protein alpha-synuclein, revealing a synergistic pathogenic mechanism where initial acid-induced compaction paradoxically stabilizes the protein against subsequent thermal stress. This work provides a powerful new tool for dissecting the complex interplay of environmental stressors that trigger protein misfolding and disease.