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

Abstract

Abstract 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.