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

Area of Science:

• Proteomics
• Biophysical Chemistry
• Cell Biology

Background:

• Observing protein dynamics in cellular environments is vital for biology.
• Mass spectrometry faces challenges due to matrix interference, limiting direct cellular analysis.

Purpose of the Study:

• To introduce a novel platform for time-resolved protein analysis directly in crude lysates and living cells.
• To overcome matrix suppression and achieve sub-second temporal resolution in complex biological samples.

Main Methods:

• Development of the RapiBOX-IM-MS platform.
• Integration of rapid online buffer exchange with pulsed electrophoretic separation.
• Utilizing a nano-electrospray emitter for analysis.

Main Results:

• Demonstrated capture of transient intermediates in hemoglobin refolding.
• Visualized lobe-specific calcium binding dynamics of in-cell calmodulin.
• Revealed a synergistic pathogenic mechanism in alpha-synuclein, showing acid-induced compaction stabilizes against thermal stress.

Conclusions:

• The RapiBOX-IM-MS platform offers a powerful new tool for dissecting protein structural dynamics in complex cellular milieus.
• The study provides insights into the interplay of environmental stressors in protein misfolding and disease.
• This technology enables direct, time-resolved investigation of proteins within their native cellular context.