Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned...

Area of Science:

• Biochemistry and Biophysics
• Analytical Chemistry
• Structural Biology

Background:

• Native mass spectrometry (MS) is crucial for studying protein interactions and functions.
• Analyzing membrane proteins and screening compound libraries with native MS yields complex spectra.
• Unambiguous peak assignment in native MS requires native top-down analysis, necessitating detergent removal.

Purpose of the Study:

• To develop a method for improved native mass spectrometry analysis of complex protein samples.
• To enable efficient detergent removal and isolation of protein-ligand complexes for top-down analysis.
• To facilitate the identification of novel therapeutic targets and drug compounds.

Main Methods:

• Implementation of a novel enhanced declustering (ED) device in a cyclic ion mobility spectrometry (IMS)-enabled mass spectrometry platform.
• Utilizing an oscillating electric field for ion declustering and liberation of membrane proteins from detergent micelles.
• Employing quadrupole selection for isolating protein-ligand complexes and collision-induced dissociation for fragmentation.

Main Results:

• The ED device effectively declusters ions and liberates membrane proteins prior to MS analysis.
• Ion mobility (IM) significantly aids in assigning top-down spectra by correlating fragments with parent ions via drift time.
• Successful identification of a novel hit compound against PfMATE from multiplexed ligand libraries.

Conclusions:

• The novel ED device enhances native MS capabilities for analyzing complex protein assemblies and interactions.
• This approach simplifies the interpretation of complex spectra, enabling unambiguous peak assignment in top-down analysis.
• The developed method facilitates drug discovery by enabling efficient screening and identification of protein-ligand interactions.