Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry

Area of Science:

• Biochemistry and Biophysics
• Structural Biology
• Mass Spectrometry

Background:

• Protein oligomerization is vital for physiological processes, but its dysregulation causes disease.
• Understanding protein assembly pathways and thermodynamics is key for biological insight and therapeutics.
• Existing binding assays have limitations in mass precision and model simplicity.

Purpose of the Study:

• To introduce an extended slow mixing mode native mass spectrometry (SLOMO-nMS) for quantifying thermodynamics in multistep protein association reactions.
• To validate the new SLOMO-nMS method using known protein systems and investigate novel interactions.

Main Methods:

• Extended SLOMO-nMS technique for simultaneous quantification of thermodynamics in complex association reactions.
• Application to homo-oligomerization of concanavalin A and insulin.
• Utilized SLOMO-nMS with charge detection for analyzing recombinant human angiotensin-converting enzyme 2 and SARS-CoV-2 spike protein binding.

Main Results:

• The extended SLOMO-nMS reliably quantified thermodynamics for multistep protein assembly.
• Confirmed known assembly pathways for concanavalin A and insulin, revealing new details.
• Uncovered novel assembly pathways and quantified affinities for the ACE2-Spike protein interaction, crucial for host cell infection.

Conclusions:

• SLOMO-nMS is a powerful tool for characterizing complex protein assembly pathways and thermodynamics.
• This method accelerates fundamental biological understanding and facilitates the development of therapeutics targeting protein oligomerization.
• The findings highlight SLOMO-nMS's potential to advance research in structural biology and disease mechanisms.