Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Membrane proteins are crucial drug targets but challenging to analyze using native mass spectrometry (MS) due to complex solubilization methods.
• Current native MS workflows involve laborious offline buffer exchange, requiring high sample amounts and extensive optimization.
• Analyzing membrane proteins in various mimetics like detergent micelles and nanodiscs presents unique analytical hurdles.

Purpose of the Study:

• To develop a streamlined method for analyzing membrane proteins in diverse membrane mimetics using native MS.
• To improve the efficiency and throughput of membrane protein analysis by automating buffer exchange.
• To enable rapid assessment of membrane protein integrity prior to structural studies.

Main Methods:

• Development of online buffer exchange coupled to native mass spectrometry (OBE-nMS).
• Screening of detergents and mobile phases (ammonium acetate with lauryl-dimethylamine oxide) for optimal membrane protein characterization.
• Implementation of a novel switching electrospray approach to preserve nanodisc integrity during analysis.

Main Results:

• OBE-nMS successfully analyzes membrane proteins in both detergent micelles and nanodiscs.
• A universal mobile phase composition was identified for detergent-based analyses of bacterial and mammalian membrane proteins.
• Ammonium acetate alone is sufficient for nanodisc-based analyses, and a switching electrospray preserves nanodisc structure.
• The OBE-nMS method significantly reduces analysis time to minutes per sample, enhancing throughput.

Conclusions:

• OBE-nMS offers a rapid, efficient, and versatile platform for analyzing membrane proteins in various mimetics.
• This method overcomes limitations of traditional offline buffer exchange, enabling higher-throughput assessment of membrane protein integrity.
• OBE-nMS facilitates quicker structural elucidation of membrane proteins by providing rapid pre-analysis integrity checks.