Nanoflow Size Exclusion Chromatography-Native Mass Spectrometry of Intact Proteoforms and Protein Complexes

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Native size-exclusion chromatography coupled with native mass spectrometry (SEC-nMS) is crucial for characterizing proteins and complexes.
• Conventional methods use wide columns and harsh ionization, requiring large sample amounts and risking denaturation.
• Limitations include low throughput, co-ionization bias, and the need for extensive sample preparation.

Purpose of the Study:

• To develop a sensitive nanoflow SEC-nMS method for protein complex analysis.
• To enable buffer exchange, oligomer separation, and mild ionization conditions.
• To improve throughput and reduce sample requirements for nMS experiments.

Main Methods:

• Development of a nanoflow SEC-nMS system utilizing 200 μm I.D. columns and a flow rate of 500 nL/min.
• Evaluation of three injection strategies: large volume, nanovolume, and online mixed-bed ion-exchange capillary trap.
• Application of the optimized method to model proteins, protein complexes, and a biological sample.

Main Results:

• The nanoflow method achieved a 4-fold increase in MS peak intensity compared to microflow.
• Significantly reduced sample concentration (20x less) was used in the nanoflow approach.
• Mild ionization conditions were maintained, preserving protein integrity.

Conclusions:

• Nanoflow SEC-nMS offers enhanced sensitivity and efficiency for protein complex characterization.
• The method is suitable for analyzing limited sample quantities and complex biological mixtures.
• This technique advances the capabilities of native mass spectrometry in biochemical research.