Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Native proteomics using native mass spectrometry (nMS) offers insights into cellular proteome dynamics.
• Current nMS methods are limited in analyzing complex samples like whole cell lysates.
• Characterizing intact proteoforms and protein complexes is crucial for understanding biological processes.

Purpose of the Study:

• To pioneer native proteomics measurements of large proteoforms and protein complexes from complex proteomes.
• To develop a method for analyzing intact protein assemblies in near-physiological conditions.
• To advance the capabilities of native mass spectrometry for comprehensive proteome analysis.

Main Methods:

• Online coupling of native capillary zone electrophoresis (nCZE) with an ultra-high mass range (UHMR) Orbitrap mass spectrometer.
• Utilizing nCZE-MS to analyze proteoforms and protein complexes in complex biological samples.
• Employing mass photometry for comparative analysis of detected proteoforms and complexes.

Main Results:

• Successfully measured large proteoforms/complexes up to 400 kDa from complex proteomes.
• The nCZE-MS technique accurately measured a 115-kDa protein complex using only 0.1 ng of material.
• Analysis of E. coli lysate detected 72 proteoforms/complexes (30-400 kDa) in a single run with 50 ng of protein.
• Detected mass distribution correlated well with mass photometry data.

Conclusions:

• This work represents a technical breakthrough in native proteomics.
• The developed nCZE-MS method enables sensitive and comprehensive analysis of complex proteomes.
• This advancement facilitates a more accurate understanding of proteome dynamics in native states.