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 under physiological conditions.
• Current nMS applications are limited to well-purified protein complexes, with few trials on complex samples like whole cell lysates.
• Characterizing large proteoforms and protein complexes in complex biological samples remains a significant challenge.

Purpose of the Study:

• To develop and validate a novel native proteomics approach for analyzing large proteoforms and protein complexes directly from complex proteomes.
• To establish a sensitive and efficient method for comprehensive proteome analysis using native capillary zone electrophoresis coupled to mass spectrometry (nCZE-MS).
• To demonstrate the capability of nCZE-MS in identifying and quantifying native protein species in a complex biological matrix.

Main Methods:

• Online coupling of native capillary zone electrophoresis (nCZE) with an ultra-high mass range (UHMR) Orbitrap mass spectrometer for native proteomics.
• Utilizing nCZE-MS to analyze protein complexes and proteoforms in complex samples, such as whole cell lysates.
• Employing mass photometry for comparative analysis of detected proteoform and protein complex mass distributions.

Main Results:

• The nCZE-MS technique successfully measured a 115-kDa standard protein complex using only 0.1 ng of protein.
• Analysis of an *E. coli* cell lysate detected 72 proteoforms or protein complexes within the 30-400 kDa mass range, using just 50 ng of protein material.
• The mass distribution of identified native protein species closely matched results from mass photometry measurements.

Conclusions:

• This work presents a technical breakthrough in native proteomics, enabling the measurement of large proteoforms and protein complexes from complex proteomes.
• The developed nCZE-MS method offers high sensitivity and broad applicability for studying native protein assemblies in biological systems.
• This advancement significantly enhances the ability to capture a comprehensive view of proteome dynamics in near-physiological states.