Native Top-Down Proteomics of Endogenous Protein Complexes Enabled by Online Two-Dimensional Liquid Chromatography

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Protein complexes are crucial for biological functions but challenging to study structurally.
• Native top-down mass spectrometry (nTDMS) is powerful for purified complexes but difficult for endogenous ones.
• Current methods for endogenous complexes are often slow and require extensive sample preparation.

Purpose of the Study:

• To develop a high-throughput method for structural analysis of endogenous protein complexes.
• To enable direct analysis of protein complexes from complex biological mixtures without extensive prefractionation.
• To advance the field of native top-down proteomics (nTDP).

Main Methods:

• Developed a nondenaturing online two-dimensional liquid chromatography (2D-LC) system.
• Integrated size-exclusion and mixed-bed ion-exchange chromatography for automated separation.
• Employed a multistage native top-down mass spectrometry (nTDMS) approach for comprehensive analysis.

Main Results:

• Achieved high coverage of endogenous protein complexes using the automated online 2D-LC nTDP system.
• Successfully captured intact noncovalent complexes, released subunits/cofactors, and backbone fragments.
• Detected 133 native proteoforms and endogenous protein complexes (up to 350 kDa) from human heart tissue in under 2 hours.

Conclusions:

• The developed online 2D-LC nTDP method significantly enhances throughput for endogenous protein complex structural characterization.
• This technique allows direct analysis of complex biological samples, overcoming limitations of previous methods.
• Represents a major advancement for the high-throughput structural study of endogenous protein complexes in their native state.