Native top-down proteomics is a rapid expanding field for characterization of protein complexes, antibodies and virus assemblies under near-physiological conditions. Compared with bottom-up and denatured top-down strategies, which will destroy noncovalent interaction among proteins and cofactors during sample preparation and reversed-phase LC separation, native top-down proteomics will allow us to accurately understand the dynamics of proteins. Capillary zone electrophoresis (CZE)-ESI-MS/MS has shown great potential for large-scale native proteomics due to its high separation efficiency for large biomolecules, mature CE-MS interfaces and high-resolution separation and high-sensitive detection of protein complexes in native condition. We established a native size-exclusion chromatography (SEC)-CZE-ESI-MS/MS platform for large-scale native top-down proteomics of complex proteome. In our previous work, we applied the platform for Escherichia coli (E. coli) cell lysate and identified 144 proteins, 672 proteoforms, and 23 protein complexes. In this work, we further improved our platform and moved forward to a more complex sample, the mouse brain proteome. Each SEC fraction was split to two aliquots, and analyzed with both the native platform and a denatured approach. If a protein complex is detected in both experiments, we could recognize the corresponding mass shifts should represent covalent modifications. The direct comparison of the same fraction could increase the identification accuracy. So far, we identified 65 gene products and 421 proteoforms from the native top-down approach. Protein complexes like the homodimer of Dynein light chain 2 and cytochrome c-heme complex were observed. Only small proteins were detected due to the limitation of our mass spectrometer, so we will apply the platform to a TOF instrument in the future. To extend the identification, other orthogonal separation methods like ion exchange chromatography will be employed to further decrease the sample complexity. In-capillary preconcentration method under native condition will be developed to increase loading capacity and sensitivity of the platform.

Native top-down proteomics for mouse brain proteome with capillary zone electrophoresis-tandem mass spectrometry

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.