Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry

Abstract

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Keywords: Capillary zone electrophoresis; Mass photometry; Native mass spectrometry; Native proteomics; Protein complex.

Figure 1.

Flow chart of nCZE-ESI-MS for native proteomics of an E. coli cell lysate. The Figure is created using the BioRender and used here with permission.

Figure 2.

Summary of detected proteoforms or protein complexes from an E. coli cell lysate using nCZE-ESI-UHMR. (A) Representative electropherogram of nCZE-ESI-UHMR analyses of the E. coli cell lysate. (B)–(F) Mass spectra of five examples of large proteoforms/protein complexes detected. The charge states and deconvolved mass of each proteoform/protein complex are labelled. (G) Linear correlation between the most abundant charges and theoretical Rayleigh charges (Z_R) of all proteoforms/protein complexes detected in single-shot nCZE-UHMR. (H) Alignment of the mass distribution of proteoforms/protein complexes in the E. coli cell lysate from mass photometry (black dash line) and nCZE-UHMR (red line) analyses.