Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography–Capillary Zone Electrophoresis–Tandem Mass Spectrometry

Abstract

Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size-exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for the characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics.

Identification and Quantification of Proteoforms by Mass Spectrometry

“…A detailed protocol for CZE‐MS/MS‐based top‐down proteomics has been provided to facilitate adoption of this technique . More recently, the Sun lab developed an SEC‐CZE‐MS/MS system for native top‐down proteomics which identified 23 protein complexes in E. coli . This work showed the first example of native top‐down proteomic analysis of a complex proteome using online liquid‐phase separation.…”

Proteoforms: General Concepts and Methodological Process for Identification

“…The technique utilizes non-denaturing and non-reducingbuffer conditions during the electrospray ionization process which helps preserve the primary and quaternary compositions of proteins and their complexes for MS [19,20]. In native proteoform approach, analytical platforms for high-resolution and liquid-phase separation of protein complexes are required prior to native mass spectrometry (MS) and MS/MS [21]. During, Escherichia coli proteome analysis, 144 proteins, 672 proteoforms, and 23 protein complexes were identified, coupling the size-exclusion chromatography and capillary zone electrophoresis-MS/ MS [21].…”

“…In native proteoform approach, analytical platforms for high-resolution and liquid-phase separation of protein complexes are required prior to native mass spectrometry (MS) and MS/MS [21]. During, Escherichia coli proteome analysis, 144 proteins, 672 proteoforms, and 23 protein complexes were identified, coupling the size-exclusion chromatography and capillary zone electrophoresis-MS/ MS [21]. Other separation techniques have been combined; coupled off-line ionexchange chromatography or gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), which demonstrates the compatibility of native GELFrEE with native and tandem mass spectrometry [21,22].…”

“…During, Escherichia coli proteome analysis, 144 proteins, 672 proteoforms, and 23 protein complexes were identified, coupling the size-exclusion chromatography and capillary zone electrophoresis-MS/ MS [21]. Other separation techniques have been combined; coupled off-line ionexchange chromatography or gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), which demonstrates the compatibility of native GELFrEE with native and tandem mass spectrometry [21,22]. An illustrative scheme of the elucidation of the proteoform structures by the bottom-up and top-down techniques is presented in Figure 2.…”

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015–mid 2018)

“…To achieve more exhaustive proteome coverage, multidimensional separation strategies combining orthogonal modes of separation are recommended for proteomic analysis . Multidimensional separations are performed either in on‐line or off‐line modes.…”