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.

Figure 1.

(A) The SEC-CZE-ESI-MS/MS platform for native proteomics. (B) An example base peak electropherogram of an SEC fraction of the E. coli lysate after CZE-MS/MS analysis. (C) The mass distribution of the identified proteoforms from the E. coli proteome.

Figure 2.

(A) One deconvolved spectrum of the identified RNA polymerase-binding transcription factor DksA-zinc complex. The averaged mass spectrum across the peak of the complex was used for the mass deconvolution with the Xtract software (Thermo Fisher Scientific) using the default settings. The x-axis is molecular weight (MW). (B) The crystal structure of glutamine-binding periplasmic protein bound with a glutamine molecule. The image of the crystal structure was obtained from the Protein Data Bank in Europe (https://www.ebi.ac.uk/pdbe/). (C) The sequence, observed fragmentation pattern, and detected mass shift of the 50S ribosomal protein L31 through the database search. The location of the mass shift and the cysteine amino acids are highlighted. (D) The molecular function distribution of the identified metalloproteins. The Retrieve/ID mapping tool on the UniProt website (http://www.uniprot.org/uploadlists/) was used to obtain the molecular function information. (E) The metal binding stoichiometry of some identified metalloproteins. The detailed information is shown in Table S3. The error bars for “Others” represent the standard deviations of relative abundance and cysteine count from 13 metalloproteins.