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

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 characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380–550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems