Native Capillary Electrophoresis–Mass Spectrometry of Near 1 MDa Non‐Covalent GroEL/GroES/Substrate Protein Complexes

Abstract

Protein complexes are essential forproteins' folding and biological function. Currently, native analysis of largemultimeric protein complexes remains challenging. Structural biology techniquesare time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reportedto characterize, under near‐physiological conditions, the conformationalrearrangements of ∽1 MDa GroEL upon complexation with binding partners involvedin a protein folding cycle. The developed CE‐MS method is fast (30min per run), highly sensitive (low‐amol level), and requires ∽10 000‐foldfewer samples compared to biochemical/biophysical techniques. The methodsuccessfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of naturalsubstrate proteins with GroEL14 can be detected and quantified.The technique allows monitoring of GroEL14 conformational changesupon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). NativeCE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants resultin up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus directinfusion ESI–MS. This study shows the CE–MS potential to provide information onbinding stoichiometry and kinetics for various protein complexes.

Coupling online size exclusion chromatography with charge detection-mass spectrometry using Hadamard transform multiplexing

“…HT multiplexing can be broadly applied to any chromatography system that uses isocratic gradients and that is capable of handling multiple injections, making it a generalized and versatile technique for generating high-quality LC-CD-MS data. For example, isocratic HIC has been applied to antibodies, 38 and CE-MS has been successfully used with proteins as large as GroEL, 39 both of which would be compatible with multiplexed injections and CD-MS data acquisition. The two primary limitations of the method are that it does not work with gradient separations and that it cannot be used for online sample clean up because contaminants from one injection could overlap with the analyte in another injection.…”

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry

“…[25] More recently, direct infusion nM was employed to measure protein complexes from a hum heart tissue lysate using a Fourier-transform ion cyclotro resonance (FTICR) mass spectrometer with the identification a handful of protein complexes about 30 kDa or smaller. Native CZE-MS (nCZE-MS) has high separation efficiency an high detection sensitivity for protein complexes and has bee applied to analyzing low-complexity protein samples, i. monoclonal antibodies, [27] large protein complexes like GroE (near 1MDa), [28][29][30] ribosomes, [31] and nucleosomes. [32] Nati SEC fractionation and online nCZE-MS analysis of an E. coli c lysate identified 23 protein complexes smaller than 30 kD representing the first native proteomics study of a compl proteome using online liquid-phase separation-MS. [33] Howeve those native proteomics studies are either too time and labo consuming or only able to detect small proteoforms/prote complexes from complex proteomes.…”