Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

Abstract

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g. specialized ribosomes).

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

“…Examples include size exclusion chromatography (SEC), ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and capillary electrophoresis (CE). 16–19 On-line separations offer notable advantages to infusion-based native MS applications. MS spectra can be correlated with retention time (RT) to provide orthogonal confirmation of isoform assignments.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Nevertheless, detection of aggregates is an important advantage of the analytical technique for characterization of mAbs. A previous study out of our lab has also reported on dimers detected in native CZE-MS analysis for a different mAb [2]. In addition to aggregates, species corresponding to dissociated light chain and light chain dimers, as well as intact mAb with a loss of two light chains were also observed.…”

“…A trace quantity of the mAb is thereby composed of light chain domains bound via non-covalent interactions instead of disulfide bonds. The presence of such species has been reported previously, and such fragments are known to occur under native conditions [2,[286][287][288].…”

“…A recent study from our laboratory using the CESI interface has demonstrated the separation of recombinant human interferon beta-1 (rhINF-β1, ~23,000 Da), where 138 proteoforms were detected and 55 quantitated [1]. In another study, mixtures of native proteins and protein complexes, a monoclonal antibody, and the protein extract from the E. coli ribosomes were characterized using CESI interface in one of the first examples of native CZE coupled online to ESI-MS [2].…”

“…Native CZE-MS was demonstrated as a viable technique for the analysis of single protein isolates, such as mAbs, simple mixtures of proteins and protein complexes, and for the protein isolate from an entire cellular organelle -the ribosome. This chapter is based on a 2017 publication in the Journal of the American Society for Mass Spectrometry [2].…”