Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry

Abstract

Membrane proteins represent the majority of clinical drug targets and are actively involved in a range of cellular processes. However, the complexity of membrane mimetics for membrane protein solubilization poses challenges for native mass spectrometry (MS) analyses. The most common approach for native MS analyses of membrane proteins remains offline buffer exchange into native MS-compatible buffers prior to manual sample loading into static nano-ESI emitters. This laborious process requires relatively high sample consumption and optimization for the individual proteins. Here, we developed online buffer exchange coupled to native mass spectrometry (OBE-nMS) for analyzing membrane proteins in different membrane mimetics, including detergent micelles and nanodiscs. Detergent screening for OBE-nMS reveals that mobile phases containing ammonium acetate with lauryl-dimethylamine oxide are most universal for characterizing both bacterial and mammalian membrane proteins in detergent. Membrane proteins in nanodiscs simply require ammonium acetate as the mobile phase. To preserve the intact nanodiscs, a novel switching electrospray approach was used to capture the high-flow separation on the column with a low-flow injection to MS. Rapid OBE-nMS completes each membrane protein measurement within minutes and thus enables higher-throughput assessment of membrane protein integrity prior to its structural elucidation.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

“…This is a short SEC column purposely designed for rapid buffer exchange and subsequent elution with very fast runtimes (<5 min), and thus not well-suited for analyte separation. 40 Because of the very narrow chromatographic peaks arising from the OBE column, it represents a likely "worst case scenario" with respect to rapidly changing ion flux and its associated challenges. We tested OBE-CDMS using a fixed ion injection time to assess whether single ion scans can be maintained over the short chromatographic elution (Figure 2).…”

“…To demonstrate the validity of this hypothesis, we chose to initially use the NativePAC OBE-1 column (hereby referred to as OBE). This is a short SEC column purposely designed for rapid buffer exchange and subsequent elution with very fast runtimes (<5 min), and thus not well-suited for analyte separation . Because of the very narrow chromatographic peaks arising from the OBE column, it represents a likely “worst case scenario” with respect to rapidly changing ion flux and its associated challenges.…”

“…Native SEC-MS is already well integrated into R&D pipelines of biopharmaceutical companies, and by implementing SEC-CDMS the range of applications for therapeutic products might be expanded. Indeed, with the latest generation of SEC columns, it now becomes possible to tackle increasingly complex analytes of different nature and size, including membrane proteins, 40 glycoproteins, 35 plasmid DNAs, 49 mRNA, or adeno-associated viruses.…”

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

“…10 However, a recent study to evaluate the feasibility of online SEC-CD-MS produced deconvolved mass spectra that, while sufficient to measure accurate protein masses, had lower signal-to-noise ratios (SNR) and contained significantly more erroneous peaks than spectra produced from infusion experiments. 11 A second study using online buffer exchange 12,13 with CD-MS for the analysis of IgM assemblies was also able to produce sufficient mass distributions after careful optimization of MS conditions but was still limited to only several hundred ions per run. 14 A third study explored the use of capillary electrophoresis (CE) and other fluidic devices with CD-MS and observed that short elution profiles often produced low-quality CD-MS spectra.…”

An Online Native Mass Spectrometry Approach for Fast, Sensitive, and Quantitative Assessment of Adeno-Associated Virus Capsid Content Ratios

“…This method involves the direct coupling of desalting size-exclusion chromatography (SEC) with native MS for rapid online removal of nonvolatile salts and exchange into MS-compatible buffers . While this approach has proven highly effective for the analysis of proteins and protein complexes, − OBE has not yet been extended to AAV. We therefore saw an opportunity to improve the applicability of native MS for routine analysis of intact AAV by employing a fully integrated online strategy.…”