Native Mass Spectrometry: What is in the Name?

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ

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

“…Native MS utilizes 100% aqueous solutions of low-to-moderate concentrations of volatile salt (e.g., 50 mM ammonium acetate) buffered at neutral pH. 3,4 This type of analysis proceeds commonly after removal of non-volatile salts by buffer exchange followed by nanospray infusion MS. 5–7 Native MS-based intact mass analysis is required for certain classes of biotherapeutics that require preservation of non-covalent associations of protein-protein or protein-ligand complexes. 8 Native MS also affords a fundamental benefit to intact mass analysis of all types of heterogeneous samples where proteins acquire fewer charges and yield spectra at high m/z relative to denatured conditions.…”

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

“…With the growing understanding that protein-protein interactions are directly correlated to biological activity, the domain of native MS has been expanding over the past several decades [91,92]. Analysis of intact proteins under native conditions allows for the detection of any noncovalent interactions and mitigates the incorporation of artificial modifications administered during sample preparation.…”

“…However, a central topic to native MS is whether or not proteins maintain their native conformations following ESI. It was hypothesized that proteins detected by native ESI-MS may not exhibit native structures and are, in fact, partially unfolded during the conversion from the condensed to gas phases [91]. A key experiment involved the comparison of protein collisional cross-sections (Ω), established via ion mobility spectrometry (IMS), with the corresponding Stokes radii (R S ), identified via dynamic light scattering (DLS), which is a technique that allows for the measurement of analyte size distributions in the condensed phase.…”

“…In native MS, noncovalent interactions are preserved, and tertiary and quaternary structures maintained [74,295]. Recently, native MS has emerged as a powerful tool to provide information on topology, stoichiometry, function, dynamics, and composition of protein assemblies [74,91,296]. In more conventional top-down and intact MS characterization of proteins, non-covalent associations are lost as the protein is unfolded under denaturing conditions.…”

“…Currently, there are several commercial solutions for interfacing CZE online to MS that can potentially be used for native MS [60,61,65,67,313]. The ability to separate noncovalent complexes from corresponding unbound complex-forming constituents can address a long-standing debate in the field of native ESI-MS on whether protein complexes and aggregates are formed in the gas phase during electrospray ionization, desolvation and ion manipulation in the mass spectrometer or the native MS data closely represent the structures of non-covalent complexes existing in aqueous solutions of biological systems [91,314]. Such separation supports the capability of native MS to infer the structures of non-covalent macromolecular complexes existing in-solution from the results of gas-phase native ESI-MS measurements [315,316].…”