Although native MS was initially primarily used to study soluble protein assemblies, membrane protein complexes have recently entered the realm of native MS through electrospraying of these assemblies from detergent micelles, nanodiscs, or even native lipid membranes. (14) Native MS has matured substantially since then, and now many groups are applying this technology to study all sorts of proteins, their assemblies, and the interactions between proteins and ligands, including small-molecule drugs, cofactors, lipids, nucleotides, DNA, and RNA ( Figure 1). In the early years of this century, the term “native MS” was coined to describe this area of biomolecular mass spectrometry. (18,19) The first examples of that focused on the analysis of intact non-covalent complexes, which remained intact and largely retained their quaternary structures throughout their transfer from the solvent into the gas phase up until they hit the mass analyzer’s detector. However, through a plethora of experimental work over the past decades, it has become apparent that when conditions are carefully managed, electrospray ionization may provide gas-phase ions of proteins and protein complexes that retain many of their native features.
Thus, a fully native state can never be retained. (13−17) This task is not trivial, as the biomolecule is charged in the ionization process and stripped of all solvent molecules before mass analysis can occur under (ultra)high-vacuum conditions. In native MS, the aim is to bring the analyte into the mass analyzer while retaining its original native structure and inter- and intramolecular interactions as much as possible. Although the birth of native MS can be traced back to the early 1990s, (10,11) just a few years after the introduction of electrospray ionization mass spectrometry (ESI-MS), (12) MS-based technologies involving the analysis of intact proteins and protein complexes are still not as mature as their peptide-centric counterparts, mainly because of the challenges behind efficient ionization and detection of the larger intact protein ions. In contrast to the technologies outlined above, native mass spectrometry (native MS), the core focus of this review, analyzes intact proteins and their non-covalent complexes, as well as other biomolecules, in a native-like folded state. Several excellent reviews are available covering all of these distinct flavors of MS-based proteomics. Additionally, it can also be used to chart proteome-wide protein–protein interactions and various post-translational modifications (PTMs).
MS-based proteomics provides a means to measure proteome-wide protein abundances and monitor them upon perturbation of a system. Herein proteins are identified and quantified following enzymatic digestion into easily amenable smaller peptides, whose sequences can be determined by different fragmentation methods and matched by well-developed search algorithms against protein, RNA, and DNA databases. Focusing on proteins and peptides, to date the dominant portion of MS-based analysis is performed by peptide-centric proteomics. This diversity originates not only from the many different biomolecules that can be analyzed and investigated, such as proteins, peptides, lipids, DNA, RNA, carbohydrates, and metabolites, but also from the wide assortment of tools available to characterize them.
This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of─among others─biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein–ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.īiological mass spectrometry comes in many flavors.
With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained.