Traditionally, mass spectrometry has been a powerful analytical method enabling the structural analysis of small molecules, and later on peptides and proteins. With the advent of native mass spectrometry, using a combination of electrospray ionisation and time of flight analysis, mass spectrometry could also be applied to the mass determination of large protein complexes such as ribosomes and whole viruses. More recently, ion mobility has been coupled to mass spectrometry providing a new dimension in the analysis of biomolecules, with ion mobility separating ions according to differences in size and shape. In the context of native mass spectrometry, ion mobility mass spectrometry opens up avenues for the detailed structural analysis of large and heterogeneous protein complexes, providing information on the stoichiometry, topology and cross section of these assemblies and their composite subunits. With these characteristics, ion mobility mass spectrometry offers a complementary tool in the context of structural biology. Here, we critically review the development, instrumentation, approaches and applications of ion mobility in combination with mass spectrometry, focusing on the analysis of larger proteins and protein assemblies (185 references).

A large-scale screen to target SARS-CoV-2: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, M ^pro , is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to M ^pro . High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science , this issue p. 642

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of ≈3 and ≈4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Young's modulus of ≈0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.