We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the analysis of megadalton biomolecular assemblies. We establish that the signal amplitudes of individual ions scale linearly with their charge, which can be used to resolve mixed ion populations, determine charge states and thus also determine the masses of individual ions. This enables the ultrasensitive analysis of heterogeneous protein assemblies including immunoglobulin oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-associated viruses.Native mass spectrometry (MS) is a powerful tool, enabling mass analysis of intact macromolecular assemblies in the megadalton range 1 . The exact mass of the intact macromolecular complex is then used to infer its composition and the stoichiometry of subunits, post-translational modifications and ligands bound to the complex 2 . Various mass analyzers, including quadrupole time-of-flight, Fourier-transform ion cyclotron resonance and, most recently, Orbitraps, have all been adapted for native MS experiments 3 .Notably, masses are not measured directly in most MS approaches, but need to be inferred from the mass-to-charge (m/z) ratios of the detected ions. As pioneered by Mann and Fenn 4 , the charge states from a population of multiply charged ions generated by electrospray ionization (ESI) can be determined from the m/z values by matching consecutive peaks in the charge state distribution to calculate accurate masses. A general limitation in native MS studies then stems from the fact that the charge state, and thus also the mass, can only be accurately measured when multiple charge states of the same molecular species can be resolved and assigned. This hampers the analysis of larger heterogeneous protein assemblies, such as highly glycosylated proteins, amyloid fibrils, genomepacked viruses and membrane protein complexes decorated with multiple lipid molecules.Even small variabilities in the monomeric building blocks can result in wide distributions of masses in their larger assemblies. In combination with the often poor desolvation, these broadened mass distributions result in overlapping signals between consecutive charge states, leading to inaccurate mass assignments. A possible solution to these problems is to measure one particle (or ion) at a time, thereby avoiding the convolution of signals that stem from insufficient resolving power 5,6 . When such single-particle detection approaches can be combined with an independent measure of the

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.

To enhance the performance of charge-detection mass spectrometry, we investigated the behaviour of macromolecular single ions on their paths towards and within the Orbitrap analyser. Ions with a mass beyond one megadalton reach a plateau of stability and can be successfully trapped for seconds, travelling a path length of multiple kilometres, thereby enabling precise mass analysis with an effective resolution of greater than 100,000 at a mass-to-charge ratio of 35,000. Through monitoring the frequency of individual ions, we show that these high-mass ions, rather than being lost from the trap, can gradually lose residual solvent molecules and, in rare cases, a single elementary charge. We also demonstrate that the frequency drift of single ions due to desolvation and charge stripping can be corrected, which improves the effective ion sampling 23-fold and gives a twofold improvement in mass precision and resolution.