Explore the vast range of titles available.

In charge detection mass spectrometry (CDMS), ions are passed through a detection tube and the m / z ratio and charge are determined for each ion. The uncertainty in the charge and m / z determinations can be dramatically reduced by embedding the detection tube in an electrostatic linear ion trap (ELIT) so that ions oscillate back and forth through the detection tube. The resulting time domain signal can be analyzed by fast Fourier transforms (FFTs). The ion’s m / z is proportional to the square of the oscillation frequency, and its charge is derived from the FFT magnitude. The ion oscillation frequency is dependent on the physical dimensions of the trap as well as the ion energy. A new ELIT has been designed for CDMS using the central particle method. In the new design, the kinetic energy dependence of the ion oscillation frequency is reduced by an order of magnitude. An order of magnitude reduction in energy dependence should have led to an order of magnitude reduction in the uncertainty of the m / z determination. In practice, a factor of four improvements was achieved. This discrepancy is probably mainly due to the trajectory dependence of the ion oscillation frequency. The new ELIT design uses a duty cycle of 50%. We show that a 50% duty cycle produces the lowest uncertainty in the charge determination. This is due to the absence of even-numbered harmonics in the FFT, which in turn leads to an increase in the magnitude of the peak at the fundamental frequency. Graphical Abstract ᅟ

Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a capsid with different surface chemistries. Viruses have been re-engineered for many applications but the ability to build asymmetric capsids in vitro has been lacking. Here, the authors report the design of asymmetric subunits with different growth rates leading to capsids with discrete patches and potential for distinct surface chemistries.

Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin β (Impα and Impβ), which bind to the core protein's C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impβ without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impβ. Impβ density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impβ per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impβ molecules. Cryo-EM of capsids incubated with excess Impβ shows a population of damaged particles and a population of \"dark\" particles with internal density, suggesting that Impβ is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impβ. Though the in vitro complexes with great excess of Impβ are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.

Spontaneous mass and charge losses from individual multi-megadalton ions have been observed with charge detection mass spectrometry (CDMS) by trapping single hepatitis B virus (HBV) capsids for 3 s. Gradual increases in the oscillation frequency of single ions in the ion trap are attributed mainly to mass loss (probably solvent, water, and/or salt). The total mass lost during the 3 s trapping period peaks at around 20 kDa for 4 MDa HBV T = 4 capsids. Discrete frequency drops punctuate the gradual increases in the oscillation frequencies. The drops are attributed to a sudden loss of charge. In most cases a single positive charge is lost along with some mass (on average around 1000 Da). Charge loss occurs for over 40% of the trapped ions. It usually occurs near the beginning of the trapping event, and it occurs preferentially in regions of the trap with strong electric fields, indicating that external electric fields promote charge loss. This process may contribute to the decrease in m / z resolution that often occurs with megadalton ions. Graphical Abstract ᅟ

This article provides a review of recent studies of the properties of unsolvated (and partially solvated) peptides and proteins. The methods used to produce vapor-phase peptide and protein ions are described along with some of the techniques used to study them, such as H/D exchange, blackbody infrared radiative dissociation, and ion mobility measurements. Studies of unsolvated peptides and proteins provide information about their intrinsic intramolecular interactions. The topics covered include the role of zwitterions and salt bridges in the vapor phase, Coulomb interactions in multiply charged ions, the unfolding and refolding of vapor-phase proteins, and the stability of unsolvated helices and sheets. Finally, dehydration and rehydration studies of proteins in the vapor phase are described. These can provide exquisitely detailed information about hydration interactions, such as the enthalpy and entropy changes associated with adsorbing individual water molecules.

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex. The structure of the yeast nuclear pore complex, determined at sub-nanometre precision using an integrative approach that combines a wide range of data, reveals details of its architecture, transport mechanism and evolutionary origins. Structure of yeast nuclear pore complex Nuclear pore complexes (NPCs) are the gateways for the transport of RNA and proteins into and out of the nucleus. Although previous work has provided structural insights into parts of NPCs, getting a full picture of these complexes has proved difficult owing to their large size and dynamic nature. A multidisciplinary team of researchers now report the structure of the entire 552-protein NPC of the yeast at a sub-nanometre level using an integrative approach including mass spectrometry, cryo-electron tomography and chemical crosslinking. The structure provides a detailed view of the functional elements of the NPC and clarifies its architecture, mechanism of action and evolutionary origins.

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly.

Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5′ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5′ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5′ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

An atmospheric pressure interface transports ions from ambient pressure to the low-pressure environment of a mass spectrometer. A capillary coupled to an ion funnel is widely used. However, conventional ion funnels do little to negate the large amount of energy picked up by high-mass ions from the gas flow through the capillary. There has been little work done on the effects of gas flow on ion transmission, and the previous studies have all been limited to low-mass, low-charge ions. In this work, we account for the effects of gas flow, diffusion, and electric fields (static and oscillating) on ion trajectories and use simulations to design a new hybrid ion funnel-ion carpet (FUNPET) interface that transmits a broad mass range with a single set of instrument conditions. The design incorporates a virtual jet disruptor where pressure buildup and counter flow dissipate the supersonic jet that results from gas flow into the interface. This, and the small exit aperture that can be used with the FUNPET, reduces the gas flow into the next stage of differential pumping. The virtual jet disruptor thermalizes ions with a broad range of masses (1 kDa to 1 GDa), and once thermalized, they are transmitted into next region of the mass spectrometer with low excess kinetic energy. The FUNPET interface is easy to fabricate from flexible printed circuit board and a support frame made by 3D printing. The performance of the interface was evaluated using charge detection mass spectrometry. Graphical Abstract ᅟ