Multiple Ion Charge Extraction (MICE) for High-Throughput Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CD-MS) is a single-particle technique, where the masses of individual ions are determined from simultaneous measurements of their mass-to-charge ratio (m/z) and charge. The ions are trapped in an electrostatic linear ion trap (ELIT) and oscillate back and forth through a conducting cylinder connected to a charge-sensitive amplifier. The oscillating ions generate a periodic signal that is processed with fast Fourier transforms (FFTs) to obtain the oscillation frequency (which is related to m/z) and magnitude (which is proportional to the charge). The simultaneous trapping of two or more ions is a way to increase throughput. However, when multiple ions are trapped, it is possible that some of them have overlapping oscillation frequencies, which can lead to an error in the charge determination. To avoid this error, results from overlapping ions are usually discarded. When measurements are performed with many trapped ions, the most abundant m/z species are discarded at a higher rate, which affects the relative abundances in the mass distribution. Here, we report the development of a post-processing method called multiple ion charge extraction (MICE) that uses a statistical approach to assign charges to ions with overlapping frequencies. MICE recovers single-ion information from high signal measurements and makes the relative abundances more resilient to the signal intensity. This approach corrects for high signal m/z biasing, allowing analysis to be faster and more reliable. Using MICE, CD-MS measurements were made at rates of 120 ions/s with little m/z biasing.

Single-Ion Mass Spectrometry for Heterogeneous and High Molecular Weight Samples.

In charge detection mass spectrometry (CD-MS) the mass of each individual ion is determined from the measurement of its mass to charge ratio (m/z) and charge. Performing this measurement for thousands of ions allows mass distributions to be measured for heterogeneous and high mass samples that cannot be analyzed by conventional mass spectrometry (MS). CD-MS opens the door to accurate mass measurements for samples into the giga-Dalton regime, vastly expanding the reach of MS and allowing mass distributions to be determined for viruses, gene therapies, and vaccines. Following the success of CD-MS, single-ion mass measurements have recently been performed on an Orbitrap. CD-MS and Orbitrap individual ion mass spectrometry (I2MS) are described. Illustrative examples are provided, and the prospects for higher resolution measurements discussed. In the case of CD-MS, computer simulations indicate that much higher resolving powers are within reach. The ability to perform high-resolution CD-MS analysis of heterogeneous samples will be enabling and disruptive in top-down MS as high-resolution m/z and accurate charge measurements will allow very complex m/z spectra to be unraveled.

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR.

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of novel symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple point mutation to various amino acids, and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent the first example of tetrahedral geometry across all characterised encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in protein sequence.

Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core.

Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/ß1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Partial genome content within rAAVs impacts performance in a cell assay-dependent manner.

Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance.

CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†.

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

Antibody Binding to Recombinant Adeno Associated Virus Monitored by Charge Detection Mass Spectrometry.

Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector. However, neutralizing antibodies reduce its efficacy. Traditional methods used to investigate antibody binding provide limited information. Here, charge detection mass spectrometry (CD-MS) was used to investigate the binding of monoclonal antibody ADK8 to AAV serotype 8 (AAV8). CD-MS provides a label-free approach to antibody binding. Individual binding events can be monitored as each event is indicated by a shift of the antibody-antigen complex to a higher mass. Unlike other methods, the CD-MS approach reveals the distribution of antibodies bound on capsids, allowing AAV8 subpopulations with different affinities to be identified. The charge state generated by the electrospray of large ions is normally correlated with the structure, and the charge is expected to increase when an antibody binds to the capsid exterior. Surprisingly, binding of the first ADK8 to AAV8 causes a substantial decrease in the charge, suggesting that the first antibody binding event causes a significant structural change. The charge increases for subsequent binding events. Finally, high ADK8 concentrations cause agglutination, where ADK8 links AAV capsids to form dimers and higher order multimers.

Electrostatic Linear Ion Trap Optimization Strategy for High Resolution Charge Detection Mass Spectrometry.

Single ion mass measurements allow mass distributions to be recorded for heterogeneous samples that cannot be analyzed by conventional mass spectrometry. In charge detection mass spectrometry (CD-MS), ions are detected using a conducting cylinder coupled to a charge sensitive amplifier. For optimum performance, the detection cylinder is embedded in an electrostatic linear ion trap (ELIT) where trapped ions oscillate between end-caps that act as opposing ion mirrors. The oscillating ions generate a periodic signal that is analyzed by fast Fourier transforms. The frequency yields the m/z, and the magnitude provides the charge. With a charge precision of 0.2 elementary charges, ions can be assigned to their correct charge states with a low error rate, and the m/z resolving power determines the mass resolving power. Previously, the best mass resolving power achieved with CD-MS was 300. We have recently increased the mass resolving power to 700, through the better optimization of the end-cap potentials. To make a more dramatic improvement in the m/z resolving power, it is necessary to find an ELIT geometry and end-cap potentials that can simultaneously make the ion oscillation frequency independent of both the ion energy and ion trajectory (angular divergence and radial offset) of the entering ion. We describe an optimization strategy that allows these conditions to be met while also adjusting the signal duty cycle to 50% to maximize the signal-to-noise ratio for the charge measurement. The optimized ELIT provides an m/z resolving power of over 300 000 in simulations. Coupled with the high precision charge determination available with CD-MS, this will yield a mass resolving power of 300 000. Such a high mass resolving power will be transformative for the analysis of heterogeneous samples.

Analysis of Megadalton-Sized DNA by Charge Detection Mass Spectrometry: Entropic Trapping and Shearing in Nanoelectrospray.

The analysis of nucleic acids by conventional mass spectrometry is complicated by counter ions which cause mass heterogeneity and limit the size of the DNA that can be analyzed. In this work, we overcome this limitation using charge detection mass spectrometry to analyze megadalton-sized DNA. Using positive mode electrospray, we find two dramatically different charge distributions for DNA plasmids. A low charge population that charges like compact DNA origami and a much higher charge population, with charges that extend over a broad range. For the high-charge population, the deviation between the measured mass and mass expected from the DNA sequence is consistently around 1%. For the low-charge population, the deviation is larger and more variable. The high-charge population is attributed to the supercoiled plasmid in a random coil configuration, with the broad charge distribution resulting from the rich variety of geometries the random coil can adopt. High-resolution measurements show that the mass distribution shifts to slightly lower mass with increasing charge. The low-charge population is attributed to a condensed form of the plasmid. We suggest that the condensed form results from entropic trapping where the random coil must undergo a geometry change to squeeze through the Taylor cone and enter an electrospray droplet. For the larger plasmids, shearing (mechanical breakup) occurs during electrospray or in the electrospray interface. Shearing is reduced by lowering the salt concentration.

Stability of 20S Proteasome Configurations: Preopening the Axial Gate.

Mass spectrometry studies of the stability of the S. cerevisiae 20S proteasome from 11 to 55 °C reveal a series of related configurations and coupled transitions that appear to be associated with opening of the proteolytic core. We find no evidence for dissociation, and all transitions are reversible. A thermodynamic analysis indicates that configurations fall into three general types of structures: enthalpically stabilized, tightly closed (observed as the +54 to +58 charge states) configurations; high-entropy (+60 to +66) states that are proposed as precursors to pore opening; and larger (+70 to +79) partially and fully open pore structures. In the absence of the 19S regulatory unit, the mechanism for opening the 20S pore appears to involve a charge-priming process that loosens the closed-pore configuration. Only a small fraction (≤2%) of these 20S precursor configurations appear to open and thus expose the catalytic cavity.

Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations.

Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

Charge detection mass spectrometry for the analysis of viruses and virus-like particles.

Heterogeneity usually restricts conventional mass spectrometry to molecular weights less than around a megadalton. As a single-particle technique, charge detection mass spectrometry (CDMS) overcomes this limitation. In CDMS, the mass-to-charge (m/z) ratio and charge are measured simultaneously for individual ions, giving a direct mass measurement for each ion. Recent applications include the analysis of viruses, virus-like particles, vaccines, heavily glycosylated proteins, and gene therapy vectors.

Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal.

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.

Analysis of thermally driven structural changes, genome release, disassembly, and aggregation of recombinant AAV by CDMS.

Charge detection mass spectrometry (CDMS) was used to analyze recombinant adeno-associated virus serotype 8 (rAAV8) vectors after incubation at elevated temperatures. rAAV8 vectors with a range of genomes of interest (GOIs) from 2.22 to 4.84 kb were investigated. For the shorter GOIs, GOI release occurred at surprisingly low temperatures (15 min at 45°C for cytomegalovirus [CMV]-GFP). The released DNA and intermediates with the GOI extruded from the capsid were detected. The temperature required to release the short GOIs is well below the 65°C incubation temperature required to disassemble the empty rAAV8 capsid. The temperature for GOI release increased with its GOI length. With the longer GOIs, the GOI stabilized the capsid so that it remained intact under conditions that would disassemble the empty particle. After incubation at 65°C, the main species in the CDMS mass distributions for the longer GOIs was the vector with the GOI. However, for GOIs longer than the wild-type genome (∼4.7 kb), the stability diminished, and genome release occurred at a lower temperature. Heterogeneous DNA fragments from the host cells or plasmids is released at a lower temperature than the longer GOIs, suggesting that the GOIs have a feature that resists early release.

Tryptophan Residues Are Critical for Portal Protein Assembly and Incorporation in Bacteriophage P22.

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.

Analysis of Keratinocytic Exosomes from Diabetic and Nondiabetic Mice by Charge Detection Mass Spectrometry.

Unresolved inflammation compromises diabetic wound healing. Recently, we reported that inadequate RNA packaging in murine wound-edge keratinocyte-originated exosomes (Exoκ) leads to persistent inflammation [Zhou, X. ACS Nano 2020, 14(10), 12732-12748]. Herein, we use charge detection mass spectrometry (CDMS) to analyze intact Exoκ isolated from a 5 day old wound-edge tissue of diabetic mice and a heterozygous nondiabetic littermate control group. In CDMS, the charge (z) and mass-to-charge ratio (m/z) of individual exosome particles are measured simultaneously, enabling the direct analysis of masses in the 1-200 MDa range anticipated for exosomes. These measurements reveal a broad mass range for Exoκ from ∼10 to >100 MDa. The m and z values for these exosomes appear to fall into families (subpopulations); a statistical modeling analysis partially resolves ∼10-20 Exoκ subpopulations. Complementary proteomics, immunofluorescence, and electron microscopy studies support the CDMS results that Exoκ from diabetic and nondiabetic mice vary substantially. Subpopulations having high z (>650) and high m (>44 MDa) are more abundant in nondiabetic animals. We propose that these high m and z particles may arise from differences in cargo packaging. The veracity of this idea is discussed in light of other recent CDMS results involving genome packaging in vaccines, as well as exosome imaging experiments. Characterization of intact exosome particles based on the physical properties of m and z provides a new means of investigating wound healing and suggests that CDMS may be useful for other pathologies.

Hysteresis in Hepatitis B Virus (HBV) Requires Assembly of Near-Perfect Capsids.

The hepatitis B virus (HBV) must release its contents to initiate infection, making capsid disassembly critical to the viral life cycle. Capsid assembly proceeds through a cascade of weak interactions between copies of capsid protein (Cp) to yield uniform particles. However, there is a hysteresis to capsid dissociation that allows capsids to persist under conditions where they could not assemble. In this study, we have sought to define the basis of hysteresis by examining urea-induced dissociation of in vitro-assembled HBV capsids. In general, capsid samples show a mixture of two pools, differentiated by stability. Labile capsid dissociation corresponds to an ∼5 µM pseudocritical concentration of assembly (pcc), the same as that observed in assembly reactions. Dissociation of the stable pool corresponds to a subfemtomolar pcc, indicative of hysteresis. The fraction of stable capsids in an assembly reaction increases with the integrity of the Cp preparation and when association is performed at a higher ionic strength, which modifies the Cp conformation. Labile complexes are more prevalent when assembly conditions yield many kinetically trapped (incomplete and overgrown) products. Cp isolated from stable capsids reassembles into a mixture of stable and labile capsids. These results suggest that hysteresis arises from an ideal capsid lattice, even when some of the substituents in that lattice have defects. Consistent with structural studies that show a subtle difference between Cp dimers and Cp in capsid, we propose that hysteresis arises when HBV capsids undergo a lattice-dependent structural transition.