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

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes 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 unique 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 cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new 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 a unique example of tetrahedral geometry when surveying all characterized 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 the protein sequence.

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.

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.

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.

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology.

Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of their mass-to-charge ratio (m/z) and charge. Masses are determined for thousands of individual ions, and then the results are binned to give a mass spectrum. Using this approach, accurate mass distributions can be measured for heterogeneous and high-molecular-weight samples that are usually not amenable to analysis by conventional mass spectrometry. Recent applications include heavily glycosylated proteins, protein complexes, protein aggregates such as amyloid fibers, infectious viruses, gene therapies, vaccines, and vesicles such as exosomes.

Integrative structure and functional anatomy of a nuclear pore complex.

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.

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.

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.

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.

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.

Core Protein-Directed Antivirals and Importin ß Can Synergistically Disrupt Hepatitis B Virus Capsids.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impß) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

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.

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

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.

Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry.

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by "top-down" CDMS measurements are 35-47% larger than those obtained from the "bottom-up" glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host's immune system.

Characterization of Classical Vaccines by Charge Detection Mass Spectrometry.

Vaccines induce immunity by presenting disease antigens through several platforms ranging from individual protein subunits to whole viruses. Due to the large difference in antigen size, the analytical techniques employed for vaccine characterization are often platform-specific. A single, robust analytical technique capable of widespread, cross-platform use would be of great benefit and allow for comparisons across manufacturing processes. One method that spans the antigen mass range is charge detection mass spectrometry (CDMS). CDMS is a single-ion approach where the mass-to-charge ratio (m/z) and charge are measured simultaneously, allowing accurate mass distributions to be measured for heterogeneous analytes over a broad size range. In this work, CDMS was used to characterize the antigens from three classical multivalent vaccines, inactivated poliomyelitis vaccine (IPOL), RotaTeq, and Gardasil-9, directly from commercial samples. For each vaccine, the antigen purity was inspected, and in the whole virus vaccines, empty virus particles were detected. For IPOL, information on the extent of formaldehyde cross-linking was obtained. RotaTeq shows a narrow peak at 61.06 MDa. This is at a slightly lower mass than expected for the double-layer particle, suggesting that around 10 pentonal trimers are missing. For Gardasil-9, buffer exchange of the vaccine resulted in very broad mass distributions. However, removal of the virus-like particles from the aluminum adjuvant using a displacement reaction generated a spectrum with narrow peaks.

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the Stabilitome.

The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an Escherichia coli lysate is provided to extend this approach to characterize the thermal stability of a proteome. As the solution temperature is increased, proteins and protein complexes undergo structural and organizational transitions; for each species, the folded ↔ unfolded and assembled ↔ disassembled populations are monitored based on changes in vT-ESI-MS charge state distributions and masses. The robustness of the approach illustrates a step toward the proteome-wide characterization of thermal stabilities and structural transitions-the stabilitome.

Determination of Antibody Population Distributions for Virus-Antibody Conjugates by Charge Detection Mass Spectrometry.

Virus-like particle (VLP) conjugates are being developed for biomedical applications; however, there is a lack of quantitative analytical methods to measure the extent of conjugation and modification of VLP based therapeutics. Charge detection mass spectrometry (CDMS) can measure mass distributions for large and heterogeneous complexes and is emerging as a valuable tool in the analysis of biologics. In this study, CDMS is used to characterize the stoichiometry and population distribution of antibodies covalently conjugated to the surface of a bacteriophage MS2 VLP. Initial CDMS analysis of the unconjugated MS2 particles suggested that they had packaged a broad distribution of exogenous genomic material. We developed procedures to remove the undesired genomic material from the VLP preparation and observed that, for the samples where the genomic fragments were removed, the antibody coupling reaction efficiency increased by almost a factor of 2. This meant there were (1) fewer VLPs with no antibodies bound, which is an important consideration for the efficacy of a targeted therapeutic and (2) fewer antibodies were wasted during the coupling reaction. CDMS could be employed in a similar manner as a tool to characterize coupling reaction product distributions and precursors and help inform the development of the next generation of conjugate-based therapies.

Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry is a single particle technique where the masses of individual ions are determined from simultaneous measurements of each ion's m/z ratio and charge. The ions pass through a conducting cylinder, and the charge induced on the cylinder is detected. The cylinder is usually placed inside an electrostatic linear ion trap so that the ions oscillate back and forth through the cylinder. The resulting time domain signal is analyzed by fast Fourier transformation; the oscillation frequency yields the m/z, and the charge is determined from the magnitudes. The mass resolving power depends on the uncertainties in both quantities. In previous work, the mass resolving power was modest, around 30-40. In this work we report around an order of magnitude improvement. The improvement was achieved by coupling high-accuracy charge measurements (obtained with dynamic calibration) with higher resolution m/z measurements. The performance was benchmarked by monitoring the assembly of the hepatitis B virus (HBV) capsid. The HBV capsid assembly reaction can result in a heterogeneous mixture of intermediates extending from the capsid protein dimer to the icosahedral T = 4 capsid with 120 dimers. Intermediates of all possible sizes were resolved, as well as some overgrown species. Despite the improved mass resolving power, the measured peak widths are still dominated by instrumental resolution. Heterogeneity makes only a small contribution. Resonances were observed in some of the m/z spectra. They result from ions with different masses and charges having similar m/z values. Analogous resonances are expected whenever the sample is a heterogeneous mixture assembled from a common building block.

Charge Detection Mass Spectrometry Measurements of Exosomes and other Extracellular Particles Enriched from Bovine Milk.

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle's mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.