Charge detection mass spectrometry: weighing heavier things.

Charge detection mass spectrometry (CDMS) is a single molecule method where the mass of each ion is directly determined from individual measurements of its mass-to-charge ratio and charge. CDMS is particularly valuable for the analysis of high mass and heterogeneous analytes, where conventional MS methods are often confounded. In the last few years, CDMS has received a renaissance. Technical developments have improved the resolution and dramatically increased the breadth of problems that can be addressed. These improvements have moved CDMS more into the mainstream as interest in the application of mass spectrometry to high molecular weight species has grown. In the article, the three main variants of CDMS are described, along with an overview of recent applications.

Spontaneous Mass and Charge Losses from Single Multi-Megadalton Ions Studied by Charge Detection Mass Spectrometry.

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.

Single-molecule mass spectrometry.

In single-molecule mass spectrometry, the mass of each ion is measured individually; making it suitable for the analysis of very large, heterogeneous objects that cannot be analyzed by conventional means. A range of single-molecule mass spectrometry techniques has been developed, including time-of-flight with cryogenic detectors, a quadrupole ion trap with optical detection, single-molecule Fourier transform ion cyclotron resonance, charge detection mass spectrometry, quadrupole ion traps coupled to charge detector plates, and nanomechanical oscillators. In addition to providing information on mass and heterogeneity, these techniques have been used to study impact craters from cosmic dust, monitor the assembly of viruses, elucidate the fluorescence dynamics of quantum dots, and much more. This review focuses on the merits of each of these technologies, their limitations, and their applications. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:715-733, 2017.

Measurement of the accurate mass of a 50 MDa infectious virus.

RATIONALE: Bacteriophage P22 is believed to contain a total of 521 copies of 9 different proteins and a 41,724 base pair genome. Despite its enormous size and complexity, phage P22 can be electrosprayed, and it remains intact in ultra-high vacuum where its molar mass distribution has been measured. METHODS: Phage P22 virions were generated by complementation in Salmonella enterica and purified. They were transferred into 100 mM ammonium acetate and then electrosprayed. The masses of individual virions were determined using charge detection mass spectrometry. RESULTS: The stoichiometry of the protein components of phage P22 is sufficiently well known that the theoretical molar mass can be determined to within a narrow range. The measured average molar mass of phage P22, 52,180 ± 59 kDa, is consistent with the theoretical molar mass and supports the proposed stoichiometry of the components. The intrinsic width of the phage P22 mass distribution can be accounted for by the distribution of DNA packaged by the headful mechanism. CONCLUSIONS: At over 50 MDa, phage P22 is the largest object with a well-defined molar mass to be analyzed by mass spectrometry. The narrow measured mass distribution indicates that the virions survive the transition into the gas phase intact. Copyright © 2016 John Wiley & Sons, Ltd.

Importin ß Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.

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 ß (Impα and Impß), 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 Impß 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 Impß. Impß density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impß per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impß molecules. Cryo-EM of capsids incubated with excess Impß shows a population of damaged particles and a population of "dark" particles with internal density, suggesting that Impß is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impß. Though the in vitro complexes with great excess of Impß 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.

Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry.

Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector purity are inefficient and low resolution. Here, we show that charge detection mass spectrometry (CDMS) can resolve capsids that contain the entire vector genome from those that contain partial genomes and from empty capsids. Measurements were performed for both single-stranded and self-complementary genomes. The self-complementary AAV vector preparation appears to contain particles with partially truncated genomes averaging at half the genome length. Comparison to results from electron microscopy with manual particle counting shows that CDMS has no significant mass discrimination in the relevant mass range (after a correction for the ion velocity is taken into account). Empty AAV capsids are intrinsically heterogeneous, and capsids from different sources have slightly different masses. However, the average masses of both the empty and full capsids are in close agreement with expected values. Mass differences between the empty and full capsids for both single-stranded and self-complementary AAV vectors indicate that the genomes are largely packaged without counterions.

Acquiring Structural Information on Virus Particles with Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CDMS) is a single-molecule technique particularly well-suited to measuring the mass and charge distributions of heterogeneous, MDa-sized ions. In this work, CDMS has been used to analyze the assembly products of two coat protein variants of bacteriophage P22. The assembly products show broad mass distributions extending from 5 to 15 MDa for A285Y and 5 to 25 MDa for A285T coat protein variants. Because the charge of large ions generated by electrospray ionization depends on their size, the charge can be used to distinguish hollow shells from more compact structures. A285T was found to form T = 4 and T = 7 procapsids, and A285Y makes a small number of T = 3 and T = 4 procapsids. Owing to the decreased stability of the A285Y and A285T particles, chemical cross-linking was required to stabilize them for electrospray CDMS.Graphical Abstract.

Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids.

Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices.

Charge Detection Mass Spectrometry with Almost Perfect Charge Accuracy.

Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's mass-to-charge ratio (m/z) and charge. CDMS has many desirable features: it has no upper mass limit, no mass discrimination, and it can analyze complex mixtures. However, the charge is measured directly, and the poor accuracy of the charge measurement has severely limited the mass resolution achievable with CDMS. Since the charge is quantized, it needs to be measured with sufficient accuracy to assign each ion to its correct charge state. This goal has now been largely achieved. By reducing the pressure to extend the trapping time and by implementing a novel analysis method that improves the signal-to-noise ratio and compensates for imperfections in the charge measurement, the uncertainty has been reduced to less than 0.20 e rmsd (root-mean-square deviation). With this unprecedented precision peaks due to different charge states are resolved in the charge spectrum. Further improvement can be achieved by quantizing the charge (rounding the measured charge to the nearest integer) and culling ions with measured charges midway between the integral values. After ions with charges more than one standard deviation from the mean are culled, the fraction of ions assigned to the wrong charge state is estimated to be 6.4 × 10(-5) (i.e., less than 1 in 15 000). Since almost all remaining ions are assigned to their correct charge state, the uncertainty in the mass is now almost entirely limited by the uncertainty in the m/z measurement.

Charge Detection Mass Spectrometry for Single Ions with an Uncertainty in the Charge Measurement of 0.65 e.

Charge detection mass spectrometry (CDMS) provides a direct measure of the mass of individual ions through nondestructive, simultaneous measurements of the mass to charge ratio and the charge. To improve the accuracy of the charge measurement, ions are trapped and recirculated through the charge detector. By substantially extending the trapping time, the uncertainty in the charge determination has been reduced by a factor of two, from 1.3 elementary charges (e) to 0.65 e. The limit of detection (the smallest charge that can be reliably measured) has been reduced by about the same proportion, from 13 to 7 e. The more precise charge measurements enable a substantial improvement in the mass resolution, which is critical for applications of CDMS to mixtures of high mass ions.

Structurally similar woodchuck and human hepadnavirus core proteins have distinctly different temperature dependences of assembly.

UNLABELLED: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 µM and 43.3 µM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host. IMPORTANCE: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Charge detection mass spectrometry of bacteriophage P22 procapsid distributions above 20 MDa.

RATIONALE: Charge state resolution is required to determine the masses of ions in electrospray mass spectrometry, a feat which becomes increasingly difficult as the mass increases. Charge detection mass spectrometry (CDMS) circumvents this limitation by simultaneously measuring the charge and the m/z of individual ions. In this work, we have used electrospray CDMS to determine the number of scaffolding proteins associated with bacteriophage P22 procapsids. METHODS: P22 procapsids containing a native cargo of scaffolding protein were assembled in E. coli and purified via differential centrifugation. Electrospray CDMS was used to measure their mass distribution. RESULTS: The procapsid peak was centered at 23.60 MDa, which indicates that they contain an average of ~112 scaffolding proteins. The distribution is relatively narrow, less than 31 scaffolding proteins wide. In addition, a peak at 19.84 MDa with a relative abundance of ~15% is attributed to empty capsids. Despite having the same sizes in solution, the empty capsid and the procapsid have significantly different average charges. CONCLUSIONS: The detection of empty capsids is unexpected and the process that leads to them is unknown. The average charge on the empty capsids is significantly lower than expected from the charge residue model, which probably indicates that the empty capsids have contracted in the gas phase. The scaffolding protein presumably limits the contraction of the procapsids. This work shows that electrospray CDMS can provide valuable information for masses greater than 20 MDa.

Charge detection mass spectrometry with resolved charge states.

Charge detection mass spectrometry (CDMS) measurements have been performed for cytochrome c and alcohol dehydrogenase (ADH) monomer using a modified cone trap incorporating a cryogenically cooled JFET. Cooling the JFET increases its transconductance and lowers thermal noise, improving the signal to noise (S/N) ratio. Single ions with as few as 9 elementary charges (e) have been detected. According to simulations, the detection efficiency for ions with a charge of 13 e is 75%, and for charges above 13 e the detection efficiency rapidly approaches 95%. With the low limit of detection achieved here, adjacent charge states are easily resolved in the m/z spectrum, so the accuracy and precision of the image charge measurements can be directly evaluated by comparing the measured image charge to the charge deduced from the m/z spectrum. For ADH monomer ions with 32 to 43 charges, the root mean square deviation of the measured image charge is around 2.2 e. Ions were trapped for over 1500 cycles. The number of cycles detected appears to be limited mainly by collisions with the background gas.