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.
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.
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.
For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment. Coarse-grained models suggest that the reaction occurs on a downhill energy landscape, so intermediates are expected to be fleeting. In this work, charge detection mass spectrometry (CDMS) has been used to track assembly of the hepatitis B virus (HBV) capsid in real time. The icosahedral T = 4 capsid of HBV is assembled from 120 capsid protein dimers. Our results indicate that there are multiple pathways for assembly. Under conditions that favor a modest association energy there is no accumulation of large intermediates, which indicates that available pathways include ones on a downhill energy surface. Under higher salt conditions, where subunit interactions are strengthened, around half of the products of the initial assembly reaction have masses close to the T = 4 capsid and the other half are stalled intermediates which emerge abruptly at around 90 dimers, indicating a bifurcation in the ensemble of assembly paths. When incubated at room temperature, the 90-dimer intermediates accumulate dimers and gradually shift to higher mass and merge with the capsid peak. Though free subunits are present in solution, the stalled intermediates indicate the presence of a local minima on the energy landscape. Some intermediates may result from hole closure, where the growing capsid distorts to close the hole due to the missing capsid proteins or from a species where subsequent additions are particularly labile.
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.
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