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Review
. 2022 Apr 27;122(8):7415-7441.
doi: 10.1021/acs.chemrev.1c00377. Epub 2021 Oct 12.

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology

Martin F Jarrold  1
Affiliations
Review

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology

Martin F Jarrold. Chem Rev. .

Abstract

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.

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Conflict of interest statement

The author declares the following competing financial interest(s): The author is a shareholder in Megadalton Solutions, a company that is engaged in commercializing CDMS.

Figures

Figure 1.
Figure 1.
How CDMS measurements are performed. (a) Schematic diagram showing the main components of a state-of-the-art ion-trap CDMS instrument. (b) Cross section through the ELIT showing the detection cylinder between the two end-caps. The white line shows the trajectory of a trapped ion oscillating on the trap axis. (c) Time sequence of the potentials applied to the end-caps to trap ions. (d) Time domain signal resulting from a 0.1 s trapping event for a single ion. The inset shows an expanded view of the signal between 0.041 and 0.042 s (represented by the two closely spaced black vertical lines near the center of the plot). Oscillations in the signal due to the ion passing back and forth through the detection cylinder are apparent. (e) FFT of the full trapping event. The fundamental is at ~10 kHz, and the sequence of higher-frequency peaks is due to harmonics. The odd-numbered harmonics have much larger magnitudes than the even-numbered ones. (a) Adapted with permission from ref . Copyright 2019 American Chemical Society.
Figure 2.
Figure 2.
Charge-state resolution in CDMS. (a) Simulated charge spectra for three different charge RMSDs. The top spectrum has an RMSD of 0.1 e, the middle spectrum is 0.2 e, and the bottom spectrum is 0.4 e. The black line in the bottom spectrum is the sum. An RMSD of ~0.2 e is needed for good charge-state resolution. (b) RMSD after charge quantization (i.e., after assigning each ion to the nearest charge state) plotted against the RMSD of the initial distribution (red line). The black line is a guide showing the situation where the two RMSDs are equal. (c) Fraction of ions assigned to the wrong charge state plotted against the initial RMSD (red line). The blue line shows the reduction in the fraction misassigned when ions with charges near the midpoints between two integer charge states are discarded. In this case, ions within ±0.15 e of the midpoints were culled. (d) Schematic of a charge-sensitive amplifier configured for low charge signals. (e) Charge RMSD plotted against m/z. Results are shown for an Amptek A250 charge-sensitive amplifier with a 2SK152 JFET at the input (black line) and for an implementation of the Bertuccio design where the feedback resistor is removed (blue line). The input JFETs for both amplifiers were cryogenically cooled to 130–140 K, and in both cases the measurement time was 1.5 s. (b, c) Adapted with permission from ref . Copyright 2015 American Chemical Society. (d) Reproduced with permission from ref . Copyright 2020 American Chemical Society. (e) Adapted with permission from ref . Copyright 2020 American Chemical Society.
Figure 3.
Figure 3.
CDMS measurements for HBV capsids. (a) Mass distribution showing prominent peaks for icosahedral capsids with 90 Cp dimers (T = 3) at ~3 MDa and with 120 Cp dimers (T = 4) at ~4 MDa. The insets show the icosahedral geometries of the T = 3 and T = 4 capsids. (b) Charge spectrum measured for the T = 4 capsid showing good charge-state resolution. (c) Mass distribution for an HBV assembly reaction that stalled, leaving many trapped intermediates. (a, c) Adapted with permission from ref . Copyright 2020 American Chemical Society.
Figure 4.
Figure 4.
Resolution of charge states in both charge and m/z dimensions by CDMS allowing for deconstruction of the m/z spectrum. (a) m/z spectrum for the mass distribution in Figure 3c. The inset shows an expanded view of the resonance at an m/z of ~16 750 Da. (b) Charge versus m/z scatter plot for the ions in the m/z spectrum in (a). Each ion is represented by a point. The points are clustered into groups where ions with the same charge states are resolved in both the m/z and charge dimensions. The inset shows an expanded view of a portion of the scatter plot that shows the groups more clearly, and the green circle shows an expanded view of a portion of the inset. Two of the groups of ions are encompassed by red circles (see text). The red lines are lines of constant mass (1.412 MDa) that correspond to the mass of the (dimer)42 oligomer. The groups of points falling on the red line make up the m/z envelope for this oligomer mass. The red arrow in (b) shows the location in the inset of the resonance in (a) at an m/z of ~16 750. Adapted with permission from ref . Copyright 2020 American Chemical Society.
Figure 5.
Figure 5.
CDMS spectrum measured for the heavily glycosylated SARS-CoV-2 S protein trimer (blue line). The gray line shows the mass distribution measured for β-galactosidase under identical conditions. The dashed vertical line shows the sequence mass of the unglycosylated trimer (414.2 kDa). The red line shows the mass distribution predicted for a statistical distribution using the glycan populations determined from glycoproteomic measurements (see text). The orange inset shows the measured charge distribution, which shows good resolution of the charge states (RMSD = 0.191 e). Adapted with permission from ref . Copyright 2021 American Chemical Society.
Figure 6.
Figure 6.
Tau amyloid fibers. (a) CDMS mass distribution for tau amyloid fibers plotted using 5 MDa bins. Low-mass and high-mass populations are distinguished by dark and light shading, respectively. (b) Charge versus mass heat map for tau amyloid fibers. The dashed lines are guides indicating how the charge depends on mass for the two populations. (c) Transmission electron microscopy (TEM) image of tau amyloid fibers. Scale bar: 200 nm. Features assigned to spherical oligomers are shown with white arrows. Adapted with permission from ref . Copyright 2018 Royal Society of Chemistry under Creative Commons Attribution 3.0 Unported License (CC By 3.0), https://creativecommons.org/licenses/by/3.0/legalcode.
Figure 7.
Figure 7.
Time-resolved CDMS spectra showing the progression of HBV capsid assembly over the first 90 min: (a) for a reaction mixture containing an initial dimer concentration of 5 μM in 210 mM ammonium acetate; (b) for a 10 μM dimer in 210 mM ammonium acetate; and (c) for a 10 μM dimer in 510 mM ammonium acetate. The earliest time interval (0–10 min) is at the front. The spectra were generated using 20 kDa bins. Each spectrum is normalized by the sum of all ions measured for each time point. Adapted with permission from ref . Copyright 2017 American Chemical Society.
Figure 8.
Figure 8.
CDMS spectrum measured for BMV capsid proteins assembled with 52 nt polyadenine oligonucleotides. The insets show the H8 and H15 geometries determined from the CDMS and cryo-EM studies. The peak at 2.5 MDa is assigned to the H8 structure, and the one at 3.4 MDa is assigned to the H15. Adapted with permission from ref . Copyright 2020 John Wiley and Sons.
Figure 9.
Figure 9.
CDMS measurements for mature bacteriophage P22. (a) Main components of P22. (b) CDMS mass distribution. The inset shows an expanded view of the main peak in the spectrum. Adapted with permission from ref . Copyright 2016 John Wiley and Sons.
Figure 10.
Figure 10.
CDMS mass distribution for AAV gene-therapy vectors. (a) CDMS mass distribution measured for a sample of empty AAV8 particles. The red dashed line shows the calculated mass distribution. An external view of the 25 nm AAV8 capsid is shown in the inset. (b) CDMS mass spectrum for the ATCC AAV8 Reference Standard (blue line). The yellow line shows the spectrum × 10 and offset vertically. The dashed red line shows the distribution expected when the sample heterogeneity and experimental resolution are included. The red arrow shows the location of the empty AAV8 particle. Adapted with permission from ref . Copyright 2021 Elsevier under Creative Commons Attribution 4.0 International Public License (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/legalcode.
Figure 11.
Figure 11.
CDMS measurements for adenovirus. (a) CDMS mass spectrum measured for HAdV5. An external view of HAdV5 is shown in the inset. The hexons are purple, the pentons are blue, and the fibers are green. (b) Charge versus mass heat map for the data in (a). Warmer colors indicate higher intensity. Adapted with permission ref . Copyright 2021 American Chemical Society.
Figure 12.
Figure 12.
CDMS analysis of classical vaccines. (a) Mass spectrum measured by CDMS for IPOL vaccine. The expected masses of the C- and D-antigen particles (see text) are shown by the purple and green lines, respectively. The bin size is 20 kDa. (b) Charge spectra of IPOL (black) and the AAV8 reference standard (red). The bin size is 1 e. (c) Structure of the triple-layered rotavirus. (d) Mass spectrum measured by CDMS for the RotaTeq vaccine. Colored vertical lines show expected masses of the DLP with all pentonal trimers (blue) and the DLP with all pentonal trimers but missing the genome (green). The bin size is 100 kDa. (a, b, d) Adapted with permission from ref . Copyright 2021 American Chemical Society. (c) Adapted with permission from ref . Copyright 2014 Elsevier under Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License (CC By-NC-ND 3.0) https://creativecommons.org/licenses/by-nc-nd/3.0/legalcode.
Figure 13.
Figure 13.
Fab binding to CPV capsids. (a) Mass distributions measured for CPV at FAB E concentrations of 0, 1.5, and 10 μM. The average mass shift provides information on the average number of Fabs bound, and the peak width provides information on the distribution of bound Fabs. The insets show cryo-EM images of CPV without Fabs (top) and saturated with 60 Fabs (bottom). (b) Plot of the average number of Fab E bound to CPV as a function of Fab E concentration. (c) Similar plot for Fab F. The black points are the measured values determined by CDMS. (d) Width of the Fab E distribution plotted against the Fab E concentration. (e) Similar plot for Fab F. The red lines show the predictions of the standard (Langmuir) model for ligand binding, where all sites have the same intrinsic affinity. The blue lines show the prediction of a model where there are CPV subpopulations with different affinities. In (b) and (c) the red and blue lines are coincident. Adapted with permission from ref . Copyright 2018 American Chemical Society.
Figure 14.
Figure 14.
CDMS mass spectra measured for lipoproteins. (a) Mass distribution measured for HDL. (b) Mass distribution for LDL. The peak in the LDL spectrum at 230 kDa is probably an HDL impurity. (c) Mass distribution measured for VLDL. The insets show structures of the lipoprotein particles. Adapted with permission from ref . Copyright 2018 American Chemical Society.
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