Higher Resolution Charge Detection Mass Spectrometry

Abstract

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.

Figure 1.

Higher resolving power CDMS mass spectrum measured for an HBV Cp149 assembly reaction. There are prominent peaks attributable to the icosahedral T = 3 and T = 4 capsids. The bin size is 2.5 kDa.

Figure 2.

Higher resolving power CDMS mass spectrum measured for another HBV Cp149 assembly reaction. In this case, there appear to be many trapped intermediates between the dimer at 33.54 kDa and the T = 4 capsid at 4.04 MDa and beyond. (a) Full composite spectrum assembled from three overlapping spectra covering the low-mass region (0–200 kDa), middle -mass region (200–2000 kDa), and high-mass region (2–5 MDa). (b) Expanded view of the high-mass regime. The bin size is 2.5 kDa.

Figure 3.

m/z spectrum corresponding to the mass distribution in Figure 2. The bin size is 20 Da. The red and blue scales mark the expected positions of resonances due to ions with different masses and charges having the same m/z values (see text).

Figure 4.

Charge versus m/z scatter plot for the mass distribution shown in Figure 2. Each ion is represented by a point. The points fall into clusters that are resolved m/z charge states. The insert is an expanded view of a portion of the scatter plot to show the resolved m/z charge states more clearly. The red line shows a line of constant mass corresponding to the (dimer)₂₂ oligomer. The clusters of points falling on this line make up the m/z envelope for this oligomer mass.

Figure 5.

Expanded view of a portion on the m/z spectrum in Figure 3 showing the resonance centered on around 16 770 Da (black line). The bin size is 20 Da. The m/z spectrum is overlaid with a portion of the charge versus m/z scatter plot from Figure 4 (blue points). Note how the clusters of ions in the m/z scatter plot align vertically to generate the resonances in the m/z spectrum.

Figure 6.

Plot of the mass deviation (measured mass minus expected mass using a dimer mass of 33 541 Da) against the number of capsid protein dimers. The red and green lines are guides showing regions where the mass deviation shows a close to linear increase. Below around 60 dimers the rate of increase is 100 Da/dimer. Above around 60 dimers the rate of increase is 340 Da/dimer.