Advancing high-resolution charge… (R01GM155650) – Project Grant to Trustees Of Indiana University

Abstract: Charge detection mass spectrometry (CD-MS) is an emerging technology that allows accurate mass distributions to be measured for heterogeneous and high mass samples. IT is a single ION approach where the mass to charge ratio (m/z) and charge are measured simultaneously for each ION, and then multiplied to give the ION's mass. Measurements are performed for thousands of ions, which are then binned into a mass distribution. The m/z ratio and charge are measured using a detection cylinder embedded in an electrostatic linear ION trap (elit). Trapped ions oscillate back and forth through the detection cylinder and the signal from the oscillating ions is detected by a charge sensitive amplifier. The oscillation frequency gives the m/z ratio, and the charge is obtained from the signal amplitude. CD-MS is early enough in its development cycle that substantial technical improvements are still occurring. In this project we address the two main limitations of CD-MS: 1) its moderate resolving power, and 2) the relatively long time needed to measure a spectrum. The mass resolving power is limited by the precision of the m/z determination. The best m/z resolving power reported to date is 700. Using computer simulations, we have designed elits with resolving powers over 300,000. However, to perform at this level, the elits need to be perfectly aligned. Computer simulations indicate we can overcome the alignment problem by segmenting some of the elit electrodes and applying slightly different voltages to the segments to recover the high resolution. Our second goal is to substantially reduce the time needed to measure a spectrum. To achieve this goal, we will a) reduce the trapping time needed to resolve charge states, and b) reduce ION-ION interactions so that more ions can be trapped and measured at the same time. The trapping time required for charge state resolution can be reduced by lowering electrical noise, which will be achieved by implementing a novel design for the charge sensitive amplifier. ION-ION interactions will be reduced by optimizing elit designs so that ions are trapped in trajectories that do not interact. Combining these advances, we expect to perform high-resolution CD-MS measurements at 500-1000 ions/s. The advances described above will be transformative for CD-MS and are expected to have a broad impact. We will explore three applications. 1) high resolution CD- MS analysis of the adeno-associated virus (AAV) gene therapy vectors will reveal subpopulations with different combinations of capsid proteins. The subpopulation relative abundances, and any correlations with post translational modifications, may help understand lot-to-lot variability in AAV preparations. 2) heteroaryldihydropyrimidines (haps) lead to aberrant assembly of hepatitis b virus (HBV) capsids, and they are being investigated as potential HBV antivirals. High resolution CD-MS measurements will be performed to monitor HAP binding to HBV capsids and assembly intermediates to probe the nature of the binding interactions. 3) to explore the use of high resolution and high throughput CD-MS to detect and monitor biomarkers, we will use CD-MS to measure intact urinary proteins to determine how many can be directly detected.

Action	Obligated	Date
Amendment	$274,622	Aug 20, 2026
Amendment	$270,289	Aug 14, 2025
Original award	$265,939	Sep 23, 2024

Advancing high-resolution charge detection-mass spectrometry

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