Setting the STORI Straight: Improved CDMS Results Via misSTORI Analysis

Importance of the Topic

Charge detection mass spectrometry (CDMS) is a powerful technique for determining the mass of very large ions, such as viral capsids, that are difficult to analyze by conventional mass spectrometry. However, extended acquisition times increase the chance of ion collisions and desolvation events that shift an ion’s mass‐to‐charge ratio (m/z) during detection, leading to underestimated charge and biased mass measurements. The misSTORI approach addresses these challenges by correcting for frequency shifts, enabling more accurate mass assignments and revealing underlying ion behavior.

Objectives and Overview of the Study

The primary goals of this work were to compare traditional STORI and misSTORI processing modes for CDMS data, evaluate the impact of acquisition length on charge precision and full/empty viral capsid ratios, and demonstrate how misSTORI analysis provides novel insights into ion desolvation and collision events inside the mass analyzer.

Methodology and Instrumentation

Data Collection and Processing:

• Instrument: Thermo Fisher Q Exactive UHMR Hybrid Quadrupole‐Orbitrap in Direct Mass Technology (DMT) mode.
• Data processing: Custom Python scripts and STORIboard software implementing traditional STORI (no frequency correction) and misSTORI (dynamic correction via phase derivative).

misSTORI Algorithm:

• Initial frequency centroids generate traditional STORI curves.
• Derivative of complex STORI values yields a phase plot capturing frequency error over time.
• Linear segments fitted to the phase allow dynamic adjustment of frequency and improved charge determination.

Main Results and Discussion

Charge Precision:

• At 0.5 s and 1.0 s acquisitions, both algorithms perform similarly.
• At 2.0 s acquisitions, traditional STORI quality degrades due to uncorrected m/z shifts, whereas misSTORI continues to improve charge precision.

Full/Empty Capsid Ratios:

• misSTORI yields consistent full:empty AAV2 capsid ratios across 1, 2, and 4 s transients.
• Traditional STORI misrepresents ratios beyond 1 s, making quantification unreliable.

Ion Desolvation and Mass Shifts:

• Under poor desolvation, “empty” AAV8 capsids lose thousands of daltons, while “full” capsids lose only hundreds, indicating residual solvent in nominally empty particles.
• misSTORI detects rare mass gains (~132 Da) in AAV ions, attributed to collision and capture of Xenon atoms, demonstrating its sensitivity to collision events.

Comparison with STFT:

• Short‐time Fourier transform (STFT) methods struggle to pinpoint the timing of small frequency changes due to lower precision in short windows.
• misSTORI phase analysis using the entire transient provides clearer identification of frequency jumps.

Benefits and Practical Applications

• Enables use of longer acquisition times to improve signal‐to‐noise and charge precision without bias from m/z shifts.
• Provides accurate full/empty viral capsid quantification crucial for gene therapy quality control.
• Offers mechanistic insights into solvent loss, collision capture events, and ion stability within the analyzer.

Future Trends and Applications

• Extending misSTORI to other large biomolecules, complexes, and nanoparticles.
• Integration with real‐time processing and machine learning for on‐the‐fly correction and classification.
• Further exploration of collision dynamics by varying buffer gases and temperature conditions.
• Incorporation into standard CDMS workflows for industrial and clinical laboratories.

Conclusion

misSTORI represents a significant advancement over traditional STORI processing by dynamically correcting m/z shifts during CDMS acquisitions. This results in improved charge precision, reliable quantification of viral capsids, and novel insight into ion behavior such as desolvation and neutral collisions. The approach unlocks the full potential of long‐transient measurements, paving the way for more accurate and informative analyses of large ions.

References

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5. Miller ZM et al. JASMS 2022, 33, 2129–2137.