Determination of collision cross sections of proteins using frequency domain linewidths and time domain decay profile fitting of MS1 data from an Orbitrap mass spectrometer

Significance of the Topic

Accurate determination of protein collision cross sections is essential for understanding biomolecular conformations and interactions. Traditionally measured by dedicated ion mobility instruments, CCS values provide insights into protein folding, complex assembly, and quality control in proteomics and biopharmaceutical analyses.

Goals and Study Overview

This study evaluates two approaches to derive protein CCS values directly from MS1 transients acquired on an Orbitrap mass spectrometer. The objectives are to compare CCS obtained by fitting time-domain decay profiles versus extracting full width at half maximum in the frequency domain, and to assess performance for isolated charge states and complex multi-charge ensembles.

Methodology and Instrumentation

The experimental workflow involves collecting full MS1 scans or isolating individual charge states on an Orbitrap instrument. Transients are processed by fast Fourier transform in Matlab and a narrow frequency window is selected. Two CCS algorithms are applied:

• Decay profile fitting: a time-domain exponential fit yields decay constants related to CCS.
• FWHM fitting: Lorentzian peak widths in the frequency domain are measured and converted to CCS.

Instrumental Setup

• Orbitrap mass spectrometer for transient acquisition
• Matlab for fast Fourier transform processing
• OriginLab for Lorentzian FWHM fitting

Main Results and Discussion

Both methods produce CCS values for model proteins (cytochrome C, myoglobin, ubiquitin) that closely match literature ion mobility benchmarks. Data from isolated single charge states and from unresolved multi-charge mixtures yield comparable CCS by both approaches. The decay fitting method currently delivers slightly higher precision, while the FWHM approach shows promise for samples with limited isotopic resolution if peak deconvolution is optimized.

Benefits and Practical Applications

The demonstrated workflows enable CCS measurement without specialized ion mobility hardware, leveraging standard Orbitrap MS1 data. This integration streamlines structural analyses in proteomics, biopharmaceutical characterization, and quality control, providing simultaneous mass and shape information in a single experiment.

Future Trends and Opportunities

Advances in frequency-domain peak fitting algorithms and improved transient processing may elevate the FWHM method to parity with decay fitting. Integration of CCS computation into vendor software and high-throughput pipelines will expand accessibility. Extension to other biomolecules, complexes, and coupling with top-down or native MS promises broader structural insights in life sciences and industrial analytics.

Conclusion

Protein CCS values derived from Orbitrap MS1 transients by both decay profile fitting and frequency-domain FWHM approaches agree well with ion mobility references. While decay fitting currently offers superior accuracy, the FWHM method represents a viable and evolving alternative for rapid CCS assessment in routine mass spectrometry workflows.

Reference

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