Thermo Scientific Direct Mass Technology mode

Importance of the topic

Mass spectrometry is a cornerstone of analytical chemistry for characterizing biomolecules and complex assemblies. Native and intact protein analysis provides critical insights into structure, post-translational modifications, and noncovalent interactions. However, conventional ensemble measurements often struggle with charge-state overlap, limited resolution, and the need for extensive deconvolution, particularly for large heterogeneous assemblies. The development of parallel individual ion measurement addresses these challenges by delivering direct mass determination, enhancing sensitivity, and expanding the accessible mass range up to the megadalton scale.

Objectives and Study Overview

This study introduces Thermo Scientific™ Direct Mass Technology™ mode implemented on the Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ mass spectrometer. Its goals are to demonstrate how simultaneous measurement of mass-to-charge ratio (m/z) and ion charge (z) for individual ions improves resolution, dynamic range, and accuracy. Examples cover complex glycoproteins (fetuin-A), membrane proteins (Aquaporin Z), and biotherapeutics (Etanercept), illustrating the method’s versatility and impact on structural biology and pharmaceutical analysis.

Methodology

Direct Mass Technology mode records induced charge signals on the Orbitrap’s outer electrode for each ion, generating Selective Temporal Overview of Resonant Ions (STORI) plots. The slope of each STORI trace correlates directly with ion charge. A calibration algorithm converts STORI slopes to charge values, and individual m/z and z measurements are combined to compute precise masses. Parallel acquisition captures hundreds of individual ions per spectrum, enabling high-throughput analysis without relying on isotopically resolved ensemble spectra.

Instrumentation

• Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer equipped with Direct Mass Technology mode
• STORIboard processing software for data calibration, STORI plot analysis, and spectrum reconstruction
• Automated ion population control and charge calibration workflows within instrument control and processing packages

Main Results and Discussion

Direct individual ion measurements eliminate the need for m/z-based deconvolution and deliver up to 20-fold resolution improvements over ensemble methods. Native fetuin-A analysis doubled the number of uniquely assigned glycoforms by resolving closely spaced modifications. Aquaporin Z membrane protein characterization achieved both monomeric and tetrameric mass distributions at high sensitivity, revealing adducts and isotopic patterns at the tetramer level. Etanercept profiling directly identified over 100 glycoforms and extended the observable mass distribution beyond 125 kDa, offering a more comprehensive proteoform landscape.

Benefits and Practical Applications

• Direct mass determination removes ambiguity from deconvolution and ensemble overlap
• Enhanced resolution and dynamic range enable detection of subtle modifications and high-mass complexes
• Reduced sample quantity requirements through hypersensitive individual ion detection
• Accelerated workflows for native protein complexes, membrane proteins, and therapeutic biologics
• Improved QA/QC and structural characterization in biopharmaceutical development

Future Trends and Applications

Further integration of charge detection mass spectrometry with advanced data analytics and machine learning will accelerate proteoform discovery and structural proteomics. Expanding throughput through multiplexed workflows, coupling with ion mobility, and pushing mass limits toward virus-scale assemblies are anticipated. The combination of Direct Mass Technology mode with emerging front-end separation and sample preparation techniques will unlock deeper insights into cellular machinery, virus particles, and next-generation drug modalities.

Conclusion

Direct Mass Technology mode on the Q Exactive UHMR platform represents a paradigm shift in high-mass biomolecular analysis. By measuring individual ion charge in parallel with m/z, this approach delivers precise mass assignments, dramatically improved resolution, and a broadened dynamic range. Its capability to resolve complex proteoforms, membrane assemblies, and therapeutic biologics with minimal sample investment makes it a powerful tool for proteomics, structural biology, and biopharmaceutical research.

References

1. Kafader et al., Journal of the American Society for Mass Spectrometry, 2019, 30:2200–2203
2. Lin et al., Journal of Proteome Research, 2018, 17:2861–2869
3. AlphaFold Protein Structure Database, entry P02765
4. Wohlschlager et al., Nature Communications, 2018, 9:1713