Enhanced resolution with multi-pass mode on the Orbitrap Astral Zoom mass spectrometer

Importance of the Topic

The Orbitrap Astral Zoom mass spectrometer with multi-pass mode significantly improves resolving power for low m/z analytes. This advance is essential for high-plex quantitative proteomics, such as TMT 32-plex, where baseline separation of reporter ions spaced by just 3 mDa is required.

Study Objectives and Overview

This study introduces an enhanced resolution multi-pass method on the Orbitrap Astral Zoom platform. Main aims:

• Extend the ion path to boost resolving power by more than two-fold for small m/z.
• Implement an automatic tuning routine to optimize analyzer voltages via a genetic algorithm.
• Evaluate performance in TMT HR Mode, comparing single-pass and multi-pass modes for TMT 11-plex and 32-plex samples.

Methodology and Used Instrumentation

The enhanced method uses a fast-switching relay prism deflector and asymmetric ion mirrors to trap ions for multiple drift passes. Key methodological elements include:

• Multi-pass mode extending ion path from ~30 m to ~90 m.
• Compensation of optical aberrations via dynamic focusing mirror electrode voltages.
• Automatic parameter optimization using a genetic search algorithm to maximize transmission and suppress overtones.
• Shot-to-shot switching between single-pass and three-pass modes with settling times under 2 ms.

Used Instrumentation

• Orbitrap Astral Zoom mass spectrometer with Ion Routing Multipole and HDR detector.
• Fast-switching relay prism deflector and asymmetric mirror ion optics.
• Quadrupole mass filter.
• Thermo Scientific Vanquish Neo LC system with IonOpticks Aurora Frontier XT column.

Main Results and Discussion

The multi-pass mode achieved resolutions above 120 k at m/z 138, over twice that of the standard Astral analyzer. TMT32-plex reporter ions were baseline resolved at 3 mDa spacing with average resolution >100 k and mass accuracy within ±200 ppm. Quantitative analysis of TMT 32-plex labeled HeLa digest yielded 4 299 protein groups and 22 146 peptides (94 % and 78 % coverage), demonstrating excellent consistency with expected ratios. Shot-to-shot voltage switching showed stable performance and rapid settling, supporting high-throughput acquisition.

Benefits and Practical Applications

• Enables high-multiplex TMT quantification up to 32 channels with baseline resolution.
• Improves proteome coverage and depth via increased resolving power.
• Supports high throughput (> 440 samples per day) for large-scale proteomics studies.
• Compatible with both standard and TMT HR acquisition schemes.

Future Trends and Possibilities of Use

Anticipated developments include integration of multi-pass mode with data-independent acquisition for deeper proteome profiling, further scaling to higher plex reagents, and real-time adaptive tuning for variable sample types, including single-cell proteomics.

Conclusion

The enhanced multi-pass Orbitrap Astral Zoom method more than doubles resolving power at low m/z, enabling robust TMT 32-plex quantification with high mass accuracy and throughput. This approach represents a significant advance for quantitative proteomics.

References

• Stewart H et al. Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High‐Throughput Quantitative Analysis. Anal. Chem. 2023;95(42):15656–15664.
• Stewart H et al. Proof of principle for enhanced resolution multi-pass methods for the Astral analyzer. Int. J. Mass Spectrom. 2024;498:117203.
• Kraegenbring J et al. Enhancing the resolving power on a novel Orbitrap Astral instrument for TMT35plex applications. ASMS 2025;MP 748.