State-of-the-art rapid stepped collision energy in an Orbitrap Astral Zoom Mass Spectrometer

Significance of the Topic

The development of stepped collision energy techniques in mass spectrometry accelerates data acquisition and improves sensitivity for complex biomolecule analysis such as glycopeptides. The Orbitrap Astral Zoom mass spectrometer introduces rapid stepped NCE to address the need for faster scan rates without compromising fragmentation quality, crucial for high-throughput proteomics, quantitative assays, and structural elucidation.

Objectives and Study Overview

This study evaluates the performance of rapid stepped collision energy in a Thermo Scientific Orbitrap Astral Zoom mass spectrometer. Key aims included measuring scan rate enhancements, assessing glycopeptide identification efficiency, and examining the impact on quantitative TMT-based workflows compared to the standard Orbitrap Astral MS.

Methodology and Instrumentation

• LC Setup: Thermo Scientific Vanquish Neo UHPLC system with Aurora Ultimate TS C18 column, 0.1% formic acid in water (A) and 80% acetonitrile/0.1% formic acid (B).
• Mass Spectrometry: Orbitrap Astral Zoom MS operated in positive ion mode, DDA experiments with one to three stepped collision energies (NCEs).
• Samples: 500 ng human plasma digest and 500 ng TMT11plex Yeast Digest Standard.
• Calibration: Thermo Scientific Pierce FlexMix solution for scan rate characterization.
• Data Analysis: Byonic v5.3.44 against UniProt human database, 1% FDR, glycopeptide filtering (score >200).

Main Results and Discussion

• Scan Rate: At 3 ms injection, stepped NCE scan rates reached 155 Hz (two NCEs) and 135 Hz (three NCEs), more than double the Orbitrap Astral MS.
• Glycopeptide Identification: 13.3% more glycopeptides identified with three NCEs and 11.5% with two NCEs compared to the Orbitrap Astral MS.
• Quantitative TMT Analysis: Similar proteome coverage achieved with single versus stepped NCEs; stepped NCE improved reporter ion signal-to-noise without loss of PSMs, peptides, or proteins.
• Byonic Scores: Higher average scores observed with three stepped NCEs, indicating improved fragmentation quality.

Benefits and Practical Applications

• Enhanced Throughput: Faster MS2 acquisition rates enable deeper coverage in shorter runs.
• Improved Sensitivity: More glycopeptide identifications and higher-quality spectra support structural and site-localization studies.
• Reliable Quantitation: Stepped collision energy preserves quantitative accuracy in TMT experiments while enhancing reporter ion signals.

Future Trends and Applications

The integration of rapid stepped collision energy is expected to expand DIA and DDA workflows, facilitate single-cell proteomics, and support real-time method optimization with AI-driven acquisition strategies. Further hardware and software refinements may push scan rates higher and improve fragment ion coverage for large-scale glycoproteomics and PTM mapping.

Conclusion

Rapid stepped collision energy on the Orbitrap Astral Zoom MS delivers significant scan speed improvements and enhanced glycopeptide identification without compromise in quantitative workflows. This advancement offers a robust platform for high-throughput proteomics and complex biomolecular analyses.

References

• Garland JM, Arrey TN, Denisov E, et al. State-of-the-art Glycopeptide Identification Using an Orbitrap-Astral Mass Spectrometer. HUPO2024 P-I-0115; 2024.