Comparison of Orbitrap Mass Accuracy Using External and Internal Lock Mass Correction Methods

Importance of the Topic

Precise mass measurement in high-resolution mass spectrometry underpins reliable compound identification and quantitation. Orbitrap analyzers deliver ppm-level accuracy essential for proteomics, metabolomics, and complex mixture analyses. Lock mass correction counters instrumental drift, maintaining stability over extended runs.

Study Objectives and Overview

This work evaluates internal (scan-to-scan) versus external (periodic) lock mass correction on an Orbitrap Eclipse™ Tribrid instrument. It compares mass accuracy and throughput across infusion and LC-MS/MS experiments to define performance trade-offs.

Methodology and Instrumentation

The study employed:

• Orbitrap Eclipse™ Tribrid mass spectrometer with Easy-ETD ion source
• Internal lock mass: continuous fluoranthene (m/z 202) injection per scan
• External lock mass: scheduled SIM scans of fluoranthene at 120k resolution
• Infusion experiments: FlexMix calibration solution at 4 µL/min via ESI, varied AGC targets/resolutions
• LC-MS/MS experiments: 1 µg HeLa digest on Easy-nLC 1200, 120 min gradient, 60k resolving power; data processed in Proteome Discoverer 2.4

Main Results and Discussion

• External correction achieved <5 ppm RMS intra-scan accuracy across diverse conditions; internal correction delivered ~3 ppm.
• External method remained within ~2 ppm of internal results under nominal conditions.
• Even with induced +15 ppm uncorrected drift, external correction maintained <5 ppm RMS accuracy.
• Peptide spectral match error distributions showed ±2.5 ppm (2σ) for external correction under +10 ppm drift.

Benefits and Practical Applications

• Removes ~15 ms per-scan lock mass injection, improving MS/MS duty cycle by up to 15 min over a 120 min gradient.
• Reduces potential interferences and simplifies method setup.
• Enables higher throughput in proteomics, QA/QC, and complex sample workflows.

Future Trends and Applications

Advanced scheduling of external lock mass scans, real-time calibration algorithms, and machine learning-driven drift prediction will further narrow the accuracy gap. Integration into automated, multi-instrument platforms and expansion into metabolomics, lipidomics, and environmental analysis will broaden adoption.

Conclusion

External lock mass correction offers mass accuracy within ~2 ppm of internal methods while significantly increasing scan throughput. Its robustness against drift and reduced injection overhead make it a compelling choice for high-throughput analytical labs.

References

• Makarov A, Denisov E, Lange O, Horning S. High-resolution Orbitrap mass analyzer performance. J Am Soc Mass Spectrom. 2006;17:977–982.
• Olsen JV et al. Internal lock mass correction in proteomics workflows. Mol Cell Proteomics. 2011;4(12):2010–2021.