Evaluation of high dynamic range MS1 acquisition methods on a Hybrid Orbitrap mass spectrometer.

Importance of the Topic

Proteomic analysis requires accurate quantification across a wide abundance range. Low-abundance proteins are challenging to detect and quantify due to dynamic range limitations in mass spectrometry. High dynamic range (HDR) MS1 scanning addresses this issue by boosting the signal-to-noise ratio of weak ions, thereby improving detection of low-intensity species in complex mixtures.

Objectives and Study Overview

This study evaluated a novel HDR MS1 acquisition scheme on an Orbitrap Exploris 480 mass spectrometer within a data-independent acquisition (DIA) workflow. The primary goal was to compare standard full-scan MS1 with the HDR mode to assess gains in dynamic range, signal detection, and quantitative precision in shotgun proteomics.

Methodology and Instrumentation

Alternating full-scan experiments were implemented: regular MS1 versus HDR MS1 at 120 000 resolution. The HDR approach divided the precursor mass range into 12 overlapping windows, with a survey scan determining optimized injection times for each window. A DIA method followed, using MS1 resolution of 90 000 and MS2 resolution of 30 000, covering m/z 400–900 (MS1) and 145–1450 (MS2).

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system
• 25 cm × 75 µm Aurora Ultimate column with internal emitter
• Thermo Fisher EASY-Spray Source
• Orbitrap Exploris 480 mass spectrometer configured for HDR MS1 and DIA

Main Results and Discussion

• Low-abundance ions exhibited a substantial increase in signal-to-noise with HDR scans, while high-intensity signals remained robust.
• Detected signals increased by 116 %, LCMS features by 119 %, and features with charge state > 1 by 72 % compared to regular MS1.
• The total number of identifications (precursors, peptides, protein groups) was unchanged despite a slight decrease in scan count.
• Quantitative precision improved: peptides with CV < 20 % rose by 17 % and CV < 10 % by 15 %; protein groups showed similar gains (13 % and 19 %, respectively).
• The enhancement was most pronounced for low-intensity analytes, confirming the effectiveness of extended injection times in boosting weak signals.

Benefits and Practical Applications

The HDR MS1 strategy enables more reliable quantification of low-abundance proteins, essential for studies with limited sample input or single-cell analyses. By doubling the number of detectable features without compromising identification rates, this method supports sensitive biomarker discovery, quality control in pharmaceutical development, and clinical proteomics.

Future Trends and Possibilities

• Integration of HDR scanning with real-time data-dependent decision algorithms to further enhance selectivity.
• Application to single-cell and spatial proteomics workflows where sample amounts are minimal.
• Refinement of mass range segmentation and injection time algorithms for higher throughput.
• Extension of HDR principles to additional mass spectrometer platforms for broader adoption.

Conclusion

HDR MS1 scans on an Orbitrap Exploris 480 significantly improve the detection and quantification of low-abundance peptides in DIA workflows. The approach enhances signal-to-noise ratios, increases feature recovery, and refines quantitative precision without affecting overall identification performance, making it a valuable tool for advanced proteomic analyses.

References

1. Meier F, Geyer PE, Virreira Winter S et al. BoxCar acquisition method enables single-shot proteomics at a depth of 10 000 proteins in 100 minutes. Nat Methods. 2018;15:440–448.
2. Jenkins C, Orsburn B. BoxCar Assisted MS Fragmentation (BAMF). BioRxiv. 2019. doi:10.1101/860858.