Enabling Deep Proteome Coverage at Ultra- High-Throughput Using Stepped-quadrupole DIA (SONAR Pulse) on the Xevo™ MRT P10 Benchtop Multi-reflecting TOF Mass Spectrometer

Importance of the topic

Proteomics workflows that combine high depth with high sample throughput are critical for large-scale biological studies, clinical cohorts, and industrial quality control. Traditional data-independent acquisition (DIA) methods face a three-way trade-off between proteome depth, analytical throughput, and precursor specificity when chromatographic gradients are shortened. The application of a stepped-quadrupole DIA approach (SONAR Pulse) on a high-duty-cycle benchtop multi-reflecting TOF seeks to overcome these constraints by preserving MS/MS sensitivity and spectral quality at very high scan rates, enabling deep proteome coverage without sacrificing throughput.

Objectives and study overview

This application note demonstrates a high-throughput DIA workflow implemented on the Xevo MRT P10 mass spectrometer using SONAR Pulse (stepped-quadrupole DIA). The study aims to show that narrow, sequential quadrupole isolation windows combined with very fast MS/MS acquisition and a high TOF duty cycle can:

• Maintain deep proteome coverage on short LC gradients;
• Improve precursor specificity in fast DIA by using many narrow isolation windows per cycle;
• Preserve resolving power and mass accuracy at high acquisition speeds to support confident identification and robust quantification.

Methodology

• Sample: K562 human cell line tryptic digest used as a complex benchmark to evaluate identification depth across fast gradients and different loadings (50 ng and 500 ng).
• Chromatography: High-throughput separations used Evosep Eno standardized short gradients (e.g., up to 500 samples per day, SPD); longer separations used ACQUITY UPLC M-Class for comparison (e.g., 12 SPD and longer).
• Acquisition strategy: SONAR Pulse (stepped-quadrupole DIA) cycle composed of a short MS1 followed by 100 MS/MS scans. Representative acquisition parameters: MS1 50 ms covering m/z 400–900; MS2 acquired at high rate (example 100 Hz) with sequential 5 Da quadrupole isolation windows stepping across m/z 400–900; TOF MS2 detection range m/z 50–1200; total cycle time ~1.05 s.
• Key instrument performance concepts leveraged: high acquisition-rate operation (>100 Hz), minimized interscan delay, wideband EDC synchronization of ion exit with TOF pusher to maximize ion utilization (higher duty cycle and MS/MS sensitivity), and preserved high resolving power at speed.
• Data processing: Raw data exported via waters_connect to mzML and analyzed with DIA-NN2 using a custom spectral library. Search settings included fixed carbamidomethylation (C), variable oxidation (M), 1% FDR, library search mode; other parameters at defaults.

Used instrumentation

• Mass spectrometer: Xevo MRT P10 Benchtop Multi-Reflecting Time-of-Flight (TOF) Mass Spectrometer operated in SONAR Pulse (stepped-quadrupole DIA) mode.
• Chromatography systems: Evosep Eno for high-throughput standardized gradients; ACQUITY UPLC M-Class for extended chromatographic separations.
• Software: waters_connect for mzML export; DIA-NN2 for DIA search and peptide/protein identification.

Main results and discussion

• SONAR Pulse improves the classic DIA trade-offs by enabling many narrow isolation windows per cycle while keeping cycle time short (~1.05 s). The example method used 100 MS/MS windows of ~5 Da across m/z 400–900, producing information-rich fragment spectra even on short gradients.
• MS/MS sensitivity at high scan rates was maintained through hardware and timing optimizations (reduced interscan delays and wideband EDC synchronization), allowing acquisition rates >100 Hz without severe loss of fragment signal. Representative comparisons showed spectral clarity preserved between low and high MS2 scan rates (e.g., 50 Hz versus 200 Hz) and adequate numbers of scans across chromatographic peaks at high speed.
• Resolving power and mass accuracy were preserved at high MS/MS rates, with reported resolving power up to ~100,000 FWHM at speed. This high resolving power reduces spectral interference, enhances fragment ion assignment confidence, and improves quantitative robustness in narrow-window DIA spectra.
• Proteome coverage scaling: Using K562 digests across different SPD settings, protein identifications scaled predictably with separation time. Example outcomes for 500 ng injection: ~4,500 protein groups at 500 SPD (fastest regime) up to ~9,000 proteins on the longest chromatography tested (12 SPD). For 50 ng injection: ~3,200 proteins at 500 SPD and ~8,000 at 12 SPD. These results indicate that a true high-throughput regime can retain substantial proteome depth when MS/MS sensitivity and duty cycle are sufficient.
• Overall, the SONAR Pulse stepped-quadrupole approach delivered improved precursor specificity (narrow windows) and sustained depth and quantitative performance at ultra-high-throughput conditions.

Benefits and practical applications

• High-throughput proteomic studies: Enables large-cohort studies, clinical screening, and population-scale experiments without forcing a severe loss of proteome coverage.
• Rapid method development: Standardized short-gradient protocols (Evosep Eno) become more viable for in-depth DIA when combined with high-rate TOF acquisition and narrow-window DIA.
• Quantitative robustness: Preserved mass accuracy and high resolving power at speed reduce interference-driven quantitation errors common in fast DIA approaches with wide windows.
• Flexible scaling: The workflow scales with sample load and gradient length, allowing users to trade minimal depth for significant gains in throughput when needed.

Future trends and potential applications

• Tighter integration of hardware and software: Continued advances in instrument timing, ion transmission optimization, and DIA-specific acquisition schemes will further increase window counts per cycle and reduce spectral interference.
• AI-driven data interpretation: Machine learning tools (e.g., neural-network based DIA search engines) will continue to improve identification and interference correction in ultra-fast DIA datasets.
• Clinical and regulated environments: High-throughput, high-depth DIA workflows on benchtop platforms could accelerate adoption in clinical proteomics and regulated laboratories, provided robustness and reproducibility are demonstrated across batches and sites.
• Expanded multiplexing and sample formats: Combining rapid DIA with sample multiplexing strategies (e.g., isobaric labeling adapted for DIA) and microflow or nanoflow innovations may increase throughput further while preserving depth.

Conclusion

The SONAR Pulse stepped-quadrupole DIA implementation on the Xevo MRT P10 demonstrates that narrow, sequential isolation windows combined with very fast MS/MS acquisition and a high TOF duty cycle enable deep proteome coverage at ultra-high throughput. Key enablers include hardware timing optimizations that maintain MS/MS sensitivity at >100 Hz, preservation of high resolving power and mass accuracy at speed, and workflow compatibility with standardized short gradients. The approach allows laboratories to increase sample throughput without a commensurate loss of identification depth or quantitative confidence.

References

1. Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods. 2020 Jan;17(1):41-44. doi:10.1038/s41592-019-0638-x.
2. Verenchikov AN, Wildgoose J, Kirillov SN, Vorobyev AV, Makarov VV, Gethings LA, Tonge RP, Daly ME, Johnson WJ, Langridge JI. A Novel Compact Multi-Reflecting Time-of-Flight Mass Spectrometer. J Am Soc Mass Spectrom. 2026 Mar 4;37(3):601-611. doi:10.1021/jasms.5c00321.