Rapid Proteome Analysis Using DIA and Super-Resolution Orbitrap Mass Spectrometry

Importance of the Topic

Rapid proteomic workflows support large-scale studies in clinical and industrial settings by delivering high sensitivity and reproducibility within short run times. Maintaining depth of coverage under time constraints requires novel computational and instrumental strategies to enhance mass resolution without extending acquisition cycles.

Objectives and Study Overview

This study evaluates real-time application of the Phase Constrained Spectrum Deconvolution Method phiSDM to full-range mass spectra in data independent acquisition mode on an Orbitrap Exploris 480 coupled to the EvoSep One system. The goal is to double effective resolving power at constant transient times, enabling faster short gradient analyses and improving quantitative proteome coverage.

Methodology and Instrumentation

• Sample preparation involved HeLa cell and plasma digestion using LysC and trypsin, followed by cleanup on C18 tips.
• Chromatography was performed on EvoSep One with preformed gradients of 5, 12, 21 and 44 minutes corresponding to 200, 100, 60 and 30 samples per day.
• Mass spectrometry used Thermo Scientific Orbitrap Exploris 480 with one full MS1 scan and 39 or 53 DIA MS2 windows per cycle.
• An auxiliary GPU-equipped computer processed full-range MS1 and MS2 spectra with phiSDM in real time without adding scan overhead.

Main Results and Discussion

• phiSDM achieved the equivalent of double nominal resolution compared to enhanced Fourier transform processing in the same transient length, resolving isotopic envelopes in 32 ms scans.
• Applying phiSDM to 21 minute gradients increased precursor identifications by up to 19% and protein group identification by 10 to 15% for both library-free and library-based DIA.
• With a 5 minute gradient, nearly 3000 protein groups were identified from 100 ng HeLa digest, representing up to 31% increase in quantified proteins at stringent variability thresholds.
• Quantification consistency improved, raising the number of proteins quantified with coefficients of variation below 20% and 10%.

Benefits and Practical Applications

• Maintaining acquisition cycle times while boosting resolving power enhances proteome depth without compromising throughput.
• Short LC gradients benefit most, enabling up to 300 samples per day workflows with robust identification and quantification.
• Implementing phiSDM on an auxiliary GPU node integrates seamlessly into existing platforms without modifying instrument firmware.

Future Trends and Opportunities

Advances in GPU-accelerated processing and machine learning-driven spectral interpretation promise further gains in speed and resolution. Integration with automated sample handling and real time quality control could establish routine high-throughput clinical proteomics. Further development may extend super-resolution approaches to other mass analyzers and acquisition modes.

Conclusion

Real time full range phiSDM processing on Orbitrap instruments substantially increases resolving power at unchanged cycle times. This strategy delivers deeper and more accurate DIA proteome analyses under minute-scale gradients, paving the way for high throughput applications in research and diagnostics.

References

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