SureQuant intelligence-driven MS: a new paradigm for targeted quantitation

Importance of the Topic

The precise quantification of predefined peptide targets by LC-MS is essential in applications ranging from clinical biomarker validation to industrial QA/QC and translational biology. High sensitivity, specificity, and reproducibility are critical when measuring low-abundance biomarkers in complex biological matrices.

Study Objectives and Overview

This work introduces SureQuant, an internal standard-triggered targeted quantitation strategy implemented on Thermo Scientific Orbitrap Exploris 480, Eclipse, and legacy systems. It aims to combine the high sensitivity of PRM/SRM with large-scale multiplexing while eliminating inefficiencies associated with retention time scheduling.

Methodology and Instrumentation

SureQuant employs isotope-labeled standard (IS) peptides spiked into samples as real-time triggers. The acquisition alternates between Watch mode (fast MS1 survey and low-resolution MS2 of IS) and Quant mode (high-resolution, high fill-time MS2 of endogenous targets) upon IS detection. A preliminary 'Survey Run' determines optimal IS precursor m/z tolerances, fragment ions, and triggering thresholds (~1% of apex). Native templates in Thermo Scientific Tune software support kits and custom panels. Key instrumentation includes:

• Thermo Scientific Orbitrap Exploris 480 and Eclipse mass spectrometers
• FAIMS Pro interface
• Easy-Spray PepMap RSLC C18 nanoLC system
• Proteome Discoverer and Skyline software for survey analysis and quantification

Main Results and Discussion

Comparison of SureQuant against conventional PRM demonstrates:

• Productive MS2 scan efficiency of 80–90% versus 10–15% for time-scheduled PRM
• Multiplex capability of up to 500 heavy/light pairs in 30 min without duty cycle compromise
• Detection and quantification of 26 out of 30 AKT–mTOR pathway targets using SureQuant versus 11 with PRM at equivalent cycle times
• Robust performance under retention time shifts without missing analytes

Benefits and Practical Applications

By dynamically triggering endogenous measurements only when targets elute, SureQuant achieves:

• Enhanced sensitivity and lower limits of quantitation through longer fill times
• High multiplexing without sacrificing quantitative precision
• Reduced method development and maintenance by eliminating retention time windows
• Increased throughput and reproducibility in routine workflows

Future Trends and Potential Applications

SureQuant paves the way for turnkey, large-scale targeted assays in proteomics, with potential extension to metabolomics and small-molecule analysis. Ongoing developments may include integration with automated sample handling, real-time data processing, AI-driven assay design, and expanded clinical diagnostic panels.

Conclusion

SureQuant represents a novel paradigm in targeted LC-MS, delivering the high quantitative performance of PRM/SRM alongside unprecedented multiplexing and robustness. Its watch-and-quant approach maximizes instrument efficiency and simplifies assay deployment, enabling reliable profiling of hundreds of analytes in complex samples.

References

• Lange V., et al. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008;4:222.
• Gallien S., et al. Targeted proteomic quantification on quadrupole-orbitrap MS. Mol. Cell. Proteomics 2012;11:1709–1723.
• Peterson AC., et al. Parallel reaction monitoring for high resolution quantitative proteomics. Mol. Cell. Proteomics 2012;11:1475–1488.
• Gallien S., Kim S.Y., Domon B. Large-scale targeted proteomics using IS-PRM. Mol. Cell. Proteomics 2015;14:1630–1644.