Combining a new hybrid nominal mass platform and intelligent data acquisition to enable highly multiplexed targeted proteomics

Importance of the Topic

Targeted proteomics plays a pivotal role in clinical research, biomarker validation and quality control. Achieving high sensitivity, specificity and throughput in peptide quantitation is essential for translating discovery data into robust assays. The integration of advanced mass spectrometry platforms with intelligent data acquisition strategies addresses challenges in assay development and performance.

Objectives and Study Overview

This study evaluates the Thermo Scientific Stellar MS—a hybrid quadrupole and linear ion trap system—for high‐multiplexed, targeted proteomics. Key goals include comparing its quantitative performance against a triple quadrupole instrument, building large‐scale assays using the PQ500 reference peptide kit in plasma and E. coli digests in HeLa matrix, and assessing the benefits of the Adaptive RT alignment algorithm.

Methodology and Instrumentation

The workflow leverages PRM Conductor within Skyline to automate precursor selection and method creation. Real‐time chromatogram alignment (Adaptive RT) narrows scheduling windows down to 0.35–0.6 min, enhancing throughput. Discovery data for E. coli and HeLa mixtures were acquired by gas‐phase‐fractionation DIA on Stellar MS. Absolute quantitation used heavy peptide standards and Prosit‐predicted spectral libraries. LC separations employed a Vanquish Neo UHPLC system with gradients from 30 min at 0.45 µL/min up to 100 SPD at 1.8 µL/min.

Used Instrumentation

• Thermo Scientific Stellar MS (hybrid Q‐LIT) for PRM and DIA
• Thermo Scientific TSQ Altis Plus triple quadrupole for comparison
• Vanquish Neo UHPLC for nano-flow separation
• Skyline with PRM Conductor and Prosit integration

Main Results and Discussion

Stellar MS delivered over 10× lower limits of quantitation (LOQ) and approximately 2× lower limits of detection (LOD) compared to the triple quadrupole. Parallel accumulation in PRM provided extended ion injection times, boosting sensitivity. High multiplex assays (804 light/heavy peptides) yielded more than six data points per peak and coefficients of variation below 20%. Throughput scaled from 60 to 100 sample processing days (SPD) with only modest performance loss.

Benefits and Practical Applications

• Dramatic sensitivity gains enable detection of low‐abundance biomarkers.
• Automated method creation reduces assay development time.
• Adaptive RT ensures robust scheduling across variable chromatographic conditions.
• DIA-based discovery seamlessly feeds targeted assay design.

Future Trends and Applications

Emerging trends include deeper multiplexing via advanced parallel accumulation strategies, integration of machine learning for retention time and spectrum prediction, and expansion into clinical diagnostic workflows. Real‐time alignment and retrospective transition selection are expected to further streamline large‐scale proteomic studies.

Conclusion

The Thermo Scientific Stellar MS, combined with PRM Conductor and Adaptive RT, overcomes traditional barriers in targeted proteomics. It offers superior sensitivity, high reproducibility and rapid assay development, facilitating translation of discovery research into quantitative assays.

References

1. Serrano et al. Molecular & Cellular Proteomics, 2024, 23(5), 100760.
2. PRM Conductor Walkthrough. panoramaweb.org/prm_conductor.url.
3. Remes et al. Analytical Chemistry, 2020, 92(17), 11809.
4. Pinot et al. Journal of Proteome Research, 2020, 19(3), 1147.
5. Remes et al. BioRxiv preprint, 2024. DOI:10.1101/2024.05.31.596848.