Highly multiplex targeted proteomics assay in plasma using Stellar mass spectrometer with adaptive RT

Importance of the Topic

Targeted proteomics assays enable precise quantification of proteins across large cohorts, bridging discovery research and clinical application. High multiplexing capacity is crucial for monitoring biomarker panels in complex biological matrices such as plasma.

Objectives and Study Overview

This study describes the development of a large-scale, multiplexed targeted proteomics workflow using an adaptive retention time (RT) strategy on the Thermo Scientific Stellar mass spectrometer. The assay aims to quantify potential disease biomarkers in patient plasma, with a demonstration on colorectal cancer (CRC) samples.

Methodology and Instrumentation

A three-stage assay incorporating MS2 and MS3 parallel reaction monitoring (PRM) was established within three days using PQ500 heavy peptide standards. Adaptive RT real-time alignment allowed a 0.65-minute scheduling window for over 1600 peptide precursors. Sample preparation employed automated digestion of plasma on the AccelerOme platform. Chromatographic separation utilized a 30-minute gradient on a Vanquish Neo UHPLC with an EASY-Spray ES906A column at 55 °C.

Used Instrumentation

• Thermo Scientific Stellar Mass Spectrometer with adaptive RT mode
• Vanquish Neo UHPLC System
• Thermo Scientific EASY-Spray ES906A column
• Thermo Scientific AccelerOme Automated Sample Preparation Platform
• Skyline software for PRM method generation and data analysis

Key Findings and Discussion

The MS3 assay enhanced signal-to-noise for low-abundance or co-eluting peptides, yielding a 10.3 % increase in identified proteins compared to MS2 alone. In plasma digests from CRC patients and healthy controls, 292 endogenous proteins and 472 peptides were quantified. Over 94 % of peptides in disease plasma demonstrated coefficients of variation below 25 % and 90 % showed R2 > 0.9. The number of detected proteins increased by 73 % when loading 1 µg versus 25 ng digest.

Statistical comparison revealed 29 proteins significantly altered (>2-fold change, adj. p < 0.05) in CRC patient plasma, including acute-phase proteins such as serum amyloid A2 (SAA2), complement C9 (CO9), and alpha-2-glycoprotein (A2GL). Many of these are implicated in cancer progression.

Benefits and Practical Applications

• High multiplexing enables simultaneous quantitation of hundreds of biomarkers in a single run.
• Adaptive RT reduces method development time and minimizes scheduling errors.
• MS3 acquisition improves sensitivity for challenging targets in complex matrices.
• Workflow is suitable for large-scale clinical studies and QA/QC applications.

Future Trends and Possibilities

Integration of adaptive acquisition strategies with real-time data analysis will further expand multiplexing capacity. Continued improvements in ion-mobility separation and artificial intelligence-driven retention time prediction may enable thousands of targets per run. Application to longitudinal patient cohorts could advance precision medicine in oncology and other fields.

Conclusion

The developed multiplexed PRM assay on the Stellar platform demonstrates robust performance for large-scale biomarker quantitation in plasma. Adaptive RT and MS3 acquisition provided enhanced sensitivity and reproducibility, identifying clinically relevant changes in CRC patient samples.

Reference

1. Chantaraamporn J, et al. Proteomes. 2020;8(3):26.
2. Geary B, et al. Cancers. 2021;13(10):2443.