A Novel Hybrid High-Speed Mass Spectrometer Allows Rapid Translation From Biomarker Candidates to Targeted Clinical Tests Using 15N-Labeled Proteins.

Recent developments in affinity binder or mass spectrometry (MS)-based plasma proteomics are now producing panels of potential biomarker candidates for diagnosis or prognosis. However, clinical validation and implementation of these biomarkers remain limited by the reliance on dated triple quadrupole MS technology. Here, we evaluate a novel hybrid high-speed mass spectrometer, Stellar MS, which integrates the robustness of triple quadrupoles with the enhanced capabilities of an advanced linear ion trap analyzer. This instrument allows for extremely rapid and sensitive parallel reaction monitoring (PRM) and MS3 targeting. The Stellar MS allowed targeting thousands of peptides originally measured on Orbitrap Astral MS, achieving high reproducibility and low coefficients of variation (CV) as well as sensitivity and specificity sufficient for many of the top 1000 plasma proteins. Furthermore, we developed targeted assays for alcohol-related liver disease (ALD) biomarkers, showcasing the potential of Stellar MS in clinical applications. Absolute quantification is typically a requirement for clinical assays, and we explore the use of ¹⁵N-labeled protein standards in a rapid, streamlined, and generic manner. Our results indicate that the Stellar MS can bridge the gap between proteomics discovery and routine clinical testing, enhancing the diagnostic and prognostic utility of protein biomarkers.