Abstract
Targeted mass spectrometry (MS) methods are powerful tools for the selective and sensitive analysis of peptides identified in global discovery experiments. Selected reaction monitoring (SRM) is the most widely accepted clinical MS method due to its reliability and performance. However, SRM and parallel reaction monitoring (PRM) are limited in throughput and are typically used for assays with around 100 targets or fewer. Here we introduce a new MS platform featuring a quadrupole mass filter, collision cell, and linear ion trap architecture, capable of targeting 5000-8000 peptides per hour. This high multiplexing capability is facilitated by acquisition rates of 70-100 Hz and real-time chromatogram alignment. We present a Skyline external software tool for building targeted methods based on data-independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. Our platform demonstrates ∼10× lower limits of quantitation (LOQs) than traditional SRM on a triple quadrupole instrument for highly multiplexed assays, due to parallel product ion accumulation. Finally, we explore how analytical figures of merit vary with method duration and the number of analytes, providing insights into optimizing assay performance.