Hybrid Quadrupole\nMass Filter–Radial Ejection\nLinear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex\nTargeted Proteomics

Targeted\nmass spectrometry (MS) methods are powerful tools for\nthe selective and sensitive analysis of peptides identified in global\ndiscovery experiments. Selected reaction monitoring (SRM) is the most\nwidely accepted clinical MS method due to its reliability and performance.\nHowever, SRM and parallel reaction monitoring (PRM) are limited in\nthroughput and are typically used for assays with around 100 targets\nor fewer. Here we introduce a new MS platform featuring a quadrupole\nmass filter, collision cell, and linear ion trap architecture, capable\nof targeting 5000–8000 peptides per hour. This high multiplexing\ncapability is facilitated by acquisition rates of 70–100 Hz\nand real-time chromatogram alignment. We present a Skyline external\nsoftware tool for building targeted methods based on data-independent\nacquisition chromatogram libraries or unscheduled analysis of heavy\nlabeled standards. Our platform demonstrates ∼10× lower\nlimits of quantitation (LOQs) than traditional SRM on a triple quadrupole\ninstrument for highly multiplexed assays, due to parallel product\nion accumulation. Finally, we explore how analytical figures of merit\nvary with method duration and the number of analytes, providing insights\ninto optimizing assay performance.