The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) DIA. We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library, and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap PRM. Two applications of these proposed workflows are shown with a CSF neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma protein survey PRM assay.

Raw files are organized by instrument and experiment.
Skyline files for MMCC contain only optimal transitions for LOQ. Skyline files for the cohort assays contain transitions selected through PRM conductor.

Code for analysis is available at https://github.com/uw-maccosslab/manuscript-stellar-biofluid.

Associated preprint: Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer | bioRxiv