Fan, C.; Wang, Y.; Huang, M.; Cao, J.; Li, X.; Ouyang, X.; Wu, H.

The complete profiling of human plasma proteome is an emerse source for disease biomarker discovery. Cutting-edge mass spectrometers, like ThermoFisher\'s Orbitrap Astral, have promised unprecedented insights into the exploration of multiple protein biomarkers from human plasma samples. However, large-scale, deep profiling of the human plasma proteome in a robust and fast way remains challenging. This is largely due to the lack of fully compatible and robust workflows with automated sample prep methodologies including nanoparticle-based protein enrichment, reduction, and digestion procedures. So far, these methods have not been incorporated into a streamlined workflow to achieve reproducibility, throughput, and detection depth. Here we report a robust and standardized workflow by incorporating the Proteonano sample prep suite (Nanomics Biotech Ltd) and Orbitrap Astral mass spectrometer (ThermoFisher Scientific Co.), demonstrating reproducibility, scalable detection depth, and throughput. This harmonized method enables researchers to carry on robust, fast and large-cohort clinical plasma proteomics studies to meet the need to discovering new proteomic biomarkers.