Complete profiling of human plasma proteome is an immerse 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, especially low-abundant proteins (LAPs, <10 ng mL-1), in a robust and fast way remains challenging. This is largely due to the lack of standardized and automated workflows including LAPs enrichment, reduction, and enzymatic digestion procedures. Until now, these complex procedures have not been incorporated into a streamlined workflow to achieve reproducibility, high-throughput, and deep proteome coverage. 

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Here we report the Proteonano™️ Ultraplex Proteomics Platform for large cohort plasma proteomics studies with robustness and high throughput by standardizing workflow by incorporating the Proteonano™️ platform and high-resolution mass spectrometers, including Orbitrap Exploris™️ 480, Orbitrap Astral™️, and timsTOF Pro 2. This pipeline demonstrates excellent stability and reproducibility, with tunable balance between detection depth and throughput. We further demonstrate the utility of this platform for biomarker discovery in an Alzheimer disease related cohort. This harmonized platform enables robust, fast and large-cohort plasma proteomics studies to meet the need to discovering new biomarkers.