ABSTRACT: This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers ~90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types.
susan mockus