Automated sample preparation with SP3 for low-input clinical proteomics

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples originating from a variety of sources, including frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format, performing unbiased protein purification and digestion delivering peptides that can be directly analyzed by LCMS. AutoSP3 eliminates hands-on time and minimizes the risk of error, and we show it reduces the protein quantification variability, and improves longitudinal performance and reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1000 proteins from 100-1000 cells (<100 ng protein). Furthermore, we added a LE220-plus focused- ultrasonicator (Covaris Ltd, UK) to our pipeline to include 96-well format lysis of fresh-frozen tissue and cells. Collectively, autoSP3 provides a generic, scalable, and cost-effective pipeline for routine and standardized proteomic sample processing that should enable reproducible proteomics in broad range of clinical and non-clinical applications.