Plasma is a rich source of potential biomarkers for monitoring disease onset and progression. The implementation of non-targeted plasma proteomics in disease diagnostics promises to improve both speed and accuracy, thereby contributing to improved patient prognosis. However, the lack of reproducible, robust, and high throughput proteomics workflows is a major limiting factor in the transition from biomarker discovery to verification and ultimately clinical application. Additionally, the large dynamic range of plasma proteins prevents the identification of many proteins with bottom-up proteomics in undepleted plasma. We have implemented automated end-to-end sample preparation methods, integrated with the Evotip Pure, for profiling of undepleted plasma and deep plasma proteomics. These advancements will significantly enhance scalability for large scale cohort analysis.

For the undepleted plasma workflow, we combined on-robot reduction and alkylation with protein aggregation capture (PAC) based digestion. An approach for deeper profiling of a sub-proteome based on a MagNet enrichment step was added for deep plasma profiling to capture membrane-bound particles.

Both methods were fully automated on a Biomek i5 liquid handling robot for processing 576 samples in parallel, incorporating all steps from neat plasma to peptides loaded on Evotips, ready-to-analyze. This is driven by efficient protocols based on hyper-porous magnetic beads directly followed by concentration of the digested peptides on Evotips to minimize losses otherwise associated with additional desalting steps. The standardized workflows can robustly quantify close to 1,500 protein groups from neat plasma, increasing to more than 5,000 protein groups from the MagNet workflow with a throughput of 100 samples analyzed per day using the Orbitrap Astral MS.