Abstract Background Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid-throughput analysis for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods Optimal conditions for sample preparation and DIA-MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high-throughput or deep profile analysis with mid-throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5–6-point dilution curve. Result Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV&lt;30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV&lt;30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high-throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid-throughput workflows. Conclusion The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids