Benchmarking Plasma Proteomics Workflows and Their Correlation to Clinical Routine Protein Assays

Plasma proteomics based on mass spectrometry has great potential for biomarker discovery. Plasma is challenging for mass spectrometry due to the high dynamic range in protein abundance. Several workflows have been developed to overcome this, and in this study, we compare prominent enrichment and depletion workflows using platelet-poor plasma (PPP), platelet-rich plasma (PRP), and serum (SER). Our results show that depletion workflows including Top14 depletion and acid precipitation allow quantification of very different proteomes than methods based on enrichments of extracellular vesicles such as bead-based enrichment or ultracentrifugation. Enrichment methods are superior in terms of proteome depth and quantitative performance but may be less robust in large cohorts. There is a very high correlation between PPP and PRP samples for all methods and less to SER samples, especially with enrichment workflows. The correlation of 10 protein measurements, performed by clinical routine processes on a Cobas system, showed heterogeneous results. Low-abundant proteins with biological dynamics within a healthy cohort, including C-reactive protein and lipoprotein(a), correlated very well to proteomics-based workflows, while others, including albumin and transferrin, correlated poorly. In conclusion, the workflow for plasma proteomics should be aligned with the aim of the analysis and setup of the sample collection.