Systematic evaluation of blood contamination in nanoparticle-based plasma proteomics

Circulating blood proteomics enables minimally invasive biomarker discovery. Nanoparticle (NP)-based circulating plasma proteomics studies have reported varying number of proteins, ranging from ca 2000 to 7000 but it is not clear whether higher protein number is more informative. Here, we first develop OmniProt - a silica-NP workflow for plasma proteomics which is optimized through systematic evaluation of NP types and protein corona formation parameters. Next, we present an Astral spectral library for 10,109 protein groups expressed in human plasma. Using Astral with a throughput of 60 sample-per-day, OmniProt identifies ca 3000 to 6000 protein groups from human plasma. Notably, we found that platelet/erythrocyte/coagulation-related contamination artificially elevates protein identifications and compromises quantification accuracy for circulating plasma proteins in NP-enriched samples. Through controlled contamination experiments, we identified and validated protein biomarkers for contaminations of platelet, erythrocyte, and coagulation, respectively, in NP-based plasma proteomics experiments. We further developed an open-access software Baize for accessing plasma proteomics contamination. Finally, we validated our OmniProt workflow and Baize software in a clinical cohort of 193 patients with CT-indistinct benign nodules or early lung cancers. On average, we identified ca 4400 protein groups for each plasma sample without significant contamination. Five samples were flagged out as contaminated. In conclusion, this study reveals that contamination of plasma samples significantly alters the protein identification and quantification in NP-based plasma proteomics experiments, and presents an open-access software Baize to evaluate plasma proteome contamination.