Standardizing Protein Corona Characterization in Nanomedicine: A Multicenter Study to Enhance Reproducibility and Data Homogeneity.

We recently revealed significant variability in protein corona characterization across various proteomics facilities, indicating that data sets are not comparable between independent studies. This heterogeneity mainly arises from differences in sample preparation protocols, mass spectrometry workflows, and raw data processing. To address this issue, we developed standardized protocols and unified sample preparation workflows, distributing uniform protein corona digests to several top-performing proteomics centers from our previous study. We also examined the influence of using similar mass spectrometry instruments on data homogeneity and standardized database search parameters and data processing workflows. Our findings reveal a remarkable stepwise improvement in protein corona data uniformity, increasing overlaps in protein identification from 11% to 40% across facilities using similar instruments and through a uniform database search. We identify the key parameters behind data heterogeneity and provide recommendations for designing experiments. Our findings should significantly advance the robustness of protein corona analysis for diagnostic and therapeutics applications.