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.