A uniform data processing pipeline enables harmonized nanoparticle protein corona analysis across proteomics core facilities.

Protein corona, a layer of biomolecules primarily comprising proteins, forms dynamically on nanoparticles in biological fluids and is crucial for predicting nanomedicine safety and efficacy. The protein composition of the corona layer is typically analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Our recent study, involving identical samples analyzed by 17 proteomics facilities, highlighted significant data variability, with only 1.8% of proteins consistently identified across these centers. Here, we implement an aggregated database search unifying parameters such as variable modifications, enzyme specificity, number of allowed missed cleavages and a stringent 1% false discovery rate at the protein and peptide levels. Such uniform search dramatically harmonizes the proteomics data, increasing the reproducibility and the percentage of consistency-identified unique proteins across distinct cores. Specifically, out of the 717 quantified proteins, 253 (35.3%) are shared among the top 5 facilities (and 16.2% among top 11 facilities). Furthermore, we note that reduction and alkylation are important steps in protein corona sample processing and as expected, omitting these steps reduces the number of total quantified peptides by around 20%. These findings underscore the need for standardized procedures in protein corona analysis, which is vital for advancing clinical applications of nanoscale biotechnologies.