Mass Spectrometry Proteomics of the Nanoparticle Corona Is Highly Dependent on Sample Preparation Protocol

ABSTRACT It is widely established that the layers of proteins bound to the surface of nanoparticles (NPs), also known as the protein corona, critically influence their behavior in biological environments. Despite numerous proteomics studies performed in different NP‐corona systems, the impact of proteomic sample preparation methods on corona profiling remains poorly understood. In this study, we systematically compared five digestion protocols, namely S‐Trap TM , iST TM , ProteaseMAX TM , RapiGest TM , and a routine in‐gel digestion protocol commonly used in several laboratories. Protein corona samples were isolated from silica NPs incubated in 80% human plasma in phosphate‐buffered saline. Protocol performance was assessed in terms of digestion efficiency, protein and peptide yield, reproducibility, and identification bias. Proteomic characterization revealed marked differences across protocols, highlighting the influence of protocol on protein digestion, recovery, and representation. Our findings emphasize the need for method standardization and tailored protocol selection in corona studies to ensure accurate and reproducible proteomic profiling.