Mass spectrometry-based top-down proteomics in nanomedicine: Proteoform-specific measurement of protein corona

Conventional bottom-up proteomics (BUP) analysis of protein corona (an evolving layer of proteins formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids, e.g., human plasma) enabled nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails pinpoint the specific proteoforms, distinct forms of protein molecules from the same gene, which is crucial for the prediction of the biological fate and pharmacokinetics of nanomedicines. We recently have pioneered a robust and reproducible MS-based top-down proteomics (TDP) technique for precisely characterizing proteoforms in the protein corona. Our TDP approach has successfully identified hundreds of proteoforms in the protein corona of polystyrene NPs, ranging from 3-70 kDa, revealing over 20 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations, which BUP could not fully discern. This MS-based TDP technique offers a more comprehensive and accurate characterization of NP protein coronas, deepening our understanding of NPs' biological identities and potentially revolutionizing the field of nanomedicine.