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

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails pinpoint the specific proteoforms—distinct molecular variants of proteins, which is essential for prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers 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—details that BUP could not fully discern. This advancement in MS-based TDP offers a more comprehensive and exact characterization of NP protein coronas, deepening our understanding of NPs’ biological identities and potentially revolutionizing the field of nanomedicine.