Integrated top-down and bottom-up proteomics enables precise characterization of proteoforms within the protein...

Precise characterization of proteoforms within the protein corona is essential for developing safer and more effective nanomedicines for diagnostic and therapeutic applications. Here, we advance the characterization of proteoforms within the protein corona by integrating mass spectrometry (MS)-based top-down proteomics (TDP) and bottom-up proteomics (BUP). TDP of the protein corona formed on polystyrene nanoparticles (PSNPs) identifies over 5000 proteoforms of 400 genes from breast cancer plasma samples, creating one of the largest human plasma TDP datasets and the most comprehensive proteoform database of the protein corona. Combining TDP and BUP improves the characterization quality for about 35% of identified proteoforms containing mass shifts, producing a more precise proteoform landscape of protein corona. It also enables the discovery and precise characterization of potential proteoform biomarkers of breast cancer. The approach will eventually enhance our understanding of the protein corona, offer valuable insights into nanoparticle-biosystem interactions, and advance proteoform biomarker discovery.