In the rapidly evolving field of nanomedicine, understanding the interactions between nanoparticles (NPs) and biological systems is crucial. A pivotal aspect of these interactions is the formation of a protein corona when NPs are exposed to biological fluids (e.g., human plasma), which significantly influences their behavior and functionality. This study introduces an advanced capillary isoelectric focusing tandem mass spectrometry (cIEF-MS/MS) platform designed to enable high-throughput and reproducible top-down proteomic analysis of protein corona. Our cIEF-MS/MS technique completed each analysis within 30 min. It produced reproducible proteoform measurements of protein corona for at least 50 runs regarding the proteoforms' migration time [relative standard deviations (RSDs) <4%], the proteoforms' intensity (Pearson's correlation coefficients between any two runs >0.90), the number of proteoform identifications (71 ± 10), and the number of proteoform-spectrum matches (PrSMs) (196 ± 30). Of the 53 identified genes, 33 are potential biomarkers of various diseases (e.g., cancer, cardiovascular disease, and Alzheimer's disease). We identified 1-102 proteoforms per potential protein biomarker, containing various sequence variations or post-translational modifications. Delineating proteoforms in protein corona by our cIEF-MS/MS in a reproducible and high-throughput fashion will benefit our understanding of nanobiointeractions and advance both diagnostic and therapeutic nanomedicine technologies.
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