Top-Down Proteomic Profiling of Protein Corona by High-Throughput Capillary Isoelectric Focusing-Mass Spectrometry

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.