Compositions, methods and systems for protein corona analysis and uses thereof
Inventors
XIA, Hongwei • HESTERBERG, Lyndal • Figa, Michael • Zhao, Xiaoyan • Troiano, Gregory • Manning, William • Blume, John • Farokhzad, Omid • McLean, Matthew • Stolarczyk, Craig • KO, Marwin • Platt, Theodore
Assignees
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Publication Number
US-12287331-B2
Publication Date
2025-04-29
Expiration Date
Abstract
Compositions, methods, and systems for analyzing the protein corona are described herein, as well as its application in the discovery of advanced diagnostic tools as well as therapeutic targets.
Core Innovation
The invention provides a high-throughput method of preparing to identify proteins in a biological sample by forming protein coronas using magnetic microparticles or magnetic particles. The method incubates the magnetic microparticles or magnetic particles with the biological sample to form protein coronas comprising proteins, and the particles are configured to form protein coronas having at least 200 unique protein groups, or at least 400 unique protein groups for the multi-particle-type embodiment, that can be identified using mass spectrometry.
After corona formation, the method magnetically isolates the magnetic microparticles or particles from unbound protein by applying an external magnetic field to enrich the proteins in the protein coronas. The enriched proteins are washed after magnetically isolating the magnetic microparticles or particles, and the enriched proteins in the protein coronas are incubated with a denaturing reagent and a digestion reagent to generate digested peptides.
The generated digested peptides are purified for protein identification, including purification comprising binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides. In one embodiment, the biological sample is plasma or serum, and in another embodiment the magnetic particles comprise at least two distinct particle types including silica with defined negative surface charges and a second particle type with positive surface charges.
Claims Coverage
Two independent claims are provided: clm-00001 and clm-00014. Together they cover a high-throughput corona-based protein enrichment workflow using magnetically isolatable particles, defined requirements for protein-group coverage, and downstream denaturing, digestion, and purification for mass-spectrometry identification.
High-throughput corona proteomics workflow with silica carboxylate magnetic microparticles
Incubating magnetic microparticles with a biological sample to form protein coronas comprising proteins, where the magnetic microparticles comprise silica, carboxylate functional groups, negative surface charges from −30 mV to −10 mV in zeta potential, heterogeneous sizes from about 1 μm to about 10 μm in diameter, and are configured to form protein coronas having at least 200 unique protein groups that can be identified using mass spectrometry; magnetically isolating from unbound protein by applying an external magnetic field; washing after magnetically isolating; incubating in the protein coronas by adding a denaturing reagent and a digestion reagent to generate digested peptides; and purifying the digested peptides.
High-throughput corona proteomics workflow with multi-type magnetic particles and solid-phase peptide purification
Incubating magnetic particles with the biological sample to form protein coronas comprising proteins, where the magnetic particles comprise at least two distinct particle types including a first particle type comprising silica with negative surface charges from −30 mV to −10 mV in zeta potential and a second particle type comprising positive surface charges, and are configured to form protein coronas having at least 400 unique protein groups identifiable using mass spectrometry; magnetically isolating by applying an external magnetic field; washing the enriched proteins and the magnetic particles at least two times; incubating with a denaturing reagent and digesting to generate digested peptides; purifying the digested peptides by binding to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides; wherein the biological sample is plasma or serum.
The claims focus on a magnetically isolated protein-corona enrichment workflow coupled to denaturing, digestion to peptides, and purification, with explicit minimum requirements for unique protein groups identifiable by mass spectrometry, and with a multi-particle-type embodiment that includes solid-phase binding, washing, and elution of digested peptides for plasma or serum protein identification.
Stated Advantages
High-throughput method for preparing proteins in a biological sample for identification using mass spectrometry by enriching proteins in protein coronas.
Allows identification of at least 200, or at least 400, unique protein groups via mass spectrometry from corona-bound proteins.
Purification of digested peptides includes solid-phase binding, washing, and elution for preparing peptides for identification.
Documented Applications
Corona-based enrichment enabling proteomic profiling and identification of proteins from plasma and serum using mass spectrometry.
Cancer-related use cases, including cancer classification using a liposome panel and NSCLC serum marker discovery with differential feature groups.
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