Compositions, methods and systems for protein corona analysis and uses thereof

Abstract

To provide compositions, methods, and systems for protein corona analysis and their uses.SOLUTION: Compositions, methods, and systems for analyzing protein corona are described, as well as their application in the discovery of advanced diagnostic tools as well as therapeutic targets. A panel of nanoparticles for detection of a wide range of diseases and disorders and determination of disease states in a subject is provided. Creating and characterizing protein corona has also been performed in the field, but most of the experimentation has been done using nonmagnetic particles, such as liposome and polymeric nanoparticles, or other particle types that may be used for targeted drug delivery.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 756,960, filed November 7, 2018, U.S. Provisional Patent Application No. 62 / 824,278, filed March 26, 2019, and U.S. Provisional Patent Application No. 62 / 874,862, filed July 16, 2019, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] The widespread implementation of proteomic information into science and medicine has lagged behind genomics in large part due to the inherent complexity of the protein molecules themselves, which requires complex workflows that limit the scalability of such analyses. Disclosed herein are compositions and methods for the rapid processing of proteomic data and the identification of key biomarkers associated with disease. Summary of the Invention [Means for solving the problem]

[0003] The present invention provides a panel of nanoparticles for the detection and pathology assessment of a wide range of diseases and disorders in subjects.

[0004] Although the creation and characterization of protein coronas has been performed in the art, the majority of experiments have been performed with non-magnetic particles, such as liposomes and polymeric nanoparticles, or other particle types that can be used for targeted drug delivery.

[0005] A problem with current (typically academic) assays for protein corona formation and characterization is that the particles used in the assay must be isolated for corona recovery, making them unsuitable for high-throughput and / or automated formats. The advantage of SPMNPs used for protein coronas is their rapid magnetic response, allowing them to be easily separated from the suspension mixture by applying an external magnetic field. This type of magnetic particle is a good platform for further chemical modification with different functional groups, which can help fine-tune the particle's interaction with plasma proteins.

[0006] In some aspects, the present disclosure provides a method for identifying proteins in a sample, the method comprising: incubating a particle panel with the sample to form a plurality of distinct biomolecular coronas corresponding to the distinct particle types of the particle panel; magnetically isolating the particle panel from unbound proteins in the sample to enrich proteins in the plurality of distinct biomolecular coronas; and assaying the plurality of distinct biomolecular coronas to identify the enriched proteins.

[0007] In some embodiments, the assaying step can identify between 1 and 20,000 protein groups. In further embodiments, the assaying step can identify between 1,000 and 10,000 protein groups. In further embodiments, the assaying step can identify between 1,000 and 5,000 protein groups. In still further embodiments, the assaying step can identify between 1,200 and 2,200 protein groups. In some embodiments, the protein groups comprise peptide sequences having a minimum length of 7 amino acid residues. In some further cases, the assaying step can identify between 1,000 and 10,000 proteins. In some still further cases, the assaying step can identify between 1,800 and 5,000 proteins.

[0008] In some embodiments, the sample comprises a plurality of samples. In some embodiments, the plurality of samples comprises at least two or more spatially separated samples. In further embodiments, the incubating step comprises simultaneously contacting at least two or more spatially separated samples with the particle panel. In further embodiments, the magnetically isolating step comprises simultaneously magnetically isolating the particle panel from unbound proteins in at least two or more spatially separated samples of the plurality of samples. In further embodiments, the assaying step comprises assaying a plurality of distinct biomolecular coronas to simultaneously identify proteins in at least two or more spatially separated samples.

[0009] In some embodiments, the method further comprises repeating the method described herein, wherein when repeated, the incubating, isolating, and assaying steps produce a quantile normalized coefficient of variation (QNCV) percentage of 20% or less, as determined by comparing peptide mass spectrometry features from at least three full assay replicates for each particle type in the particle panel. In some embodiments, when repeated, the incubating, isolating, and assaying steps produce a quantile normalized coefficient of variation (QNCV) percentage of 10% or less, as determined by comparing peptide mass spectrometry features from at least three full assay replicates for each particle type in the particle panel. In some embodiments, the assaying step can identify proteins over a dynamic range of at least 7, at least 8, at least 9, or at least 10.

[0010] In some embodiments, the method further comprises washing the particle panel at least once or at least twice after magnetically isolating the particle panel from unbound proteins. In some embodiments, the method further comprises lysing proteins in the plurality of distinct biomolecular coronas after assaying.

[0011] In some embodiments, the method further comprises digesting proteins in the plurality of distinct biomolecular coronas to produce digested peptides.

[0012] In some embodiments, the method further comprises purifying the digested peptides.

[0013] In some embodiments, the assaying step comprises identifying proteins in the sample using mass spectrometry. In some embodiments, the assay is performed within about 2 to about 4 hours. In some embodiments, the method is performed within about 1 to about 20 hours. In some embodiments, the method is performed within about 2 to about 10 hours. In some embodiments, the method is performed within about 4 to about 6 hours. In some embodiments, the isolating step takes about 30 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 2 minutes or less. In some embodiments, the plurality of samples comprises at least 10 spatially separated samples, at least 50 spatially separated samples, at least 100 spatially separated samples, at least 150 spatially separated samples, at least 200 spatially separated samples, at least 250 spatially separated samples, or at least 300 spatially separated samples. In further embodiments, the plurality of samples comprises at least 96 samples.

[0014] In some embodiments, the particle panel comprises at least two distinct particle types, at least three distinct particle types, at least four distinct particle types, at least five distinct particle types, at least six distinct particle types, at least seven distinct particle types, at least eight distinct particle types, at least nine distinct particle types, at least ten distinct particle types, at least eleven distinct particle types, at least thirteen distinct particle types, at least fourteen distinct particle types, at least fifteen distinct particle types, at least twenty distinct particle types, at least twenty-five distinct particle types, or at least thirty distinct particle types. In further embodiments, the particle panel comprises at least ten distinct particle types. In further embodiments, at least two spatially separated samples differ in at least one physicochemical property.

[0015] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, where the first distinct particle type and the second distinct particle type share at least one physicochemical property but differ in at least one physicochemical property, and thus the first distinct particle type and the second distinct particle type are different. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, where the first distinct particle type and the second distinct particle type share at least two physicochemical properties but differ in at least two physicochemical properties, and thus the first distinct particle type and the second distinct particle type are different. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, where the first distinct particle type and the second distinct particle type share at least one physicochemical property but differ in at least two physicochemical properties, and thus the first distinct particle type and the second distinct particle type are different.

[0016] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type share at least two physicochemical properties but differ in at least one physicochemical property, and thus the first distinct particle type and the second distinct particle type are different. In further embodiments, the physicochemical property comprises size, charge, core material, shell material, porosity, or surface hydrophobicity. In further embodiments, the size is a diameter or radius measured by dynamic light scattering, SEM, TEM, or any combination thereof.

[0017] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type comprising a carboxylate material, the first distinct particles being microparticles, and the second distinct particle type being nanoparticles. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type having surface charges of 0 mV and -50 mV, and the first distinct particle type having a diameter less than 200 nm and the second distinct particle type having a diameter greater than 200 nm.

[0018] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type having diameters between 100 and 400 nm, the first distinct particle type having a positive surface charge and the second distinct particle type having a neutral surface charge. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type being nanoparticles, the first distinct particle type having a surface charge less than -20 mV and the second distinct particle type having a surface charge greater than -20 mV.

[0019] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type are microparticles, the first distinct particle type having a negative surface charge, and the second distinct particle type having a positive surface charge. In some embodiments, the particle panel comprises a subset of negatively charged nanoparticles, each particle of the subset differing in at least one surface chemical group. In some embodiments, the particle panel comprises a first distinct particle type, a second particle, and a third distinct particle type, wherein the first distinct particle type, the second distinct particle type, and the third distinct particle type comprise an iron oxide core, a polymer shell, and are less than about 500 nm in diameter, the first distinct particle type having a negative charge, the second distinct particle type having a positive charge, and the third distinct particle type having a neutral charge, and the diameter is an average diameter measured by dynamic light scattering. In a further embodiment, a first distinct type of particle comprises a silica coating, a second distinct type of particle comprises poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), and a third distinct type of particle comprises a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating.

[0020] In some embodiments, at least one distinct type of particle in the particle panel is a nanoparticle. In some embodiments, at least one distinct type of particle in the particle panel is a microparticle. In some embodiments, at least one distinct type of particle in the particle panel is a superparamagnetic iron oxide particle. In some embodiments, each particle in the particle panel comprises an iron oxide material. In some embodiments, at least one distinct type of particle in the particle panel has an iron oxide core. In some embodiments, at least one distinct type of particle in the particle panel has iron oxide crystals embedded in a polystyrene core. In some embodiments, each distinct type of particle in the particle panel is a superparamagnetic iron oxide particle. In some embodiments, each distinct type of particle in the particle panel comprises an iron oxide core. In some embodiments, each distinct type of particle in the particle panel has iron oxide crystals embedded in a polystyrene core. In some embodiments, at least one distinct type of particle in the particle panel comprises a carboxylated polymer, an aminated polymer, a zwitterionic polymer, or any combination thereof. In some embodiments, at least one type of particle in the particle panel comprises an iron oxide core with a silica shell coating. In some embodiments, at least one type of particle in the particle panel comprises an iron oxide core with a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating. In some embodiments, at least one type of particle in the particle panel comprises an iron oxide core with a poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) coating.

[0021] In some embodiments, at least one distinct particle type of the particle panel has a negative surface charge. In some embodiments, at least one distinct particle type of the particle panel has a positive surface charge. In some embodiments, at least one distinct particle type of the particle panel has a neutral surface charge. In some embodiments, the particle panel comprises one or more distinct particle types selected from Table 10. In some embodiments, the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 10. In some embodiments, the particle panel comprises one or more distinct particle types selected from Table 12. In some embodiments, the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 12.

[0022] In various aspects, the present disclosure provides compositions comprising three or more distinct magnetic particle types that differ in two or more physicochemical properties, wherein a subset of the three or more distinct magnetic particle types share a physicochemical property among the two or more physicochemical properties, and wherein the subset of such particle types bind to different proteins.

[0023] In some embodiments, three or more distinct magnetic particle types adsorb proteins from a biological sample over a dynamic range of at least 7, at least 8, at least 9, or at least 10. In some embodiments, three or more distinct magnetic particle types are capable of adsorbing 1 to 20,000 protein groups from a biological sample. In some aspects, three or more distinct magnetic particle types are capable of adsorbing 1,000 to 10,000 protein groups from a biological sample. In further aspects, three or more distinct magnetic particle types are capable of adsorbing 1,000 to 5,000 protein groups from a biological sample. In further aspects, three or more distinct magnetic particle types are capable of adsorbing 1,200 to 2,200 protein groups from a biological sample. Still further In embodiments, the protein group comprises a peptide sequence having a minimum length of 7 amino acid residues. In some embodiments, three or more distinct magnetic particle types are capable of adsorbing 1 to 20,000 proteins from a biological sample. In further embodiments, three or more distinct magnetic particle types are capable of adsorbing 1,000 to 10,000 proteins from a biological sample. In yet further embodiments, three or more distinct magnetic particle types are capable of adsorbing 1,800 to 5,000 proteins from a biological sample.

[0024] In some embodiments, the composition comprises at least four distinct magnetic particle types, at least five distinct magnetic particle types, at least six distinct magnetic particle types, at least seven distinct magnetic particle types, at least eight distinct magnetic particle types, at least nine distinct magnetic particle types, at least ten distinct magnetic particle types, at least eleven distinct magnetic particle types, at least 12 distinct magnetic particle types, at least 13 distinct magnetic particle types, at least 14 distinct magnetic particle types, at least 15 distinct magnetic particle types, at least 20 distinct magnetic particle types, or at least 30 distinct magnetic particle types. In some embodiments, the composition comprises at least ten distinct magnetic particle types. In some embodiments, the composition comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type share at least two physicochemical properties but differ in at least two physicochemical properties, such that the first distinct particle type and the second distinct particle type are different.

[0025] In some embodiments, the composition comprises a first distinct particle type and a second distinct particle type, and the first distinct particle type and the second distinct particle type share at least two physicochemical properties but differ in at least one physicochemical property, thus the first distinct particle type and the second distinct particle type are different.In some embodiments, the physicochemical property comprises size, charge, core material, shell material, porosity, or surface hydrophobicity.In further embodiments, the size is the diameter or radius measured by dynamic light scattering, SEM, TEM, or any combination thereof.

[0026] In some embodiments, the composition comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type comprise a carboxylate material, the first distinct particles being microparticles, and the second distinct particle type being nanoparticles. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type have surface charges of 0 mV and -50 mV, respectively, the first distinct particle type having a diameter less than 200 nm, and the second distinct particle type having a diameter greater than 200 nm. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type have diameters between 100 and 400 nm, the first distinct particle type having a positive surface charge, and the second distinct particle type having a neutral surface charge.

[0027] In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type are nanoparticles, wherein the first distinct particle type has a surface charge less than -20 mV and the second distinct particle type has a surface charge greater than -20 mV. In some embodiments, the particle panel comprises a first distinct particle type and a second distinct particle type, wherein the first distinct particle type and the second distinct particle type are microparticles, wherein the first distinct particle type has a negative surface charge and the second distinct particle type has a positive surface charge. In some embodiments, the composition comprises a subset of negatively charged nanoparticles, wherein each particle type of the subset differs in at least one surface chemical group. In some embodiments, the composition comprises a first distinct type of particles, a second distinct type of particles, and a third distinct type of particles, wherein the first distinct type of particles, the second distinct type of particles, and the third distinct type of particles comprise an iron oxide core, a polymer shell, and are less than about 500 nm in diameter, the first distinct type of particles have a negative charge, the second distinct type of particles have a positive charge, and the third distinct type of particles have a neutral charge, and the diameter is the average diameter measured by dynamic light scattering. In further embodiments, the first distinct type of particles comprise a silica coating, the second distinct type of particles comprise poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), and the third distinct type of particles comprise a poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) coating.

[0028] In some embodiments, the three or more distinct magnetic particle types comprise nanoparticles. In some embodiments, the three or more distinct magnetic particle types comprise microparticles. In some embodiments, at least one distinct particle type of the three or more distinct magnetic particle types is a superparamagnetic iron oxide particle. In some embodiments, at least one distinct particle type of the three or more distinct magnetic particle types comprises an iron oxide material. In some embodiments, at least one distinct particle type of the three or more distinct magnetic particle types has an iron oxide core. In some embodiments, at least one distinct particle type of the three or more distinct magnetic particle types has iron oxide crystals embedded in a polystyrene core.

[0029] In some embodiments, each of the three or more distinct magnetic particle types is a superparamagnetic iron oxide particle. In some embodiments, each of the three or more distinct magnetic particle types comprises an iron oxide core. In some embodiments, each of the three or more distinct magnetic particle types comprises iron oxide crystals embedded in a polystyrene core. In some embodiments, at least one of the three or more distinct magnetic particle types comprises a polymer coating.

[0030] In some embodiments, the three or more distinct magnetic particle types comprise a carboxylated polymer, an aminated polymer, a zwitterionic polymer, or any combination thereof. In some embodiments, at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core with a silica shell coating. In some embodiments, at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core with a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating. In some embodiments, at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core with a poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) coating.

[0031] In some embodiments, at least one particle type of the three or more distinct magnetic particle types has a negative surface charge. In some embodiments, at least one particle type of the three or more distinct magnetic particle types has a positive surface charge. In some embodiments, at least one particle type of the three or more distinct magnetic particle types has a neutral surface charge.

[0032] In some embodiments, the three or more distinct magnetic particle types comprise one or more particle types from Table 10. In some embodiments, the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 10. In some embodiments, the three or more distinct magnetic particle types comprise one or more particle types from Table 12. In some embodiments, the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 12.

[0033] In some aspects, the present disclosure provides a method for determining a biological state of a sample from a subject, the method comprising the steps of generating a plurality of protein coronas by exposing the biological sample to a panel comprising a plurality of nanoparticles; generating proteomic data from the plurality of protein coronas; determining a protein profile of the plurality of protein coronas; and relating the protein profile to the biological state, wherein the panel comprises at least two different nanoparticles.

[0034] In some embodiments, the panel comprises at least three different nanoparticles. In some embodiments, the method correlates the protein profile to a biological state with at least 90% accuracy. In some embodiments, the plurality of nanoparticles comprises at least one iron oxide nanoparticle.

[0035] In some aspects, the present disclosure provides a method for selecting a panel for protein corona analysis, the method comprising selecting a plurality of nanoparticles having at least three different physicochemical properties.

[0036] In some embodiments, the different physicochemical property is selected from the group consisting of surface charge, surface chemical structure, size, and morphology. In some embodiments, the different physicochemical property comprises surface charge.

[0037] In various embodiments, the present disclosure provides a method for identifying proteins in a sample, the method comprising: incubating a panel comprising a plurality of particle types with the sample to form a plurality of protein coronas; digesting the plurality of protein coronas to generate proteomic data; and identifying the proteins in the sample by quantifying the proteomic data. In some embodiments, the sample is from a subject.

[0038] In some embodiments, the method further comprises determining a protein profile of the sample from the identifying step and associating the protein profile with a biological state of the subject. In some embodiments, the method further comprises determining a biological state of the sample from the subject by generating proteomic data by digesting a plurality of protein coronas, determining a protein profile of the plurality of protein coronas, and associating the protein profile with the biological state, wherein the panel comprises at least two different nanoparticles. In some embodiments, the association is performed by a trained classifier.

[0039] In some embodiments, a panel comprises at least three different particle types, at least four different particle types, at least five different particle types, at least six different particle types, at least seven different particle types, at least eight different particle types, at least nine different particle types, at least ten different particle types, at least eleven different particle types, at least thirteen different particle types, at least fourteen different particle types, at least fifteen different particle types, or at least twenty different particle types. In some embodiments, a panel comprises at least four different particle types. In some embodiments, at least one particle type in a panel has a different physical characteristic than a second particle type in the panel. In some embodiments, the physical characteristic is size, polydispersity index, surface charge, or morphology. In some embodiments, the size of at least one particle type of the plurality of particle types in a panel is between 10 nm and 500 nm.

[0040] In some embodiments, the polydispersity index of at least one particle type of the plurality of particle types in the panel is between 0.01 and 0.25. In some embodiments, the morphology of at least one particle type of the plurality of particle types includes spherical, colloidal, square, rod, wire, conical, pyramidal, and ellipsoidal. In some embodiments, the surface charge of at least one particle type of the plurality of particle types includes a positive surface charge. In some embodiments, the surface charge of at least one particle type of the plurality of particle types includes a negative surface charge.

[0041] In some embodiments, the surface charge of at least one particle type of the plurality of particle types comprises a neutral surface charge. In some embodiments, at least one particle type of the plurality of particle types has a different chemical characteristic from a second particle type of the panel. In some embodiments, the chemical characteristic is a surface functional chemical group. In some embodiments, the functional chemical group is an amine or a carboxylate. In some embodiments, at least one particle type of the plurality of particle types is made of a material comprising a polymer, a lipid, or a metal.

[0042] In further embodiments, the polymer comprises polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), or a copolymer of two or more polymers.

[0043] In further embodiments, the lipid is selected from the group consisting of dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lecithin, lysolecithin, le ... Sphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol , sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, or cholesterol.

[0044] In some embodiments, the metal comprises gold, silver, copper, nickel, cobalt, palladium, platinum, iridium, osmium, rhodium, ruthenium, rhenium, vanadium, chromium, manganese, niobium, molybdenum, tungsten, tantalum, iron, or cadmium. In some embodiments, at least one particle type of the plurality of particle types is surface-functionalized with polyethylene glycol. In some embodiments, the method associates the protein profile with a biological state with at least 70% accuracy, at least 75% accuracy, at least 80% accuracy, at least 85% accuracy, at least 90% accuracy, at least 92% accuracy, at least 95% accuracy, at least 96% accuracy, at least 97% accuracy, at least 98% accuracy, at least 99% accuracy, or 100% accuracy. In some embodiments, the method associates the protein profile with a biological state with at least 70% sensitivity, at least 75% sensitivity, at least 80% sensitivity, at least 85% sensitivity, at least 90% sensitivity, at least 92% sensitivity, at least 95% sensitivity, at least 96% sensitivity, at least 97% sensitivity, at least 98% sensitivity, at least 99% sensitivity, or 100% sensitivity.

[0045] In some embodiments, the method associates the protein profile with a biological state with at least 70% specificity, at least 75% specificity, at least 80% specificity, at least 85% specificity, at least 90% specificity, at least 92% specificity, at least 95% specificity, at least 96% specificity, at least 97% specificity, at least 98% specificity, at least 99% specificity, or 100% specificity. In some embodiments, the method identifies at least 100 unique proteins, at least 200 unique proteins, at least 300 unique proteins, at least 400 unique proteins, at least 500 unique proteins, at least 600 unique proteins, at least 700 unique proteins, at least 800 unique proteins, at least 900 unique proteins, at least 1000 unique proteins, at least 1100 unique proteins, at least 1200 unique proteins, at least 1300 unique proteins, at least 1400 unique proteins, at least 1500 unique proteins, at least 1600 unique proteins, at least 1700 unique proteins, at least 1800 unique proteins, at least 1900 unique proteins, or at least 2000 unique proteins. In some embodiments, at least one particle type of the plurality of particle types comprises iron oxide nanoparticles. In further embodiments, the sample is a bodily fluid. In yet further embodiments, the bodily fluid comprises plasma, serum, CSF, urine, tears, or saliva.

[0046] In various embodiments, the present disclosure provides a method for selecting a panel for protein corona analysis, the method comprising selecting a plurality of particle types having at least three distinct physicochemical properties. In some embodiments, the distinct physicochemical properties are selected from the group consisting of surface charge, surface chemistry, size, and morphology. In further embodiments, the distinct physicochemical property comprises surface charge.

[0047] In various embodiments, the present disclosure provides a composition comprising a panel of particles, the panel comprising a plurality of particle types, the plurality of particle types having at least three different physicochemical properties. In some embodiments, the different physicochemical properties are selected from the group consisting of surface charge, surface chemistry, size, and morphology. In further embodiments, the different physicochemical properties include surface charge.

[0048] In various embodiments, the present disclosure provides a system including any one of the above panels.

[0049] In various embodiments, the present disclosure provides a system including a panel, the panel comprising a plurality of particle types. In some embodiments, the plurality of particle types have at least three different physicochemical properties. In some embodiments, the panel includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different particle types. In some embodiments, the plurality of particle types can adsorb a plurality of proteins from a sample to form a plurality of protein coronas. In some embodiments, the plurality of protein coronas are digested to determine a protein profile. In some embodiments, the protein profile is associated with a biological state using a trained classifier.

[0050] In some embodiments, the sample is a biological sample. In further embodiments, the biological sample is plasma, serum, CSF, urine, tears, cell lysate, tissue lysate, cell homogenate, tissue homogenate, nipple aspirate, fecal sample, synovial fluid, and whole blood, or saliva. In some embodiments, the sample is a non-biological sample. In further embodiments, the non-biological sample is water, milk, a solvent, or a homogenate sample. Incorporation by Reference

[0051] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0052] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application document with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0053] [Figure 1] Figure 1 shows an example of the surface chemical structure of a magnetic particle (MP). In some cases, the magnetic particle may be a magnetic core nanoparticle (MNP).

[0054] [Figure 2] Figure 2A shows the formation of a protein corona on a particle. The protein corona profile depends on protein-particle, protein-protein, and protein concentration factors. Figure 2B shows the formation of a protein corona on three different particles. In some cases, the particles may be nanoparticles. The properties of these particles result in different protein corona profiles.

[0055] [Figure 3] Figure 3 shows some examples of particle types and some examples of ways in which the particle surface can be functionalized. In some cases, the particles can be nanoparticles.

[0056] [Figure 4]Figure 4 shows the preparation of superparamagnetic iron oxide nanoparticles (SPIONs) from the residual solution. As shown in the left-hand photograph, the SPIONs are dispersed into the solution before or upon application of a magnet to the side of the vial, appearing as a dark, opaque solution in the glass vial. Within 30 seconds of applying the magnet to the side of the vial, the SPIONs separate from the solution, as illustrated by the accumulation of dark particles next to the magnet and increased solution transparency in the right-hand photograph. When the separated solution, shown in the right image, is shaken, the particles return to their dispersed state, shown in the left-hand image, within 5 seconds. SPIONs are fast-responding.

[0057] [Figure 5] FIG. 5 provides an example of the process for generating proteomic data and for panel selection.

[0058] [Figure 6] FIG. 6 shows some examples of different properties of particles and how to characterize them.

[0059] [Figure 7] Figure 7 shows an example of the size distribution of nanoparticles characterized by dynamic light scattering. Figure 7 shows a dynamic light scattering overlay of two particle types: SP-002 (phenol-formaldehyde coated particles) and SP-010 (carboxylate, PAA coated particles), both of which have iron oxide cores. Dynamic light scattering can also be used to measure the size distribution of larger sized particles, including microparticles.

[0060] [Figure 8]Figures 8A and 8B show the characterization of nanoparticles with different functionalization before corona formation. Figure 8A shows a transmission electron microscopy (TEM) of SP-002 (particles with a phenol-formaldehyde coating). Figure 8B shows a TEM of SP-339 (polystyrene carboxyl particles). TEM can also be used to characterize larger sized particles, including microparticles.

[0061] [Figure 9] Figure 9 shows Fe 2p / 3 spectra from various nanoparticles, including SP-333 (carboxylate), SP-339 (polystyrene carboxylate), SP-356 (silica amino), SP-374 (silica silanol), HX-20 (SP-003) (silica coated), HX-42 (SP-006) (silica coated, amine), and HX-74 (SP-007) (PDMPAPMA coated, dimethylamine). The spectra were obtained from XPS (X-ray photoelectron spectroscopy), which provides a chemical fingerprint of the particle surface (measuring the percentage of various elements on the surface). XPS can also be used to measure spectra of larger sized particles, including microparticles.

[0062] [Figure 10] Figure 10 shows an example of the disclosed process for proteome analysis. The process shown is optimized for high throughput and automation, allowing it to be performed simultaneously across multiple samples within a few hours. The process includes particle-matrix association, particle washing (x3), protein corona formation, in-plate digestion, and mass spectrometry. Using this process, a batch of 96 samples can take only 4-6 hours. Typically, one particle type is incubated with the sample at a time.

[0063] [Figure 11]Figure 11 shows protein counts (number of proteins identified from corona analysis) for panel sizes ranging from 1 particle type to 12 particle types. Each particle in the panel can be unique in base material, surface functionalization, and / or physical properties (e.g., size or shape). A single pooled plasma representative of a pool of healthy subjects was used. Counts are the number of unique proteins observed across a panel of 12 particle types in an approximately 2-hour mass spectrometry (MS) run. 1,318 proteins were identified using a panel size of 12 particle types. As used herein, a "feature" identified by mass spectrometry includes a signal at a specific combination of retention time and m / z (mass-to-charge ratio), with each feature having an associated intensity. Some features are further fragmented in a second mass spectrometry analysis (MS2) for identification.

[0064] [Figure 12] Figure 12 shows gel electrophoresis analysis of multiple particle types after incubation in plasma over multiple days. Each individual assay typically ran for approximately 1 hour, with triplicate runs performed by different operators on three different days to demonstrate assay precision. From left to right, the gel shows a ladder, three consecutive rows of SP-339 (polystyrene carboxyl), three consecutive rows of SP-374 (silica silanol), a DNA ladder, and three consecutive rows of HX56 (SP-007) (silica amino).

[0065] [Figure 13] Figure 13 shows mass spectrometry analysis of protein identification across three separate assays of one particle type (SP-339, polystyrene carboxyl). Across the three separate assays, 180 proteins were commonly identified.

[0066] [Figure 14A]Figures 14A and 14B show the concentration response to spiked protein compared to the control. The spikes vary with concentration. The endogenous protein control did not vary with concentration. Figure 14A shows data from a CRP spike recovery experiment. Protein was spiked at four levels: 2x, 5x, 10x, and 100x. HX-42 (SP-006) (left) and HX-97 (right, same as SP-007) were used. Figure 14B shows the slope of the response for the spike and control. The slopes from the regression model were fit to the MS-enzyme-linked immunosorbent assay (ELISA) data. [Figure 14B] Figures 14A and 14B show the concentration response to spiked protein compared to the control. The spikes vary with concentration. The endogenous protein control did not vary with concentration. Figure 14A shows data from a CRP spike recovery experiment. Protein was spiked at four levels: 2x, 5x, 10x, and 100x. HX-42 (SP-006) (left) and HX-97 (right, same as SP-007) were used. Figure 14B shows the slope of the response for the spike and control. The slopes from the regression model were fit to the MS-enzyme-linked immunosorbent assay (ELISA) data.

[0067] [Figure 15] Figure 15 shows a comparative study to evaluate particle types for panel selection. 56 plasma samples were obtained from 28 diseased subjects and 28 control subjects. The diseased subjects had confirmed stage IV non-small cell lung cancer (NSCLC), comorbidities, and treatments, such as diabetes, cardiovascular disease, and hypertension. The control subjects were age- and sex-matched to the diseased subjects to reduce bias. This study was conducted to evaluate particle types between groups with significant differences, showing age and sex matching. Seven particles were used in this study, as shown in Figure 16A.

[0068] [Figure 16A]Figure 16A shows a comparison of samples (28 affected vs. 28 controls) using a panel of 7 particle types including SP-339, HX74 (SP-007), SP-356, SP-333, HX20 (SP-003), SP-364, and HX42 (SP-006). 14,481 filtered MS features were compared, with 120 (0.8%) different. [Figure 16B] Figure 16B shows the top hits from SP-339 in Figure 16A. The differences detected confirm that corona can differentiate between sample types and ultimately build a classifier to define disease versus health.

[0069] [Figure 17] Figure 17A shows a schematic diagram of the process described herein, including sample collection from healthy and cancer patients, isolation of plasma from the samples, incubation with uncoated liposomes to form a protein corona, and enrichment of select plasma proteins. Proteins in the samples were assayed using a particle-type panel with distinct particle types to enrich proteins in distinct biomolecular coronas formed on the distinct particle types. Protein corona formation was specific to the physicochemical properties of the particles. Figure 17B shows corona analysis using an embodiment of the present disclosure, Proteograph, for a three-particle-type panel. Plasma was collected from 45 subjects (eight from each of five cancers, including glioblastoma, lung cancer, meningioma, myeloma, and pancreatic cancer, and five healthy controls). Corona analysis output, Proteograph, was generated for each particle in the three-particle-type panel. Random forest models were constructed in each of 1000 rounds of cross-validation, providing strong evidence of robust corona analysis signals. A first preliminary analysis was performed by principal component analysis (PCA) on the proteins detected from the combination of the three particles.

[0070] [Figure 18]Figures 18A and 18B show that early cancers can be isolated up to 8 years before symptom onset. The Golestan cohort enrolled 50,000 healthy subjects between 2004 and 2008. Stored plasma from the enrollment was tested, as shown in Figure 18A. Eight years after enrollment, approximately 1,000 patients developed cancer. Figure 18B shows the classification of stored plasma from Figure 18A. Coronal analysis of stored plasma from the enrollment date correctly classified cancer in 15 of the 15 subjects tested (5 patients for each of the three cancer types).

[0071] [Figure 19] Figure 19 shows the robust classification of five cancer types with an overall accuracy of 95% using three-particle corona analysis on the Proteograph. The data show that adding particle type diversity improves performance. Three different liposomes with negative, neutral, and positive net surface charges (pH 7.4) were used.

[0072] [Figure 20] Figure 20 shows an example of scaling particle biosensor production. This platform can be used across multiple assays and samples.

[0073] [Figure 21] Figure 21 shows examples of particle types of the present disclosure. Particle types can include nanoparticles (NPs) and microparticles.

[0074] [Figure 22A]Figures 22A-B show schematic diagrams of particle protein corona formation (Figure 22A) and the Proteograph platform workflow (Figure 22B) of an embodiment of the present disclosure based on a multi-particle protein corona approach and mass spectrometry for plasma proteome analysis. Figure 22A shows three distinct particle types (depicted in the center of the figure, with the top, middle, and bottom spheres representing the three distinct particle types) that differ from each other in at least one physicochemical property, leading to the formation of different protein corona compositions on the particle surface. Figure 22B shows the corona analysis workflow using the Proteograph, including (1) particle-plasma incubation and protein corona formation, (2) particle protein corona purification by magnet, (3) digestion of corona proteins, and (4) analysis by mass spectrometry. [Figure 22B] Figures 22A-B show schematic diagrams of particle protein corona formation (Figure 22A) and the Proteograph platform workflow (Figure 22B) of an embodiment of the present disclosure based on a multi-particle protein corona approach and mass spectrometry for plasma proteome analysis. Figure 22A shows three distinct particle types (depicted in the center of the figure, with the top, middle, and bottom spheres representing the three distinct particle types) that differ from each other in at least one physicochemical property, leading to the formation of different protein corona compositions on the particle surface. Figure 22B shows the corona analysis workflow using the Proteograph, including (1) particle-plasma incubation and protein corona formation, (2) particle protein corona purification by magnet, (3) digestion of corona proteins, and (4) analysis by mass spectrometry.

[0075] [Figure 23]FIG. 23 shows the characterization of three superparamagnetic iron oxide nanoparticles (SPIONs) shown in the first column on the far left—from top to bottom: a silica-coated SPION (SP-003), a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated SPION (SP-007), and a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-coated SPION (SP-011)—by the following methods: scanning electron microscopy (SEM, second column of images), dynamic light scattering (DLS, third column of images), transmission electron microscopy (TEM, fourth column of images), high-resolution transmission electron microscopy (HRTEM, fifth column), and X-ray photoelectron spectroscopy (XPS, sixth column). DLS shows three replicates of each particle type. HRTEM photographs were recorded on the surface of individual SP-003, SP-007, and SP-011 particle types, respectively, with arrows pointing to regions of amorphous SiO2 (top HRTEM image) and amorphous SiO2 / polymer coating (middle and bottom HRTEM images) on the particle surface.

[0076] [Figure 24] Figure 24 shows the dynamic range for proteins observed in raw plasma for SP-003, SP-007, and SP-011 particles compared to a compiled database (top panel) from Keshishian et al. (Mol Cell Proteomics. 2015 Sep;14(9):2375-93. doi: 10.1074 / mcp.M114.046813. Epub 2015 Feb 27.).

[0077] [Figure 25] FIG. 25 shows the correlation of maximum intensity of particle corona proteins relative to plasma proteins with the reported concentrations of the same proteins.

[0078] [Figure 26]FIG. 26 shows the reproducibility of particle corona intensity for each particle type (SP-003, SP-007, and SP-011) demonstrated by three replicate experiments using the same plasma sample.

[0079] [Figure 27] Figure 27 shows altered features in a non-small cell lung cancer (NSCLC) pilot study using SP-007 particles. Seven MS features were identified as statistically significantly different between 28 subjects with stage IV NSCLC (with associated comorbidities and treatment effects) and 28 age- and sex-matched apparently healthy subjects. The table below lists the seven proteins with significant differences, including five known proteins and two unknown proteins. If a peptide-spectrum match was found to the MS2 data associated with the feature, the peptide sequence (and alteration) and potential parent protein are shown; if no MS2 match was associated with the feature, both the peptide and protein are marked as "unknown."

[0080] [Figure 28] Figure 28A shows a schematic diagram for the synthesis of a SPION core. Figure 28B shows a schematic diagram for the synthesis of a silica-coated SPION (SP-003). Figure 28C shows a schematic diagram for the synthesis of a vinyl-functionalized SPION. Figure 28D shows a schematic diagram for the synthesis of a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated SPION (SP-007). Figure 28E shows a schematic diagram for the synthesis of a poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA)-coated SPION (SP-011).

[0081] [Figure 29]Figure 29 shows the distribution of median-normalized MS feature intensities, filtered for presence and filtered for cluster quality, for a 56-sample NSCLC comparative study. Each line represents the density of log2 feature intensities for either diseased or control samples. Densities from 0.00 to 0.15 are plotted on the y-axis, and log2 feature intensities from 15 to 35 are plotted on the x-axis. At the highest peak, located near a log2 feature intensity of approximately 28, where the density ranges from approximately 0.13 to approximately 0.17, the two highest traces correspond to control samples, while the lowest trace corresponds to a diseased sample. The remaining control and diseased traces are distributed between the highest and lowest traces. At a log2 feature intensity of approximately 20 and two shoulder peaks present at a log2 feature intensity of approximately 23, at a log2 feature intensity of 20, the two highest traces are control traces, and the two lowest traces are control traces; at a log2 feature intensity of 23, the highest trace is a diseased trace.

[0082] [Figure 30] FIG. 30 shows the measurement accuracy for C-reactive protein (CRP) for SP-007 nanoparticles in four different peptide spike recovery experiments.

[0083] [Figure 31] FIG. 31 shows the accuracy of measuring peptide characteristics of angiogenin in spike recovery experiments.

[0084] [Figure 32-1] FIG. 32 shows the accuracy of measuring peptide signatures of S10A8 in spike recovery experiments. [Figure 32-2] FIG. 32 shows the accuracy of measuring peptide signatures of S10A8 in spike recovery experiments.

[0085] [Figure 33-1] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0086] [Figure 33-2] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0087] [Figure 33-3] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0088] [Figure 33-4] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0089] [Figure 33-5] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0090] [Figure 33-6] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0091] [Figure 33-7] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0092] [Figure 33-8] FIG. 33 shows the accuracy of measuring peptide signatures of S10A9 in spike recovery experiments.

[0093] [Figure 34] FIG. 34 shows the accuracy of measuring peptide features of C-reactive protein (CRP) in spike recovery experiments.

[0094] [Figure 35-1] FIG. 35 shows the accuracy of measurement of protein features in spike recovery experiments. [Figure 35-2] FIG. 35 shows the accuracy of measurement of protein features in spike recovery experiments.

[0095] [Figure 36] Figure 36 shows the matching and coverage of a particle panel of 10 distinct particle types against a 5,304 plasma protein database of MS intensities. The ranked intensities of the database proteins are shown in the top panel ("Database"), the intensities of proteins from a simple plasma MS assessment are shown in the second panel ("Plasma"), and the intensities of the optimal 10-particle type panel are shown in the remaining panels. The plasma protein intensity database is from Keshishian et al. (2015). Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields: Novel Candidates for Early Myocardial Injury. Molecular & Cellular Proteomics, 14(9), 2375-2393.

[0096] [Figure 37] Figure 37 shows various particle types that may be included in the panels disclosed herein. Figure 37A shows a schematic of a hollow magnetic particle. Figure 37B shows a schematic of a particle with a hydrophobic pocket. Figure 37C shows a schematic of an eggshell-yolk type SPION microgel hybrid particle.

[0097] [Figure 38] FIG. 38 shows a schematic diagram of particle surfaces engineered to capture proteins and / or peptides, taken from Mol. Cells 2019, 42(5), 386-396. DETAILED DESCRIPTION OF THE INVENTION

[0098] Disclosed herein are compositions and methods for using them to assay peptides and proteins in a sample in a simple and high-throughput manner. The disclosure provides particle panels of multiple distinct particle types that concentrate proteins from a sample onto distinct biomolecular coronas formed on the surfaces of the distinct particle types. The particle panels disclosed herein can be used in corona analysis methods to detect thousands of proteins over a wide dynamic range in a few hours.

[0099] Currently, only a small number of protein-based biomarkers are currently used in clinical diagnostics, and despite extensive efforts to analyze the plasma proteome to expand the marker pool, relatively few new candidates have been accepted as clinically useful diagnostics. The plasma proteome contains >10,000 proteins and potentially contains an extraordinary number of protein isoforms with concentrations spanning more than 10 orders of magnitude (mg / mL to pg / mL). These characteristics, combined with the lack of traditional molecular tools (e.g., copying or amplification mechanisms) for protein analysis tasks, make comprehensive studies of the plasma proteome extremely challenging. Approaches to overcome the wide dynamic range of proteins in biological samples remain for robust identification and quantification against a background of thousands of unique proteins and even more protein variants. However, no technology exists that can simultaneously measure proteins across the entire plasma concentration range in a format with sufficient throughput and a realistic cost profile to enable studies of appropriate size with robust prospects for validation and replication. These challenges not only limit the discovery of protein-based biomarkers for disease but also hinder the more rapid adoption of proteogenomics and protein annotation of genomic variants. Advances in mass spectrometry (MS) methods, along with the development of improved data analysis, have provided tools for deep and broad proteome analysis. Several efforts have been made to substantially improve the detection of low-abundance proteins, such as depletion of high-abundance proteins, plasma fractionation, peptide fractionation, and label-based quantification. However, current methods are extremely complex and time-consuming (days to weeks), thus requiring a trade-off between protein coverage depth and sample throughput. Consequently, simple and robust strategies for comprehensive and rapid analysis of the wealth of available information about the proteome remain an unmet need.

[0100] In addition, the earlier a disease is diagnosed, the greater the chance of successfully curing or managing it, resulting in a better prognosis for the patient. Treating a disease early can potentially prevent or delay problems from the disease, potentially improving patient outcomes, including extending the patient's lifespan and / or quality of life. Early diagnosis of cancer is crucial because many types of cancer can be successfully treated in their early stages. For example, the 5-year survival rates for breast cancer, ovarian cancer, and lung cancer after early diagnosis and treatment are 90%, 90%, and 70%, respectively, compared with 15%, 5%, and 10% for patients diagnosed at the most advanced disease stage. Once cancer cells leave their primary tissue, successful treatment using available established therapies becomes highly unlikely. While recognizing warning signs of cancer and taking prompt countermeasures can lead to early diagnosis, the majority of cancers (e.g., lung) only show symptoms after cancer cells have already invaded surrounding tissues and metastasized throughout the body. For example, more than 60% of patients with breast, lung, colon, and ovarian cancer have occult or even metastatic colonies when their cancer is detected. Therefore, there is an urgent need to develop effective methods for early cancer detection. Such methods should have the sensitivity to identify cancer at various stages and the specificity to provide a negative result if the person undergoing the test does not have cancer. Although there have been significant efforts to develop methods for early cancer detection, and a vast number of risk factors and biomarkers have been introduced, a broadly relevant platform for the early detection of a wide range of cancers remains elusive. Because various types of cancer can alter the composition of plasma, even in their early stages, one promising approach for early detection is molecular blood analysis of biomarkers. This strategy has already been investigated for a few cancers (e.g., PSA for prostate cancer), but specific biomarkers for the early detection of the majority of cancers do not yet exist. For such cancers (e.g., lung), none of the defined circulating biomarker candidates have been clinically validated, and very few have reached late-stage clinical development.Therefore, novel approaches are urgently needed to improve our ability to detect cancer as well as other diseases at very early stages.

[0101] To meet the need for high-throughput, simple assays for detecting proteins in samples that can be used for very early detection of proteins associated with specific diseases, the present disclosure provides particle panels and methods for using the particle panels to assay for peptides and proteins in samples (e.g., complex biological samples, such as plasma) in a simple, rapid, and high-throughput manner. Specifically, the present disclosure provides particle panels of multiple distinct particle types that concentrate proteins from a sample onto distinct biomolecular coronas formed on the surfaces of the distinct particle types. The particle types included in the particle panels disclosed herein are particularly well suited for enriching multiple proteins over a wide dynamic range in an unbiased manner. The combinations of particle types selected for inclusion in the particle panels of the present disclosure vary in their physicochemical properties (e.g., size, surface charge, core material, shell material, surface chemistry, porosity, morphology, and other properties). However, particle types may share some of the physicochemical properties. For example, a particle panel disclosed herein can include a first particle type and a second particle type, where the first particle type and the second particle type share at least two physicochemical properties but differ in at least two physicochemical properties, and thus the first particle type and the second particle type are distinct. Importantly, a change in at least one physicochemical property between the first particle type and the second particle type of a particle panel can lead to the formation of distinct coronas on the corresponding distinct particle types. Thus, the selection of particle types for inclusion in a particle panel for use in the methods disclosed herein allows for a large number of proteins to be enriched in a sample (e.g., plasma) over a wide dynamic range.

[0102] The particle panels of the present disclosure can include particle types with magnetic properties that allow for easy separation after incubation in complex biological samples. For example, the present disclosure provides superparamagnetic iron oxide nanoparticles (SPIONs) that have unique magnetic properties that allow for rapid separation of biomolecules, drug delivery agents, and contrast agents in magnetic resonance imaging (MRI). The superparamagnetic particles can have a pure iron oxide core (e.g., an iron oxide core) or small iron oxide crystals embedded in a polystyrene core.

[0103] This disclosure provides a method developed for the synthesis of SPIONs. In one example, thermal decomposition of iron oleate in a nonpolar solvent can be used to synthesize small (typically <30 nm) monodisperse magnetic nanoparticles with high crystallinity. These nanoparticles are hydrophobic and require transfer to the aqueous phase by ligand exchange or chemical modification. SPIONs produced using this method can meet the requirements for biomedical use. The solvothermal method is another method for synthesizing SPIONs by the reduction of iron(III) chloride with ethylene glycol. Highly water-dispersible SPMNPs can be synthesized by a modified solvothermal method using hydrophilic ligands such as citrate, polyacrylic acid (PAA), and polyvinylpyrrolidone (PVP). The particle surface can be further modified with different functionalized silanes by the Stöber method. Surface functional groups can also be obtained by a convenient method using seeded emulsion polymerization in the absence of surfactants to form SPMNP@polymer composite particles.

[0104] The present disclosure provides compositions, systems, and methods for their use for large-scale, high-throughput, efficient, and cost-effective proteome profiling and machine learning. Disclosed herein is a scalable, parallel protein identification and quantification technique for assaying proteins in a sample using a particle panel having distinct particle types to enrich for proteins in distinct biomolecular coronas formed on the distinct particle types. As used herein, "biomolecular corona" may be used interchangeably with the term "protein corona" and refers to the formation of a layer of proteins on the surface of particles after contacting the particles with a sample (e.g., plasma). This method may also be referred to interchangeably as corona analysis, or in some instances, "proteographic" analysis (illustrated in Figure 22), which combines a multi-particle protein corona strategy with mass spectrometry (MS). The particle types included in the particle panels disclosed herein are superparamagnetic and therefore rapidly separated or isolated from unbound proteins (proteins not adsorbed to the particle surface to form a corona) in the sample after incubation of the particles in the sample.

[0105] Currently, particles are poorly characterized for high-throughput translational proteomic analysis due to steps related to processing of proteins in the protein corona (e.g., centrifugation or membrane filtration to separate corona proteins from free plasma proteins, and washing to remove loosely bound proteins from the particles), which confound data and lack reproducibility and accuracy. The corona analysis (e.g., "proteograph") method disclosed herein can integrate overlapping but distinct particle-based protein coronas with, for example, liquid chromatography-mass spectrometry (LC-MS) for potential use in large-scale efficient proteome profiling and machine learning. The particle-based platform can be unbiased (e.g., not limited to a given analyte), and the MS data collection platform can be unbiased in terms of analyte measurement, both of which may be amenable to automation. The formation of a layer of protein on the surface of particles upon contact with plasma, called the protein corona (Figure 22A), is disclosed herein as a method for identifying proteins. The composition and amount of corona proteins can depend on the physicochemical properties of the particle type, and by varying these engineered properties, proteins that differ in identity and / or amount in the corona can be reproducibly obtained.

[0106] In some embodiments, the particles disclosed herein may be superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs may be distinct from one another in that they are synthesized with distinct surface chemistries. In some embodiments, SPIONs with different surface chemistries can be used together to analyze proteins formed on their protein coronas. SPIONs, other particle types, or a combination thereof can be used together to create a panel of particle types that can be used for proteomic analysis of a sample.

[0107] In some embodiments, disclosed herein is a panel of three particle types with distinct surface chemistries that are synthesized and used to form a protein corona, allowing for rapid magnetic separation from unbound proteins. Each particle type can reproducibly generate a unique protein corona pattern by capturing both high- and low-abundance proteins. For example, by integrating distinct proteomic profiles generated from at least three particle types, it is possible to identify more than 1,500 proteins in a single pooled colorectal cancer (CRC) plasma sample, many of which may be FDA-approved / certified biomarkers. For example, in some embodiments, screening three particle types can detect more than 1,500 proteins, 65 of which are FDA-approved / certified biomarkers.

[0108] In some embodiments, the corona analysis workflow using the Proteograph (Figure 22B) can take approximately 4-6 hours to prepare a batch of 96 corona samples for analysis by MS. Identification of proteins in the CRC pool can be performed using only three full MS fractions, each approximately 1 hour long, for a total of approximately 3 hours of MS time.

[0109] In some embodiments, more than 1,500 proteins can be identified from a single pooled plasma within 8 hours, including sample preparation and LC-MS, using three distinct particle types, as opposed to less than 500 proteins using the compositions, systems, and methods disclosed herein for using a particle-type corona strategy.

[0110] This corona analysis technology can be used to identify diseases. For example, the particles and methods for their use disclosed herein can be used to analyze serum samples from patients with non-small cell lung cancer (NSCLC) and age- and sex-matched healthy subjects. MS features, including known and novel features, that can distinguish NSCLC samples from control samples can be discovered using this platform. Therefore, the corona analysis platform technology allows for larger, more robust validation and replication studies. Furthermore, the unique properties of corona analysis for high-throughput, unbiased proteome sample extraction may enable the annotation of genomic data and the application of machine learning classification methods. The multi-particulate protein corona-based platform technology described herein can facilitate efficient and comprehensive proteome profiling, enabling larger studies for biomarker discovery and validation.

[0111] In some embodiments, the compositions and methods of their use disclosed herein exhibit high assay accuracy, as demonstrated by the addition of increasing concentrations of reference C-reactive protein (CRP) to plasma samples and subsequent detection of CRP levels in the protein corona, which exhibits a slope of 0.9 (95% CI 0.81-0.98) for CRP levels in the particle corona relative to the spiked plasma. In some embodiments, the median platform precision across assay replicates can be approximately 24 CV% across 8,738 measured MS features obtained from three separate particle-type protein coronas.

[0112] SPIONs with distinct surface chemistry can be applied to protein corona analysis of a single pooled plasma sample. For example, as described in this disclosure, the resulting proteome data demonstrates that increasing the number of particle types in a particle panel can result in the identification of more proteins (especially low-abundance proteins). By adding more distinct particle types, broader and deeper proteome profiling can be obtained. The particle compositions disclosed herein, and particle type panels containing the different particle types, can be tailored to profile the proteome at different levels of depth and breadth—similar to different levels of coverage in gene sequencing—by varying the number and types of particles in the panel.

[0113] The multiparticulate protein corona-based assays disclosed herein exhibit several equally important features for plasma proteome analysis. Compared to traditional proteome techniques, which typically involve time-consuming depletion and fractionation workflows, the compositions and methods of use disclosed herein can avoid these complex workflows and be much faster. Notably, corona analysis assays can be robustly automated, further improving the accuracy and reducing the amount of time required for sample analysis, for example, in a 96-well plate format. The corona analysis platform can sensitively measure differences between samples, further reducing the dynamic range of those comparisons and thus allowing for more comparisons to be observed. Corona analysis techniques can identify new biomarkers without targeting a predetermined set of proteins. The scalability and efficiency of the corona analysis platform can be used for large-scale proteome studies, which may lead to a deeper understanding of disease and biological mechanisms. For example, by adding proteome data to multi-omic datasets and performing machine learning analysis, novel classifications can be generated and put into context genomic disease information that is currently not well understood, such as single nucleotide polymorphism (SNP) variants, changes in DNA methylation patterns, and splice variants. In addition, this technology can be extended to other body fluids, such as cerebrospinal fluid, cell lysates, and even tissue homogenates, for rapid, reliable, and accurate profiling of the proteome, which can facilitate the discovery of new biomarkers for different diseases.

[0114] Disclosed herein are methods for making superparamagnetic nanoparticles (SPMNPs) or superparamagnetic iron oxide particles (SPIONs). These particles and embodiments thereof can be used in protein corona assays.

[0115] The disclosed methods and systems improve proteome analysis by simplifying sample preparation and MS data collection, performing sample preparation in about five steps within about 0.25 days and collecting MS data for about 12 fractions within about 0.5 days per sample.

[0116] The present disclosure provides compositions for assaying samples for proteins and methods of their use. The compositions described herein include particle panels containing one or more distinct particle types. The particle panels described herein can vary in the number of particle types and the diversity of particle types within a single panel. For example, particles within a panel can vary by size, polydispersity, shape and morphology, surface charge, surface chemistry and functionalization, and base material. The panel can be incubated with a sample and analyzed for proteins and protein concentrations. Proteins in the sample adsorb to the surfaces of different particle types within the particle panel to form protein coronas. The exact proteins and protein concentrations adsorbed to a particular particle type within the particle panel can depend on the composition, size, and surface charge of the particle type. Thus, each particle type within a panel can have a different protein corona due to adsorption to a different set of proteins, different concentrations of specific proteins, or a combination thereof. Each particle type within a panel can have mutually exclusive or overlapping protein coronas. Overlapping protein coronas may have overlapping protein identifications, overlapping protein concentrations, or both.

[0117] The present disclosure also provides methods for selecting particle types for inclusion in a panel depending on the sample type. The particle types included in the panel can be a combination of particles optimized for removal of high abundance proteins. Particle types also compatible for inclusion in the panel are those selected for adsorbing a specific protein of interest. The particles can be nanoparticles. The particles can be microparticles. The particles can be a combination of nanoparticles and microparticles.

[0118] The present disclosure provides methods for selecting particle panels that represent broad coverage of proteins in biological samples (e.g., plasma samples). Particles are selected for inclusion in the particle panel using a combinatorial approach. Particles with a wide range of physicochemical properties are selected; for example, particles may differ in size, surface charge, core material, shell material, surface chemistry, porosity, morphology, and other properties. However, particles may also share some of the physicochemical properties. For example, a particle panel disclosed herein can include a first particle type and a second particle type, where the first particle type and the second particle type share at least two physicochemical properties but differ in at least two physicochemical properties, and thus the first particle type and the second particle type are different. A particle panel disclosed herein can include a first particle type and a second particle type, where the first particle type and the second particle type share at least one physicochemical property but differ in at least two physicochemical properties, and thus the first particle type and the second particle type are different. The particle panels disclosed herein can include a first particle type and a second particle type, where the first particle type and the second particle type share at least two physicochemical properties but differ in at least one physicochemical property, thus dissimilar. Non-limiting examples of physicochemical properties include size, charge, core material, shell material, porosity, or surface hydrophobicity. Importantly, a change in at least one physicochemical property between the first particle type and the second particle type of a particle panel can lead to the formation of distinct coronas on the corresponding distinct particle types. For example, a first particle type and a second particle type with different charges can each adsorb different proteins, the same protein at different concentrations, or both different proteins and the same protein at different concentrations. Thus, the first particle type and the second particle type will have distinct biomolecular coronas. Size is one example of a physicochemical property that can be varied to achieve this result.One or more physicochemical properties (e.g., size, charge, core material, shell material, porosity, or surface hydrophobicity, or any combination thereof) can be varied to produce distinct biomolecular coronas. Other optimization parameters for the selection of particle types for a panel can include any particular annotation set, such as interactome, secretome, FDA markers, or proteins with clinically significant genetic polymorphisms. As seen in Tables 10 and 12, more than one of these particles is a nanoparticle but is made with a different polymer coating. In another example, more than one particle in Tables 10 and 12 shares a similar surface charge and similar size but is made with a different material. In another example, more than one particle in Tables 10 and 12 exhibits a porous surface. In another example, more than one particle in Tables 10 and 12 exhibits a nonporous surface. In another example, more than one particle in Tables 10 and 12 exhibits a carboxylate-coated surface. In another example, more than one particle in Table 10 and Table 12 exhibits an amine-coated surface. With all of the many combinations of particles and particle types that may be present within a particle panel, it is surprising and unexpected that the particle panels disclosed herein can identify a large number of proteins (e.g., plasma proteins) in a sample (e.g., a plasma sample) in an unbiased manner over a wide dynamic range, and can be used in methods to assay proteins in a sample with high levels of reproducibility (e.g., quantile-normalized coefficient of variation <20%).

[0119] The present disclosure provides more than 200 distinct particle types, with more than 100 different surface chemical structures and more than 50 different physical properties.In particular, more than 23 particle types have been characterized for use in the method of assaying proteins in samples.Each of these particle types can be combined with other particle types in a panel designed to optimally assay specific proteins of interest or optimally identify biomarkers for diseases of interest.A panel can include any number of these particle types or any combination of particle types, and the various particle types disclosed herein can enable the assay and detection of a wide range of proteins with different physicochemical properties.

[0120] In some embodiments, the present disclosure provides a method for identifying proteins in a sample, the method comprising: incubating a panel comprising a plurality of particle types with the sample to form a plurality of protein coronas; digesting the plurality of protein coronas to generate proteomic data; and identifying the proteins in the sample by quantifying the proteomic data. particle material

[0121] The particle panels disclosed herein can include particle types made from a variety of different materials. Panels can be assembled with specific particle types to identify a wide range of proteins in a sample or selectively assay for a specific protein or set of proteins of interest. Particle types can include, for example, nanoparticles (NPs), microparticles, magnetic particles (MPs), magnetic nanoparticles (MNPs), paramagnetic iron oxide nanoparticles (SPIONs), or superparamagnetic nanoparticles (SPMNPs). The particles described herein can be magnetic particles. The magnetic particles herein can be superparamagnetic particles (SPMPs), superparamagnetic nanoparticles (SPMNPs), superparamagnetic iron oxide particles (SPIOPs), or superparamagnetic iron oxide nanoparticles (SPIONs). In some cases, SPMNPs can be SPIONs. The magnetic force can be imparted by the iron oxide core or by iron oxide crystals grafted to the particle. In some cases, the inventors refer to SPMNPs as magnetic particles herein. SPMNPs can also be synthesized to be SPMPs.

[0122] Particles can be made from a variety of materials. For example, particles can be made from polymers, lipids, metals, silica, proteins, nucleic acids, small molecules, or large molecules. For example, particle materials consistent with the present disclosure include metals, metal oxides, magnetic materials, polymers, and lipids. Examples of metal materials include gold, silver, copper, nickel, cobalt, palladium, platinum, iridium, osmium, rhodium, ruthenium, rhenium, vanadium, chromium, manganese, niobium, molybdenum, tungsten, tantalum, iron, and cadmium, or any other material described in U.S. Patent No. 7,749,299, either singly or in any combination. The metal oxide particles can be iron oxide particles or titanium oxide particles. The magnetic particles can be iron oxide nanoparticles.

[0123] Examples of polymers include any one or any combination of polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), polystyrene, or copolymers of two or more polymers, for example, copolymers of polyalkylene glycol (e.g., PEG) and polyester (e.g., PLGA). In some embodiments, the polymer is a lipid-terminated polyalkylene glycol, and polyester, or any other material disclosed in U.S. Pat. No. 9,549,901, the entirety of which is incorporated herein by reference.

[0124] Examples of lipids that can be used to form particles of the present disclosure include cationic lipids, anionic lipids, and lipids with a neutral charge. For example, the particles may be composed of dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidyl Dioleoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-transIt may be made of any one or any combination of PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, and cholesterol, or any other materials listed in U.S. Pat. No. 9,445,994, the entirety of which is incorporated herein by reference.

[0125] The particle panels disclosed herein may include specific particle types with structures that are particularly suited for sampling proteins of specific sizes present in a sample. For example, the particle panel may include hollow magnetic particles as shown in FIG. 37A. Hollow magnetic particles include nanoparticles with a hollow core and a nanoparticle shell made of smaller iron oxide primary crystals. Hollow magnetic particles are described in Cheng, Wei, et al. ("One-step synthesis of superparamagnetic monodisperse porous nanoparticles," which is incorporated herein by reference in its entirety. They can be synthesized by an autoclave reaction based on hydrothermal treatment of FeCl, citrate, polyacrylamide or sodium polyacrylate, and urea, as described in "Fe3O4 hollow and core-shell spheres." Journal of Materials Chemistry 20.9 (2010): 1799-1805). The nanoparticle shells made of smaller iron oxide primary crystals are porous, allowing for the generation of a protein corona on the surface of the nanoparticles through a size exclusion effect. The binding surface is primarily inside the pores, thus preventing larger proteins from diffusing and binding within the particle. In some cases, larger proteins may still bind on the exterior and be included in the total corona, but these hollow particles can enrich smaller proteins more than larger ones. These hollow magnetic particles can be included in any of the particle panels described herein and are also easily separated from unbound proteins using a magnet.

[0126] In another example, the particle panel may include nanoparticles with hydrophobic pockets, as shown in Figure 37B. Nanoparticles with hydrophobic pockets are particularly suitable for sampling molecules or proteins with specific solubility (e.g., poorly soluble proteins) present in a sample. Nanoparticles with hydrophobic pockets have a superparamagnetic iron oxide core structure and additionally have a polymer coating. Nanoparticles with hydrophobic pockets can be synthesized using an autoclave reaction for SPION core synthesis, as described elsewhere herein, followed by free radical polymerization to synthesize a poly(glycidyl methacrylate) coating. This can be followed by post-synthetic modification of the surface with hydrophobic amines, such as benzylamine moieties. These nanoparticles with hydrophobic pockets are particularly well suited for capturing small molecules for metabolomics. The pore size of the nanoparticles can be modulated to exclude proteins, so that only small molecules interact with the hydrophobic pockets. In some embodiments, the pore size can be adjusted during synthesis. For example, synthesis may include swelling the particles to create porosity, and the degree of swelling can affect the pore size. As another example, corrosion techniques can be used to create different pores in particle types with harder surfaces. Nanoparticles with hydrophobic pockets can also be used to sample protein targets.

[0127] In another example, the particle panel can include eggshell-yolk SPION microgel hybrid nanoparticles, as shown in Figure 37C. Eggshell-yolk SPION microgel hybrid nanoparticles have a SPION exterior and a microgel interior. Eggshell-yolk SPION microgel hybrid nanoparticles can be synthesized using an autoclave reaction based on the hydrothermal treatment of FeCl, citrate, polyacrylamide or sodium polyacrylate, and urea, as described by Cheng et al. ("One-step synthesis of superparamagnetic monodisperse porous Fe3O4 hollow and core-shell spheres," Journal of Materials Chemistry 20.9 (2010): 1799-1805) and Zou et al. ("Facile synthesis of highly water-dispersible and monodispersed Fe3O4 hollow microspheres and their application in water treatment," RSC Advances 3.45 (2013): 23327-23334). In the second step of the synthesis, a hydrogel is formed within the hollow nanoparticles.

[0128] In another example, particle panels can include particle surfaces engineered to capture proteins and / or peptides. A primary layer of proteins can form an initial corona on the particle surface, either non-covalently or covalently. Direct non-covalent interactions with the particle surface can include ionic, hydrophobic, and hydrogen bonds. Additionally, these particles can be initially modified with small chemicals to bind to other proteins in plasma after the formation of the primary protein corona. Modulation of chemical modifications present on the particle surface can be used to tailor the non-covalent interactions between the particle surface and peptides and / or proteins in the sample. Alternatively or additionally, the solution-phase composition (e.g., pH) can be modulated to tailor the non-covalent interactions between the particle and peptides and / or proteins in the sample. Covalent coupling of proteins and / or peptides to the particle surface can be achieved by introducing reactive groups (e.g., NHS esters, maleimides, carboxylates, etc.) onto the particle surface. Therefore, particles with NHS esters may be particularly suitable for sampling one or more proteins in solution. Alternatively, the coupling reagent capable of sampling and binding proteins from a complex mixture can be a photoinitiated reactive group that can react with proteins only after exposure to photons of a given wavelength (e.g., UV) and intensity. Advantages of using particles functionalized with such photoinitiated reactive groups include, but are not limited to, (1) immobilization within the primary protein corona without protein loss during particle washing steps and / or replacement with proteins of higher affinity; (2) capture of proteins with moderate binding affinity because covalent bonds can be established at any time during the binding process; (3) generation of larger surfaces than would occur at equilibrium; and (4) generation of surfaces with specific protein stoichiometries by using defined protein mixtures (instead of plasma) for binding.A defined protein mixture can be any synthetically derived or purified mixture of proteins, either recombinantly obtained or produced, or isolated and then mixed in a defined stoichiometric ratio. For example, for a particular protein of interest for protein-protein interactions (e.g., ubiquitin), the defined protein mixture can be a simple solution of that protein. As another example, the defined protein mixture can be a purified or enriched set of proteins from a particular class of interest (e.g., glycosylated proteins). Particles can also be modified to be functionalized with one half of a click chemistry reaction pair, and the non-natural point mutant protein in the sample contains an amino acid with the other half of the click chemistry reaction pair. Particles and other proteins in the sample can be functionalized with one half of a click chemistry reaction pair, and the non-natural point mutant protein in the sample contains an amino acid with the other half of the click chemistry reaction pair. Reactions are carried out in the presence of a chemical catalyst (e.g., copper) or light (e.g., photo-initiated click chemistry reagents), leading to the linkage of particles to the non-natural point mutant protein in the sample and / or the linkage of the non-natural point mutant protein in the sample to other proteins in the sample. For example, this method can begin with a simple solution of protein, which may contain mutant proteins concentrated in a sample. A key advantage of these systems is that the particle surface can be engineered with respect to stoichiometry, protein / surface orientation, and protein / protein orientation. This allows for the engineering of durable surfaces that can withstand the assay steps described elsewhere herein (e.g., extensive washing). For example, proteins with specific unnatural amino acids at positions of interest within the protein sequence are described by Lee et al. (Mol Cells. 2019 May 31;42(5):386-396. doi: 10.14348 / molcells.2019.0078.). The unnatural amino acid introduced into the protein can have one half of a click chemistry pair, which can react with the other half of the click chemistry pair on the particle surface. In this way, rather than random adsorption of the protein to the particle surface to form a corona, these modified proteins can be attached to the particle surface in a specific orientation. As a result, the corona that forms on the surface of the particle type can be tailored with engineered proteins in a controlled, specific 3D orientation. This same general methodology can be used to attach protein complexes to the surface of the particle type. Here, one or more subunits of the complex can be modified to be covalently linked to each other and then attached to the surface of the particle type in a second synthesis step or using different chemistries that can be performed later in the particle surface modification process. A schematic diagram is shown in Figure 38. Particles with surfaces engineered to non-covalently or covalently capture proteins and / or peptides can nonetheless be SPION or polymer-modified SPION particles, which can be synthesized by a variety of methods, including solvothermal methods, ligand exchange processes, silica coating processes, and / or standard SPION synthesis by initiating or installing specific reagent coupling strategies. Advantages of these systems include biosurface generation, interactome utilization, and directed corona assembly.

[0129] In another example, the particle panel can include functionalized particles for histone capture. For example, these particles can be anionic. Additionally or alternatively, assay conditions can be optimized to enrich histones in samples, and labeling methods can be optimized to improve mass spectrometric detection of histones and post-translational modifications. These functionalized particles for histone capture can be made of polymers, silica, targeting ligands, and / or any combination thereof. Functionalized particles can be synthesized using a variety of techniques, including standard SPION synthesis via solvothermal methods, ligand exchange processes, and surface-initiated polymerization. Anionic particle surfaces can be synthesized by functionalizing the surface with polymers, such as polycarboxylates, various ligands, such as dendrimers, branched ligands, or carboxylate derivatives, and / or sulfanilamido acids. Optimizing assay conditions can include buffering the binding solution to a pH of approximately 9. While many proteins will exhibit reduced binding to anionic surfaces at this pH, histones are basic proteins with primary sequences having a pI of approximately 11 (e.g., H4_human pI approximately 11.3, H3_human pI approximately 11), which will maintain an almost entirely positively charged state. As a result, under basic pH conditions, histones can strongly interact with the particle surface through ionic bonds, and therefore, histones can be selectively enriched on the particle surface. In addition, histones can be selectively enriched on the particle surface by modifying the particle surface with histone-binding proteins. Therefore, histone protein coronas can be obtained using the particles disclosed herein. Labeling optimization methodologies can help simplify the interpretation of mass spectrometry data and enable improved identification and assignment of post-translational modifications (PTMs). Because the type, location, and occupancy of post-translational modifications on histones can vary and contribute to the regulation of gene expression, these particles can be useful for selectively enriching histones in samples and interrogating PTMs, which can provide information about how genes in a sample are regulated. Consequently, this can be useful in providing information on a variety of disease states in which gene expression is dysregulated.

[0130] Examples of particle types consistent with the present disclosure are shown in FIG. 21 and in Table 1 below. [Table 1] Particle characteristics

[0131] Particles consistent with the present disclosure can be made and used in methods to form protein coronas in a wide range of sizes after incubation in biological fluids. For example, particles disclosed herein can be at least 10 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1000 nm, at least 1100 nm, at least 1200 nm, at least 1300 nm, at least 1400 nm, at least 1500 nm, at least 1600 nm, at least 1700 nm, at least 1800 nm, at least 1900 nm, at least 2100 nm, at least 2200 nm, at least 2300 nm, at least 2400 nm, at least 2500 nm, at least 2600 nm, at least 2700 nm, at least 2800 nm, at least 2900 nm, at least 3000 nm, at least 3100 nm, at least 3200 nm, at least 3300 nm, at least 3400 nm, at least 3500 nm, at least 3600 nm, at least 3700 nm, at least 3800 nm, at least 3900 nm, at least 4000 nm, at least 4100 nm, at least 4200 nm, at least 4300 nm, at least 4400 nm, at least 4500 nm, at least 4600 nm, at least 4700 nm, at least 4800 nm, at least 4900 nm, at least 5000 nm, at least 5100 nm, at least 5200 nm, at least 5300 nm, at least 5 nm, at least 1900 nm, at least 2000 nm, at least 2100 nm, at least 2200 nm, at least 2300 nm, at least 2400 nm, at least 2500 nm, at least 2600 nm, at least 2700 nm, at least 2800 nm, at least 2900 nm, at least 3000 nm, at least 3100 nm, at least 3200 nm, at least 3300 nm, at least 3400 nm, at least 3500 nm, at least 3600 nm, at least 3700 nm, at least 3800nm, at least 3900nm, at least 4000nm, at least 4100nm, at least 4200nm, at least 4300nm, at least 4400nm, at least 4500nm, at least 4600nm, at least 4700nm, at least 4800nm, at least 4900nm, at least 5000nm, at least 5100nm, at least 5200nm, at least 5300nm, at least 5400nm, at least 5500nm, at least 5600nm, at least 5700nm, 00nm, at least 5800nm, at least 5900nm, at least 6000nm, at least 6100nm, at least 6200nm, at least 6300nm, at least 6400nm, at least 6500nm, at least 6600nm, at least 6700nm, at least 6800nm, at least 6900nm, at least 7000nm, at least 7100nm, at least 7200nm, at least 7300nm, at least 7400nm, at least 7500nm, at least 7600nm,At least 7700nm, at least 7800nm, at least 7900nm, at least 8000nm, at least 8100nm, at least 8200nm, at least 8300nm, at least 8400nm, at least 8500nm, at least 8600nm, at least 8700nm, at least 8800nm, at least 8900nm, at least 9000nm, at least 9100nm, at least 9200nm, at least 9300nm, at least 9400nm, at least 9500nm, at least 9600nm, at least 970 0nm, at least 9800nm, at least 9900nm, at least 10000nm, or 10nm to 50nm, 50nm to 100nm, 100nm to 150nm, 150nm to 200nm, 200nm to 250nm, 250nm to 300nm, 300nm to 350nm, 350nm to 400nm, 400nm to 450nm, 450nm to 500nm, 500nm to 550nm, 550nm to 600nm, 600nm to 650nm, 650nm to 700nm, 700nm to 750nm, 750nm to 800nm, 800nm ​​to 850nm, 850nm to 900nm nm to 900nm, 100nm to 300nm, 150nm to 350nm, 200nm to 400nm, 250nm to 450nm, 300nm to 500nm, 350nm to 550nm, 400nm to 600nm, 450nm to 650nm, 500nm to 700nm, 550nm to 750nm, 600nm to 800nm, 650nm to 850nm, 700nm to 900nm, or 10nm to 900nm, 10 to 100nm, 100 to 200nm, 200 to 300nm, 300 to 400nm, 400 to 500nm, 500 to 600nm, 600 to 700nm , 700~800nm, 800~900nm, 900~1000nm, 1000~1100nm, 1100~1200nm, 1200~1 300nm, 1300~1400nm, 1400~1500nm, 1500~1600nm, 1600~1700nm, 1700~180 0nm, 1800~1900nm, 1900~2000nm, 2000~2100nm, 2100~2200nm, 2200~2300n m, 2300~2400nm, 2400~2500nm, 2500~2600nm, 2600~2700nm, 2700~2800nm,2800~2900nm, 2900~3000nm, 3000~3100nm, 3100~3200nm, 3200~3300nm, 3300~3400nm, 3400~3500nm, 3500~3600nm, 3600~3700nm, 3 460 0~4700nm, 4700~4800nm, 4800~4900nm, 4900~5000nm, 5000~5100nm, 5100~5200nm, 5200~5300nm, 5300~5400nm, 5400~5500nm, 5500~ 5600nm, 5600~5700nm, 5700~5800nm, 5800~5900nm, 5900~6000nm, 6000~6100nm, 6100~6200nm, 6200~6300nm, 6300~6400nm, 6400~65 00nm, 6500~6600nm, 6600~6700nm, 6700~6800nm, 6800~6900nm, 6900~7000nm, 7000~7100nm, 7100~7200nm, 7200~7300nm, 7300~740 0nm, 7400~7500nm, 7500~7600nm, 7600~7700nm, 7700~7800nm, 7800~7900nm, 7900~8000nm, 8000~8100nm, 8100~8200nm, 8200~8300n The diameters can be measured by dynamic light scattering (DLS) as an indirect measure of size. The DLS measurement may be an "intensity-weighted" average, meaning that the size distribution from which this average is calculated may be weighted by the sixth power of the radius.Sometimes referred to herein as "z-average" or "intensity average."

[0132] Alternatively, the particles disclosed herein may be at least 10 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1000 nm, at least 1100 nm, at least 1200 nm, at least 1300 nm, at least 1400 nm, at least 1500 nm, at least 1600 nm, at least 1700 nm, at least 1800 nm, at least 1900 nm, At least 2000nm, at least 2100nm, at least 2200nm, at least 2300nm, at least 2400nm, at least 2500nm, at least 2600nm, at least 2700nm, at least 2800nm, at least 2900nm, at least 3000nm, at least 3100nm, at least 3200nm, at least 3300nm, at least 3400nm, at least 3500nm, at least 3600nm, at least 3700nm, at least 3800nm, at least 3900nm, at least 400 0nm, at least 4100nm, at least 4200nm, at least 4300nm, at least 4400nm, at least 4500nm, at least 4600nm, at least 4700nm, at least 4800nm, at least 4900nm, at least 5000nm, at least 5100nm, at least 5200nm, at least 5300nm, at least 5400nm, at least 5500nm, at least 5600nm, at least 5700nm, at least 5800nm, at least 5900nm, at least 6000nm, at least Also 6100nm, at least 6200nm, at least 6300nm, at least 6400nm, at least 6500nm, at least 6600nm, at least 6700nm, at least 6800nm, at least 6900nm, at least 7000nm, at least 7100nm, at least 7200nm, at least 7300nm, at least 7400nm, at least 7500nm, at least 7600nm, at least 7700nm, at least 7800nm, at least 7900nm, at least 8000nm, at least 8100nm,at least 8200nm, at least 8300nm, at least 8400nm, at least 8500nm, at least 8600nm, at least 8700nm, at least 8800nm, at least 8900nm, at least 9000nm, at least 9100nm, at least 9200nm, at least 9300nm, at least 9400nm, at least 9500nm, at least 9600nm, at least 9700nm, at least 9800nm, at least 9900nm, at least 10000nm, or 10000nm to 1500nm, 1500nm to 1500nm 00nm, 100nm~150nm, 150nm~200nm, 200nm~250nm, 250nm~300nm, 300nm~350 nm, 350nm~400nm, 400nm~450nm, 450nm~500nm, 500nm~550nm, 550nm~600nm , 600nm~650nm, 650nm~700nm, 700nm~750nm, 750nm~800nm, 800nm~850nm, 8 50nm~900nm, 100nm~300nm, 150nm~350nm, 200nm~400nm, 250nm~450nm, 300 nm to 500nm, 350nm to 550nm, 400nm to 600nm, 450nm to 650nm, 500nm to 700nm, 550nm to 750nm, 600nm to 800nm, 650nm to 850nm, 700nm to 900nm, or 10nm to 900nm, 10 to 100nm, 100 to 200nm, 200 to 300nm, 300 to 400nm, 400 to 500nm, 500 to 600nm, 600 to 700nm, 700 to 800nm, 800 to 900nm, 900 to 1000nm, 1000 to 1100nm, 1100 to 1200nm, 1200 ~1300nm, 1300~1400nm, 1400~1500nm, 1500~1600nm, 1600~1700nm, 1700~1 800nm, 1800~1900nm, 1900~2000nm, 2000~2100nm, 2100~2200nm, 2200~230 0nm, 2300~2400nm, 2400~2500nm, 2500~2600nm, 2600~2700nm, 2700~2800n m, 2800~2900nm, 2900~3000nm, 3000~3100nm, 3100~3200nm, 3200~3300nm,3300~3400nm, 3400~3500nm, 3500~3600nm, 3600~3700nm, 3700~3800nm, 3800~3900nm, 3900~4000nm, 4000~4100nm, 4100~4 200nm, 4200~4300nm, 4300~4400nm, 4400~4500nm, 4500~4600nm, 4600~4700nm, 4700~4800nm, 4800~4900nm, 4900~5000nm, 5000~5100nm, 5100~5200nm, 5200~5300nm, 5300~5400nm, 5400~5500nm, 5500~5600nm, 5600~5700nm, 5700~5800nm, 5800~5 900nm, 5900~6000nm, 6000~6100nm, 6100~6200nm, 6200~6300nm, 6300~6400nm, 6400~6500nm, 6500~6600nm, 6600~6700nm, 6 700~6800nm, 6800~6900nm, 6900~7000nm, 7000~7100nm, 7100~7200nm, 7200~7300nm, 7300~7400nm, 7400~7500nm, 7500~76 00nm, 7600~7700nm, 7700~7800nm, 7800~7900nm, 7900~8000nm, 8000~8100nm, 8100~8200nm, 8200~8300nm, 8300~8400nm, 8 It can have a radius of 400 to 8500 nm, 8500 to 8600 nm, 8600 to 8700 nm, 8700 to 8800 nm, 8800 to 8900 nm, 8900 to 9000 nm, 9000 to 9100 nm, 9100 to 9200 nm, 9200 to 9300 nm, 9300 to 9400 nm, 9400 to 9500 nm, 9500 to 9600 nm, 9600 to 9700 nm, 9700 to 9800 nm, 9800 to 9900 nm, 9900 to 10000 nm.

[0133] In certain examples, the particles disclosed herein have a diameter of 100 nm to 400 nm. In other examples, the particles disclosed herein have a radius of 100 nm to 400 nm. Particle size can be determined by several techniques, such as dynamic light scattering or electron microscopy (e.g., SEM, TEM). The particles disclosed herein can be nanoparticles or microparticles.

[0134] In addition, particles can have a uniform size distribution or a non-uniform size distribution. Polydispersity index (PDI), which can be measured by techniques such as dynamic light scattering, is a measure of size distribution. A low PDI indicates a more uniform size distribution, and a higher PDI indicates a more non-uniform size distribution. For example, the particles disclosed herein can have a PDI of less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, or less than 0.1. In certain embodiments, the particles disclosed herein have a PDI of less than 0.1.

[0135] The particles disclosed herein can have a range of different surface charges. They can be negatively charged, positively charged, or neutral in charge. In some embodiments, the particles can have a range of different surface charges, such as -500mV to -450mV, -450mV to -400mV, -400mV to -350mV, -350mV to -300mV, -300mV to -250mV, -250mV to -200mV, -200mV to -150mV, -150mV to -100mV, -100mV to -90mV, -90mV to -80mV, or -80mV. ~-70mV, -70mV~-60mV, -60mV~-50mV, -50mV~-40mV, -40mV~-30mV, -30mV~-20mV, -20mV~-10mV , -10mV~0mV, 0mV~10mV, 10mV~20mV, 20mV~30mV, 30mV~40mV, 40mV~50mV, 50mV~60mV, 60mV~70mV , 70mV~80mV, 80mV~90mV, 90mV~100mV, 100mV~110mV, 110mV~120mV, 120mV~130mV, 130mV~140m V, 140mV~150mV, 150mV~200mV, 200mV~250mV, 250mV~300mV, 300mV~350mV, 350mV~400mV, 400m The particles have a surface charge of -450 mV, 450 mV to 500 mV, -500 mV to -400 mV, -400 mV to -300 mV, -300 mV to -200 mV, -200 mV to -100 mV, -100 mV to 0 mV, 0 mV to 100 mV, 100 mV to 200 mV, 200 mV to 300 mV, 300 mV to 400 mV, or 400 mV to 500 mV. In certain examples, the particles disclosed herein have a surface charge of -60 mV to 60 mV.

[0136] Various particle morphologies are consistent with the particle types within the panels of the present disclosure. For example, particles can be spherical, colloidal, cubic, square, rod, wire, conical, pyramidal, and ellipsoidal. The particles of the present disclosure can be solid particles, porous particles, or mesoporous particles. The particles can have small or large surface areas. The particles can have various magnetic properties, which can be measured by a SQUID, which determines magnetic force in response to an external field. In some cases, the particles have a core-shell structure or a yolk-shell structure. Particles Panel

[0137] The particle panels disclosed herein can be used to identify several proteins, peptides, or protein groups using the proteograph workflow described herein (MS analysis of distinct biomolecular coronas corresponding to distinct particle types within the particle panel). Feature intensity, as disclosed herein, refers to the intensity of distinct spikes ("features") found in a plot of mass-to-charge ratio versus intensity from a mass spectrometry run of a sample. These features may correspond to variably ionized fragments of peptides and / or proteins. Using the data analysis methods described herein, feature intensities can be sorted into protein groups. A protein group refers to two or more proteins identified by a shared peptide sequence. Alternatively, a protein group can refer to a single protein identified using a unique identifying sequence. For example, if a sample is assayed for a peptide sequence shared between two proteins (protein 1: XYZZX and protein 2: XYZYZ), the protein group can be an "XYZ protein group" with two members (protein 1 and protein 2). Alternatively, if the peptide sequence is unique to a single protein (Protein 1), the protein group could be a "ZZX" protein group with one member (Protein 1). Each protein group can be supported by more than one peptide sequence. A protein detected or identified according to the present disclosure can refer to a distinct protein detected in a sample (e.g., distinct from other proteins detected using mass spectrometry). Thus, analysis of proteins present in distinct coronas corresponding to distinct particle types within a particle panel will result in a large number of feature intensities. This number decreases as the feature intensities are processed into distinct peptides, which in turn decrease as distinct peptides are processed into distinct proteins, which in turn decrease as peptides are grouped into protein groups (two or more proteins that share a distinct peptide sequence).

[0138] The particle panels disclosed herein can be used to identify at least at least 100 proteins, at least 200 proteins, at least 300 proteins, at least 400 proteins, at least 500 proteins, at least 600 proteins, at least 700 proteins, at least 800 proteins, at least 900 proteins, at least 1000 proteins, at least 1100 proteins, at least 1200 proteins, at least 1300 proteins, at least 1400 proteins, at least 1500 proteins, at least 1600 proteins, at least 1700 proteins, at least 1800 proteins, at least 1900 proteins, at least 2000 proteins, at least 2100 proteins, at least 2200 proteins, at least 2300 proteins, at least 2400 proteins, at least 2500 proteins, at least 2600 proteins, at least 2700 proteins, at least 2800 proteins, at least 2900 proteins, at least 3000 proteins, Quality, at least 3100 proteins, at least 3200 proteins, at least 3300 proteins, at least 3400 proteins, at least 3500 proteins, at least 3600 proteins, at least 3700 proteins, at least 3800 proteins, at least 3900 proteins, at least 4000 proteins, at least 4100 proteins, at least 4200 proteins, at least 4300 proteins, at least 4400 proteins, at least 4500 proteins, at least 4 600 proteins, at least 4700 proteins, at least 4800 proteins, at least 4900 proteins, at least 5000 proteins, at least 10000 proteins, at least 20000 proteins, at least 50000 proteins, at least 100000 proteins, 100-5000 proteins, 200-4700 proteins, 300-4400 proteins, 400-4100 proteins, 500-3800 proteins, 600-3500 proteins, 700-3200 proteins,800-2900 proteins, 900-2600 proteins, 1000-2300 proteins, 1000-3000 proteins, 3000-4000 proteins, 4000-5000 proteins, 5000-6000 proteins, 6000-7000 proteins, 7000-8000 proteins, 8000-9000 proteins, 9000-10000 proteins, 10000-11000 proteins, 11000-12000 proteins, 12000-13000 proteins, 13000-14000 proteins, 14000-150 00 proteins, 15,000 to 16,000 proteins, 16,000 to 17,000 proteins, 17,000 to 18,000 proteins, 18,000 to 19,000 proteins, 19,000 to 20,000 proteins, 20,000 to 25,000 proteins, 25,000 to 30,000 proteins, 10,000 to 20,000 proteins, 10,000 to 50,000 proteins, 20,000 to 100,000 proteins, 2000 to 20,000 proteins, 1800 to 20,000 proteins, or 10,000 to 100,000 proteins can be identified.

[0139] Using the particle panels disclosed herein, it is possible to identify at least at least 100 protein groups, at least 200 protein groups, at least 300 protein groups, at least 400 protein groups, at least 500 protein groups, at least 600 protein groups, at least 700 protein groups, at least 800 protein groups, at least 900 protein groups, at least 1000 protein groups, at least 1100 protein groups, at least 1200 protein groups, at least 1300 protein groups, at least 1400 protein groups, at least 1600 protein groups, at least 1800 protein groups, at least 1900 protein groups, at least 2000 protein groups, at least 2100 protein groups, at least 2200 protein groups, at least 2300 protein groups, at least 2400 protein groups, at least 2500 protein groups, at least 2600 protein groups, at least 2700 protein groups, at least 2800 protein groups, at least 3000 protein groups, at least 3100 protein groups, at least 3200 protein groups, at least 3300 protein groups, at least 3400 protein groups, at least 3500 protein groups, at least 3600 protein groups, at least 3700 protein groups, at least 3800 protein groups, at least 3900 protein groups, at least 4000 protein groups, at least 4100 protein groups, at least 4200 protein groups, at least 4300 protein groups, at least 4400 protein groups, at least 4500 protein groups, at least 4600 protein groups, at least 4700 protein groups, at least 4800 protein groups, at least 4900 protein groups, at least 5000 protein groups, at least 5100 protein groups, at least 5200 protein groups, at least 5300 protein groups, at least 540 00 proteins, at least 1500 proteins, at least 1600 proteins, at least 1700 proteins, at least 1800 proteins, at least 1900 proteins, at least 2000 proteins, at least 2100 proteins, at least 2200 proteins, at least 2300 proteins, at least 2400 proteins, at least 2500 proteins, at least 2600 proteins, at least 2700 proteins, at least 2800 proteins , at least 2900 proteins, at least 3000 proteins, at least 3100 proteins, at least 3200 proteins, at least 3300 proteins, at least 3400 proteins, at least 3500 proteins, at least 3600 proteins, at least 3700 proteins, at least 3800 proteins, at least 3900 proteins, at least 4000 proteins, at least 4100 proteins, at least 4200 proteins, at least 430 0 protein groups, at least 4400 protein groups, at least 4500 protein groups, at least 4600 protein groups, at least 4700 protein groups, at least 4800 protein groups, at least 4900 protein groups, at least 5000 protein groups, at least 10000 protein groups, at least 20000 protein groups, at least 100000 protein groups, 100-5000 protein groups, 200-4700 protein groups, 300-4400 protein groups, 400-4100 protein groups,500-3800 protein groups, 600-3500 protein groups, 700-3200 protein groups, 800-2900 protein groups, 900-2600 protein groups, 1000-2300 protein groups, 1000-3000 protein groups, 3000-4000 protein groups, 4000-5000 protein groups, 5000-6000 protein groups, 6000-7000 protein groups, 7000-8000 protein groups, 8000-9000 protein groups, 9000-10000 protein groups, 10000-11000 protein groups, 11000-12000 protein groups, 12000-13000 protein groups, 1300 A protein group of 0 to 14,000, a protein group of 14,000 to 15,000, a protein group of 15,000 to 16,000, a protein group of 16,000 to 17,000, a protein group of 17,000 to 18,000, a protein group of 18,000 to 19,000, a protein group of 19,000 to 20,000, a protein group of 20,000 to 25,000, a protein group of 25,000 to 30,000, a protein group of 10,000 to 20,000, a protein group of 10,000 to 50,000, a protein group of 20,000 to 100,000, a protein group of 2000 to 20,000, a protein group of 1800 to 20,000, or a protein group of 10,000 to 100,000.

[0140] The particle panels disclosed herein can be used to identify a number of distinct proteins disclosed herein and / or any number of specific proteins disclosed herein across a wide dynamic range. For example, a particle panel disclosed herein comprising distinct particle types can enrich proteins in a sample and identify these proteins across the entire dynamic range over which the proteins are present in a sample (e.g., a plasma sample) using a proteograph workflow. In some embodiments, a particle panel comprising any number of distinct particle types disclosed herein enriches and identifies proteins across a dynamic range of at least 2. In some embodiments, a particle panel comprising any number of distinct particle types disclosed herein enriches and identifies proteins across a dynamic range of at least 3. In some embodiments, a particle panel comprising any number of distinct particle types disclosed herein enriches and identifies proteins across a dynamic range of at least 4. In some embodiments, a particle panel comprising any number of distinct particle types disclosed herein enriches and identifies proteins across a dynamic range of at least 5. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 6. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 7. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 8. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 9. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 10.In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 11. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 12. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 13. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 14. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 15. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of at least 20. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of 2 to 100. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of 2 to 20. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of 2 to 10. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of 2 to 5. In some embodiments, particle panels comprising any number of distinct particle types disclosed herein enrich and identify proteins over a dynamic range of 5 to 10.

[0141] A particle panel comprising any number of distinct particle types disclosed herein enriches and identifies a single protein or group of proteins. In some embodiments, the single protein or group of proteins may include proteins with different post-translational modifications. For example, a first particle type within the particle panel may enrich a protein or group of proteins with a first post-translational modification, a second particle type within the particle panel may enrich the same protein or group of proteins with a second post-translational modification, and a third particle type within the particle panel may enrich the same protein or group of proteins lacking the post-translational modification. In some embodiments, a particle panel comprising any number of distinct particle types disclosed herein enriches and identifies a single protein or group of proteins by binding to different domains, sequences, or epitopes of the single protein or group of proteins. For example, a first particle type within the particle panel may enrich a protein or group of proteins by binding to a first domain of the protein or group of proteins, and a second particle type within the particle panel may enrich the same protein or group of proteins by binding to a second domain of the protein or group of proteins.

[0142] In some embodiments, a particle panel can have more than one particle type. Increasing the number of particle types in a panel can be a way to increase the number of proteins that can be identified in a given sample. An example of how increasing the panel size can increase the number of identified proteins is shown in Figure 11. For example, as shown in Figure 11, a panel size of 1 particle type identified 419 unique proteins, a panel size of 2 particle types identified 588 proteins, a panel size of 3 particle types identified 727 proteins, a panel size of 4 particle types identified 844 proteins, a panel size of 5 particle types identified 934 proteins, a panel size of 6 particle types identified 1008 proteins, a panel size of 7 particle types identified 1075 proteins, a panel size of 8 particle types identified 1133 proteins, a panel size of 9 particle types identified 1184 proteins, a panel size of 10 particle types identified 1230 proteins, a panel size of 11 particle types identified 1275 proteins, and a panel size of 12 particle types identified 1318 proteins. Particle types may include nanoparticle types.

[0143] In some embodiments, a panel size of 1 particle can identify 200-600 unique proteins. In some embodiments, a panel size of 2 particles can identify 300-700 unique proteins. In some embodiments, a panel size of 3 particles can identify 500-900 unique proteins. In some embodiments, a panel size of 4 particles can identify 600-1000 unique proteins. In some embodiments, a panel size of 5 particles can identify 700-1100 unique proteins. In some embodiments, a panel size of 6 particles can identify 800-1200 unique proteins. In some embodiments, a panel size of 7 particles can identify 850-1250 unique proteins. In some embodiments, a panel size of 8 particles can identify 900-1300 unique proteins. In some embodiments, a panel size of 9 particles can identify 950-1350 unique proteins. In some embodiments, a panel size of 10 particles can identify 1000-1400 unique proteins. In some embodiments, a panel size of 11 particles can identify 1050-1450 unique proteins. In some embodiments, a panel size of 12 particles can identify 1100-1500 unique proteins. Particle types may include nanoparticles.

[0144] The present disclosure provides over 200 distinct particle types with over 100 different surface chemical structures and over 50 different physical properties. The distinct particle types have at least one physicochemical property that differs between a first particle type and a second particle type. For example, the present disclosure provides at least two distinct particle types, at least three distinct particle types, at least four distinct particle types, at least five distinct particle types, at least six distinct particle types, at least seven distinct particle types, at least eight distinct particle types, at least nine distinct particle types, at least ten distinct particle types, at least eleven distinct particle types, at least 12 distinct particle types, at least 13 distinct particle types, at least 14 distinct particle types, at least 15 distinct particle types, at least 20 distinct particle types, at least 25 distinct particle types, at least 30 distinct particle types, at least 35 distinct particle types, at least 40 distinct particle types, at least 45 distinct particle types, at least 50 distinct particle types. The particle panel may include at least 100 distinct particle types, at least 150 distinct particle types, at least 200 distinct particle types, at least 250 distinct particle types, at least 300 distinct particle types, at least 350 distinct particle types, at least 400 distinct particle types, at least 450 distinct particle types, at least 500 distinct particle types, 2-500 distinct particle types, 2-5 distinct particle types, 5-10 distinct particle types, 10-15 distinct particle types, 15-20 distinct particle types, 20-40 distinct particle types, 40-60 distinct particle types, 60-80 distinct particle types, 80-100 distinct particle types, 100-500 distinct particle types, 4-15 distinct particle types, or 2-20 distinct particle types. The particle types may include nanoparticle types.

[0145] In some embodiments, the present disclosure provides a method for producing at least one distinct type of particle, at least two distinct type of particle, at least three distinct type of particle, at least four distinct type of particle, at least five distinct type of particle, at least six distinct type of particle, at least seven distinct type of particle, at least eight distinct type of particle, at least nine distinct type of particle, at least ten distinct type of particle, at least eleven distinct type of particle, at least twelve distinct type of particle, at least thirteen distinct type of particle, at least fourteen distinct type of particle, at least At least 15 distinct particle types, at least 16 distinct particle types, at least 17 distinct particle types, at least 18 distinct particle types, at least 19 distinct particle types, at least 20 distinct particle types, at least 25 distinct particle types, at least 30 distinct particle types, at least 35 distinct particle types, at least 40 distinct particle types, at least 45 distinct particle types, at least 50 distinct particle types, at least 55 distinct particle types, at least 60 distinct particle types, at least 65 distinct particle types, at least 70 distinct particle types, at least 75 distinct particle types, at least 80 distinct particle types, at least 85 distinct particle types, at least 90 distinct particle types, at least 95 distinct particle types, at least 100 distinct particle types, at least 105 ... distinct particle types, at least 75 distinct particle types, at least 80 distinct particle types, at least 85 distinct particle types, at least 90 distinct particle types, at least 95 distinct particle types, at least 100 distinct particle types, 1-5 distinct particle types, 5-10 distinct particle types, 10-15 distinct particle types, 15-20 distinct particle types, 20-25 distinct particle types, 25-30 distinct particle types, 30-35 distinct particle types, 35-40 distinct particle types, 40-45 distinct particle types, 45-50 distinct particle types, 50- Panel sizes of 55 distinct particle types, 55-60 distinct particle types, 60-65 distinct particle types, 65-70 distinct particle types, 70-75 distinct particle types, 75-80 distinct particle types, 80-85 distinct particle types, 85-90 distinct particle types, 90-95 distinct particle types, 95-100 distinct particle types, 1-100 distinct particle types, 20-40 distinct particle types, 5-10 distinct particle types, 3-7 distinct particle types, 2-10 distinct particle types, 6-15 distinct particle types, or 10-20 distinct particle types are provided. In certain embodiments, the present disclosure provides panel sizes of 3-10 distinct particle types. In certain embodiments, the present disclosure provides panel sizes of 4-11 distinct particle types.In certain embodiments, the present disclosure provides a panel size of 5 to 15 distinct particle types. In certain embodiments, the present disclosure provides a panel size of 5 to 15 distinct particle types. In certain embodiments, the present disclosure provides a panel size of 8 to 12 distinct particle types. In certain embodiments, the present disclosure provides a panel size of 9 to 13 distinct particle types. In certain embodiments, the present disclosure provides a panel size of 10 distinct particle types. The particle types may include nanoparticle types.

[0146] For example, the present disclosure provides for at least two distinct particle types, at least three different surface chemistries, at least four different surface chemistries, at least five different surface chemistries, at least six different surface chemistries, at least seven different surface chemistries, at least eight different surface chemistries, at least nine different surface chemistries, at least ten different surface chemistries, at least eleven different surface chemistries, at least thirteen different surface chemistries, at least fourteen different surface chemistries, at least fifteen different surface chemistries, at least twenty different surface chemistries, at least twenty-five different surface chemistries, at least thirty-five different surface chemistries, at least thirty-five different surface chemistries, at least forty-five different surface chemistries, at least forty-five different surface chemistries, at least fifteen different surface chemistries, at least twenty-five different surface chemistries, at least thirty-five different surface chemistries, at least forty-five different surface chemistries, at least fifteen different surface chemistries, at least twenty-five ... and a particle panel having at least 100 different surface chemical structures, at least 150 different surface chemical structures, at least 200 different surface chemical structures, at least 250 different surface chemical structures, at least 300 different surface chemical structures, at least 350 different surface chemical structures, at least 400 different surface chemical structures, at least 450 different surface chemical structures, at least 500 different surface chemical structures, 2 to 500 different surface chemical structures, 2 to 5 different surface chemical structures, 5 to 10 different surface chemical structures, 10 to 15 different surface chemical structures, 15 to 20 different surface chemical structures, 20 to 40 different surface chemical structures, 40 to 60 different surface chemical structures, 60 to 80 different surface chemical structures, 80 to 100 different surface chemical structures, 100 to 500 different surface chemical structures, 4 to 15 different surface chemical structures, or 2 to 20 different surface chemical structures.

[0147] The present disclosure provides for at least two different surface chemistries, at least three different surface chemistries, at least four different surface chemistries, at least five different surface chemistries, at least six different surface chemistries, at least seven different surface chemistries, at least eight different surface chemistries, at least nine different surface chemistries, at least ten different surface chemistries, at least eleven different surface chemistries, at least thirteen different surface chemistries, at least fourteen different surface chemistries, at least fifteen different surface chemistries, at least twenty different surface chemistries, at least twenty-five different surface chemistries, at least thirty-five different surface chemistries, at least forty-five different surface chemistries, at least forty-five different surface chemistries, at least fifteen different surface chemistries, at least twenty-five different surface chemistries, at least thirty ... and a particle panel having at least 100 different surface chemistries, at least 150 different surface chemistries, at least 200 different surface chemistries, at least 250 different surface chemistries, at least 300 different surface chemistries, at least 350 different surface chemistries, at least 400 different surface chemistries, at least 450 different surface chemistries, at least 500 different surface chemistries, 2 to 500 different surface chemistries, 2 to 5 different surface chemistries, 5 to 10 different surface chemistries, 10 to 15 different surface chemistries, 15 to 20 different surface chemistries, 20 to 40 different surface chemistries, 40 to 60 different surface chemistries, 60 to 80 different surface chemistries, 80 to 100 different surface chemistries, 100 to 500 different surface chemistries, 4 to 15 different surface chemistries, or 2 to 20 different surface chemistries.

[0148] The present disclosure provides for at least two different physical properties, at least three different physical properties, at least four different physical properties, at least five different physical properties, at least six different physical properties, at least seven different physical properties, at least eight different physical properties, at least nine different physical properties, at least ten different physical properties, at least eleven different physical properties, at least thirteen different physical properties, at least fourteen different physical properties, at least fifteen different physical properties, at least twenty different physical properties, at least twenty-five different physical properties, at least thirty-five different physical properties, at least forty different physical properties, at least forty-five different physical properties, at least fifteen different physical properties, at least fifteen different physical properties, at least twenty different physical properties, at least twenty-five different physical properties, at least thirty-five different physical properties, at least forty-five different physical properties, at least fifteen different physical properties, at least twenty-five ... The particle panel may have at least 100 different physical properties, at least 150 different physical properties, at least 200 different physical properties, at least 250 different physical properties, at least 300 different physical properties, at least 350 different physical properties, at least 400 different physical properties, at least 450 different physical properties, at least 500 different physical properties, 2 to 500 different physical properties, 2 to 5 different physical properties, 5 to 10 different physical properties, 10 to 15 different physical properties, 15 to 20 different physical properties, 20 to 40 different physical properties, 40 to 60 different physical properties, 60 to 80 different physical properties, 80 to 100 different physical properties, 100 to 500 different physical properties, 4 to 15 different physical properties, or 2 to 20 different physical properties.

[0149] In some embodiments, a panel that optimally identifies proteins and optimally associates biomarkers with disease includes a panel selected from the particle types listed in Table 1. For example, a panel that optimally identifies proteins and optimally associates biomarkers with disease includes a panel that includes SP-339, HX74, SP-356, SP-333, HX20, SP-374, HX42, SP-003, SP-007, and SP-011. A particle panel that is particularly suitable for identifying a large number of proteins in a sample (e.g., more than 1,500 proteins) includes 5 to 10 distinct particle types in the assay. The number of distinct particle types included in a particle panel can be tailored for specific applications (e.g., detection of a specific subset of proteins or a group of markers associated with a particular disease). In some embodiments, a panel with physicochemically distinct particle types that optimally identifies proteins and optimally associates biomarkers with disease includes silica-coated SPIONs, acrylamide-based SPIONs, and acrylate-based SPIONs. For example, a panel of particles disclosed herein that generate information-rich proteomic data through their protein corona that can be correlated with biomarkers and disease with high sensitivity and specificity includes silica-coated SPIONs (SP-003), poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated SPIONs (SP-007), and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-coated SPIONs (SP-011).

[0150] In some embodiments, the total assay time from a single pool of plasma, including sample preparation and LC-MS, can be about 8 hours. In some embodiments, the total assay time from a single pool of plasma, including sample preparation and LC-MS, can be about at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, at least 5 to 10 minutes, at least 10 to 20 minutes, at least 20 to 30 minutes, at least 3 hours, The incubation time may be 0 to 40 minutes, at least 40 to 50 minutes, at least 50 to 60 minutes, at least 1 to 1.5 hours, at least 1.5 to 2 hours, at least 2 to 2.5 hours, at least 2.5 to 3 hours, at least 3 to 3.5 hours, at least 3.5 to 4 hours, at least 4 to 4.5 hours, at least 4.5 to 5 hours, at least 5 to 5.5 hours, at least 5.5 to 6 hours, at least 6 to 6.5 hours, at least 6.5 to 7 hours, at least 7 to 7.5 hours, at least 7.5 to 8 hours, at least 8 to 8.5 hours, at least 8.5 to 9 hours, at least 9 to 9.5 hours, or at least 9.5 to 10 hours. Early detection

[0151] The panels described herein and their methods of use can be used to detect markers in samples from subjects that are consistent with specific disease states. As shown in Figures 18A and 18B, early-stage cancers can be isolated up to eight years before symptom onset. The Golestan cohort enrolled 50,000 healthy subjects between 2004 and 2008. Stored plasma from the enrollment was tested, as shown in Figure 18A. Eight years after enrollment, approximately 1,000 patients developed cancer. Figure 18B shows the classification of three cancer types (brain, lung, and pancreatic cancer) from stored plasma using principal component analysis. Here, the corona results are plotted against three principal component axes, representing three distinct statistical populations. Corona analysis of stored plasma from the enrollment date (including measurement of multiple features to profile multiple proteins) correctly classified cancer in 15 of the 15 subjects tested (five patients for each of the three cancer types). In some embodiments, a panel of the present disclosure can be used to diagnose a condition up to 1 year, up to 2 years, up to 3 years, up to 4 years, up to 5 years, up to 6 years, up to 7 years, up to 8 years, up to 9 years, up to 10 years, up to 15 years, up to 20 years, or up to 25 years prior to the onset of symptoms of the condition.

[0152] The disclosed panels can be used to detect a wide range of disease states in a given sample. For example, the disclosed panels can be used to detect cancer. The cancer can be brain cancer, lung cancer, pancreatic cancer, glioblastoma, meningioma, myeloma, or pancreatic cancer. Corona analysis signals for these cancers are shown in Figure 17B and Figure 18.

[0153] Additionally, the panels of the present disclosure can be used to detect other cancers, including, for example, any one of the cancers listed at https: / / www.cancer.gov / types: acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML), adolescent cancer; adrenocortical carcinoma; pediatric adrenocortical carcinoma; rare childhood cancers; AIDS-related cancers; Kaposi's sarcoma (soft tissue sarcoma); AIDS-related lymphoma (lymphoma); primary CNS lymphoma (lymphoma); anal cancer; appendix cancer - see gastrointestinal carcinoid tumor; astrocytoma, childhood (brain cancer). ;Atypical teratomas / rhabdoid tumors, childhood;Central nervous system (brain cancer);Basal cell carcinoma of the skin - see Skin cancer;Bile duct cancer;Bladder cancer;Pediatric bladder cancer;Bone cancer (including Ewing's sarcoma and osteosarcoma and malignant fibrous histiocytoma);Brain tumors;Breast cancer;Pediatric breast cancer;Bronchial tumors, childhood;Burkitt's lymphoma - see Non-Hodgkin's lymphoma;Carcinoid tumors (gastrointestinal tract);Pediatric carcinoid tumors;Cancer of unknown cause;Pediatric cancer of unknown cause;Carcinoid (heart) tumors, childhood;Central nervous system;Atypical teratomas / rhabdoid tumors, childhood ( Brain Cancer); Embryonal Tumors, Childhood (Brain Cancer); Germ Cell Tumors, Childhood (Brain Cancer); Primary CNS Lymphoma; Cervical Cancer; Childhood Cervical Cancer; Childhood Cancer; Unusual Cancers of Childhood; Bile Duct Cancer - See Bile Duct Cancer; Chordoma, Childhood; Chronic Lymphocytic Leukemia (CLL); Chronic Myeloid Leukemia (CML); Myeloproliferative Neoplasms; Colorectal Cancer; Childhood Colorectal Cancer; Craniopharyngioma, Childhood (Brain Cancer); Cutaneous T-Cell Lymphoma - See Lymphoma (Mycosis Fungoides and Sézary Syndrome); Ductal Carcinoma in Situ (DCIS) - See Breast Cancer; Embryonal Tumors, Central Nervous System, Childhood (Brain Cancer ); Endometrial cancer (uterine cancer); Ependymoma, childhood (brain cancer); Esophageal cancer; Pediatric esophageal cancer; Nasal neuroblastoma (head and neck cancer); Ewing's sarcoma (bone cancer); Extracranial germ cell tumor, childhood; Extragonadal germ cell tumor; Eye cancer; Pediatric intraocular melanoma; Intraocular melanoma; Retinoblastoma; Fallopian tube cancer; Osteofibrous histiocytoma, malignant, and osteosarcoma; Gallbladder cancer; Gastric (stomach) cancer; Pediatric gastric (stomach) cancer; Gastrointestinal carcinoid tumor; Gastrointestinal stromal tumor (GIST) (soft tissue sarcoma); Pediatric gastrointestinal stromal tumor; Germ cell tumor;Pediatric central nervous system germ cell tumors (brain cancer); pediatric extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; testicular cancer; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; heart tumors, childhood; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin's lymphoma; hypopharyngeal cancer (head and neck cancer); intraocular melanoma; pediatric intraocular melanoma; pancreatic islet cell tumors, pancreatic neuroendocrine tumors; Kaposi's sarcoma (soft tissue sarcoma); kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer (head and neck cancer); leukemia; lip and oral cavity cancer (head and neck cancer); liver cancer; lung cancer (non-small cell and small cell); pediatric lung cancer; lymphoma; male breast cancer; malignant fibrous histiocytoma of bone and Osteosarcoma;Melanoma;Pediatric melanoma;Melanoma, intraocular (eye);Pediatric intraocular melanoma;Merkel cell carcinoma (skin cancer);Mesothelioma, malignant;Pediatric mesothelioma;Metastatic cancer;Metastatic squamous cell carcinoma of the neck with unknown primary (head and neck cancer);Midline carcinoma with NUT gene alterations;Oral cancer (head and neck cancer);Multiple endocrine neoplasia syndrome;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides (lymphoma);Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm;Myeloid leukemia, chronic (CML);Myeloid leukemia, acute (AML);Myeloproliferative neoplasm, chronic;Nasal cavity and sinus cancer (head and neck cancer);Nasopharyngeal carcinoma (head and neck cancer);Neuroblastoma;Non-Hodgkin's lymphoma;Non-small cell lung cancer;Oral cancer, lip and oral cavity cancer cancer) and oropharyngeal cancer (head and neck cancer); osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pediatric ovarian cancer; pancreatic cancer; pediatric pancreatic cancer; pancreatic neuroendocrine tumors (pancreatic islet cell tumors); papillomatosis (pediatric pharynx); paraganglioma; pediatric paraganglioma; paranasal sinus and nasal cavity cancer (head and neck cancer); parathyroid cancer; penile cancer; pharyngeal cancer (head and neck cancer); pheochromocytoma; pediatric pheochromocytoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; pregnancy breast cancer Cancer;Primary central nervous system (CNS) lymphoma;Primary peritoneal cancer;Prostate cancer;Rectal cancer;Recurrent cancer;Renal cell (kidney) cancer;Retinoblastoma;Rhabdomyosarcoma, childhood (soft tissue sarcoma);Salivary gland cancer (head and neck cancer);Sarcoma;Childhood rhabdomyosarcoma (soft tissue sarcoma);Childhood vascular tumor (soft tissue sarcoma);Ewing's sarcoma (bone cancer);Kaposi's sarcoma (soft tissue sarcoma);Osteosarcoma (bone cancer);Soft tissue sarcoma;Uterine sarcoma;Sezary syndrome (lymphoma);Skin cancer;Pediatric skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; cutaneous squamous cell carcinoma - see skin cancer; squamous cell carcinoma of the neck of unknown primary, metastatic (head and neck cancer); stomach (gastric) cancer; childhood stomach (gastric) cancer; T-cell lymphoma, cutaneous - see lymphoma (mycosis fungoides and Sézary syndrome); testicular cancer; childhood testicular cancer; throat cancer (head and neck cancer); nasopharyngeal cancer; oropharyngeal cancer; hypopharyngeal cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter (kidney (renal cell) cancer); cancer of unknown cause; childhood cancer of unknown cause; rare childhood cancer; ureter and renal pelvis, transitional cell carcinoma (kidney (renal cell) cancer); urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; childhood vaginal cancer; vascular tumors (soft tissue sarcomas); vulvar cancer; Wilms' tumor and other childhood kidney tumors; or cancer in young adults. Additionally, the particle panels of the present disclosure can be used to detect other diseases, such as Alzheimer's disease and multiple sclerosis. sample

[0154] Using the panels of the present disclosure, proteomic data can be generated from the protein corona, which can then be associated with any of the biological states described herein. Samples consistent with the present disclosure include biological samples from subjects. The subjects can be human or non-human animals. The biological sample can be a bodily fluid. For example, the bodily fluid can be plasma, serum, CSF, urine, tears, cell lysate, tissue lysate, cell homogenate, tissue homogenate, nipple aspirate, fecal sample, synovial fluid, whole blood, or saliva. The sample can also be a non-biological sample, such as water, milk, solvent, or anything that can be homogenized to a fluid state. The biological sample can contain multiple proteins or proteomic data, which can be analyzed after adsorption of proteins to the surfaces of various particle types within the panel and subsequent digestion of the protein corona. Proteomic data can include nucleic acids, peptides, or proteins. Any of the samples herein can contain several different analytes, which can be analyzed using the compositions and methods disclosed herein. The analyte can be a protein, peptide, small molecule, nucleic acid, metabolite, lipid, or any molecule that can potentially bind to or interact with a particulate surface.

[0155] Compositions and methods for multi-omics analysis are disclosed herein. "Multi-omics" or "multi-omics" can refer to large-scale biomolecular analysis analytical techniques in which data sets are multiple omes, such as proteomes, genomes, transcriptomes, lipidomes, and metabolomes. Non-limiting examples of multi-omics data include proteome data, genomics data, lipidomics data, glycomics data, transcriptomics data, and metabolomics data. The "biomolecule" in "biomolecular corona" can refer to any molecule or biological component that can be produced by or present within a biological organism. Non-limiting examples of biomolecules include proteins (protein corona), polypeptides, polysaccharides, sugars, lipids, lipoproteins, metabolites, oligonucleotides, nucleic acids (DNA, RNA, microRNA, plasmids, single-stranded nucleic acids, double-stranded nucleic acids), metabolomes, and small molecules, such as primary metabolites, secondary metabolites, and other natural products, or any combination thereof. In some embodiments, the biomolecule is selected from the group of a protein, a nucleic acid, a lipid, and a metabolome.

[0156] In some embodiments, the sample of the present disclosure can be a plurality of samples.At least two samples of the plurality of samples can be spatially separated.Spatially separated refers to the samples contained in different volumes.For example, spatially separated samples can refer to the samples that are in different wells in a plate or in different tubes.Spatially separated samples can also refer to the samples that are in different wells in a plate or in different tubes, and are assayed together by the same instrument. In some embodiments, the present disclosure provides for the analysis of multiple samples, e.g., at least two spatially separated samples, at least five spatially separated samples, at least 10 spatially separated samples, at least 15 spatially separated samples, at least 20 spatially separated samples, at least 25 spatially separated samples, at least 30 spatially separated samples, at least 35 spatially separated samples, at least 40 spatially separated samples, at least 45 spatially separated samples, at least 50 spatially separated samples, at least 55 spatially separated samples, at least 60 spatially separated samples, at least 65 spatially separated samples, at least 70 spatially separated samples, at least 75 spatially separated samples, at least at least 80 spatially separated samples, at least 85 spatially separated samples, at least 90 spatially separated samples, at least 95 spatially separated samples, at least 96 spatially separated samples, at least 100 spatially separated samples, at least 120 spatially separated samples, at least 140 spatially separated samples, at least 160 spatially separated samples, at least 180 spatially separated samples, at least 200 spatially separated samples, at least 220 spatially separated samples, at least 240 spatially separated samples, at least 260 spatially separated samples, at least 280 spatially separated samples, at least 300 spatially separated samples, at least 320 spatially separated samples,at least 340 spatially separated samples, at least 360 spatially separated samples, at least 380 spatially separated samples, at least 400 spatially separated samples, at least 420 spatially separated samples, at least 440 spatially separated samples, at least 460 spatially separated samples, at least 480 spatially separated samples, at least 500 spatially separated samples, at least 600 spatially separated samples, at least 700 spatially separated samples, at least 800 spatially separated samples, at least 900 spatially separated samples, at least 1000 spatially separated samples, at least 1100 spatially separated samples, at least 1200 spatially separated samples, at least 1300 spatially separated samples, at least 1400 spatially separated samples, at least 1500 spatially separated samples, at least 1600 spatially separated samples, at least 1700 spatially separated samples , at least 1800 spatially separated samples, at least 1900 spatially separated samples, at least 2000 spatially separated samples, at least 5000 spatially separated samples, at least 10000 spatially separated samples, 2-10 spatially separated samples, 2-100 spatially separated samples, 2-200 spatially separated samples, 2-300 spatially separated samples, 50-150 spatially separated samples, 10-20 spatially separated samples, 20- 30 spatially separated samples, 30-40 spatially separated samples, 40-50 spatially separated samples, 50-60 spatially separated samples, 60-70 spatially separated samples, 70-80 spatially separated samples, 80-90 spatially separated samples, 90-100 spatially separated samples, 100-150 spatially separated samples, 150-200 spatially separated samples, 200-250 spatially separated samples, 250-300 spatially separated samples,300-350 spatially separated samples, 350-400 spatially separated samples, 400-450 spatially separated samples, 450-500 spatially separated samples, 500-600 spatially separated samples, 600-700 spatially separated samples, 700-800 spatially separated samples, 800-900 spatially separated samples, 900-1000 spatially separated samples, 1000-2000 spatially separated samples, 2 The present invention provides particle panels and methods for using the same that are suitable for analyzing 1,000 to 3,000 spatially separated samples, 3,000 to 4,000 spatially separated samples, 4,000 to 5,000 spatially separated samples, 5,000 to 6,000 spatially separated samples, 6,000 to 7,000 spatially separated samples, 7,000 to 8,000 spatially separated samples, 8,000 to 9,000 spatially separated samples, or 9,000 to 10,000 spatially separated samples.

[0157] The methods disclosed herein include isolating a particle panel (having multiple particle types) from one or more samples. Particle panels having superparamagnetic particle types can be rapidly isolated or separated from a sample using a magnet. Furthermore, multiple spatially separated samples can be processed in parallel. Thus, the methods disclosed herein provide for the simultaneous isolation or separation of a particle panel from unbound proteins within multiple spatially separated panels by using a magnet. For example, a particle panel can be incubated with multiple spatially separated samples, each of which is located within a well of a well plate (e.g., a 96-well plate). After incubation, the particle panel within each well of the well plate can be separated from unbound proteins present in the spatially separated samples by placing the entire plate on a magnet. This simultaneously pulls down the superparamagnetic particles within the particle panel. The supernatant within each well can be removed to remove unbound proteins. These steps (incubating, pulling down using a magnet) can be repeated to effectively wash the particles and, thus, remove any residual background unbound proteins that may be present in the sample. This is one example, but one skilled in the art can envision numerous other scenarios in which superparamagnetic particles are rapidly isolated simultaneously from one or more spatially separated samples.

[0158] In some embodiments, the disclosed panels provide for the identification and measurement of specific proteins in a biological sample by processing proteomic data from digestion of the corona formed on the surface of particles. Examples of proteins that can be identified and measured include high-abundance proteins, moderate-abundance proteins, and low-abundance proteins. Low-abundance proteins may be present in a sample at concentrations of about 10 ng / mL or less. High-abundance proteins may be present in a sample at concentrations of about 10 μg / mL or more. Moderate-abundance proteins may be present in a sample at concentrations between about 10 ng / mL and about 10 μg / mL. Examples of high-abundance proteins include albumin, IgG, and the top 14 proteins in terms of abundance, which contribute 95% of the mass in plasma. Additionally, any protein that can be purified using a conventional depletion column can be directly detected in a sample using the particle panels disclosed herein. Examples of proteins are described in Keshishian et al. (Mol Cell Proteomics. 2015 Sep;14(9):2375-93. doi: 10.1074 / mcp.M114.046813. Epub 2015 Feb 27.), Farr et al. (J Proteome Res. 2014 Jan 3;13(1):60-75. doi: 10.1021 / pr4010037. Epub 2013 Dec 6.), or Pernemalm et al. (Expert Rev Proteomics. 2014 Aug;11(4):431-48. doi: 10.1586 / 14789450.2014.901157. Epub 2014 Mar 24.).

[0159] In some embodiments, examples of proteins that can be measured and identified using the particle panels disclosed herein include albumin, IgG, lysozyme, CEA, HER-2 / neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPigR, 14-3-3 eta, troponin I, B-type natriuretic peptide, BRCA1, c-Myc, IL-6, fibrinogen, EGFR, gastrin, PH, G-CSF, desmin, NSE, FSH, VEGF, P21, PCNA, calcitonin, PR, CA125, LH, somatostatin, S100, insulin, alpha-prolactin, ACTH, Bcl-2, ER alpha, Ki-67, p53, cathepsin D, beta-catenin, VWF, CD15, k-ras, caspase 3, EPN, CD10, FAS, BRCA2, CD30L, CD30, CGA, CRP, prothrombin, CD44, APEX, transferrin, GM-CSF, E-cadherin, IL-2, Bax, IFN-gamma, beta-2-MG, TNF-alpha, c-erbB-2, trypsin, cyclin D1, MG B, XBP-1, HG-1, YKL-40, S-gamma, NESP-55, netrin-1, geminin, GADD45A, CDK-6, CCL21, BrMS1, 17betaHDI, PDGFRA, Pcaf, CCL5, MMP3, claudin-4, and claudin-3. In some embodiments, other examples of proteins that can be measured and identified using the particle panels disclosed herein are any proteins or groups of proteins listed in the open targets database for a particular disease indication of interest (e.g., prostate cancer, lung cancer, or Alzheimer's disease). Analysis method

[0160] Several different analytical techniques can be used to identify, measure, and quantify the proteome data of a sample. For example, SDS-PAGE or any gel-based separation technique can be used to analyze the proteome data. Immunoassays such as ELISA can also be used to identify, measure, and quantify peptides and proteins. Alternatively, mass spectrometry, high-performance liquid chromatography, LC-MS / MS, Edman degradation, immunoaffinity techniques, methods disclosed in EP3548652, WO2019083856, WO2019133892 (each of which is incorporated herein by reference in its entirety), and other protein separation techniques can be used to identify, measure, and quantify the proteome data. Computer Control System

[0161] The present disclosure provides a computer-controlled system programmed to execute the disclosed methods. This determination, analysis, and statistical classification can be performed using a wide range of supervised and unsupervised data analysis and clustering techniques, including, but not limited to, hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), machine learning (also known as random forests), logistic regression, decision trees, support vector machines (SVM), k-nearest neighbors, naive Bayes, linear regression, polynomial regression, SVM for regression, k-means clustering, and hidden Markov models, among others. The computer system can perform various aspects of analyzing the protein sets or protein coronas of the present disclosure, such as comparing / analyzing the biomolecular coronas of several samples and determining, using statistical significance, what patterns are common between individual biomolecular coronas to determine protein sets associated with biological states. The computer system can also be used to develop classifiers for detecting and distinguishing different proteins or protein coronas (e.g., those specific to the composition of the protein coronas). Data collected from the sensor arrays disclosed herein can be used to train machine learning algorithms, particularly algorithms that receive array measurements from patients and output specific biomolecular corona compositions from each patient. Prior to training the algorithm, the raw data from the array can first be denoised to reduce the variability of individual variables.

[0162] Machine learning can be generalized as the ability of a learning machine to accurately perform new, unseen examples / tasks after experiencing a training dataset. Machine learning can include the following concepts and methods: supervised learning concepts such as AODE; artificial neural networks, e.g., backpropagation, autoencoders, Boltzmann machines, restricted Boltzmann machines, and spiking neural networks; Bayesian statistics, e.g., Bayesian networks and Bayesian knowledge bases; case-based reasoning; Gaussian process regression; gene expression programming; group data processing methods (GMDH); inductive logic programming; case-based learning; lazy learning; learning automata; learning vector quantization; logistic model trees; minimum message length (decision trees, decision graphs, etc.), e.g., nearest neighbor algorithms and analogical modeling; probabilistic and approximately correct learning (PAC) learning; ripple-down rules, knowledge acquisition methodologies. ; symbolic machine learning algorithms; support vector machines; random forests; ensembles of classifiers, e.g., bootstrap aggregating (bagging) and boosting (meta-algorithms); ordinal classification; information fuzzy networks (IFNs); conditional random fields; ANOVA; linear classifiers, e.g., Fisher linear discriminant, linear regression, logistic regression, multinomial logistic regression, naive Bayes classifier, perceptron, support vector machine; quadratic classifiers; k-nearest neighbors; boosting; decision trees, e.g., C4.5, random forest, ID3, CART, SLIQ, SPRINT; Bayesian networks, e.g., naive Bayes; and hidden Markov models. Unsupervised learning concepts may include expectation maximization algorithms; vector quantization; generative phase maps; information bottleneck methods; artificial neural networks, e.g., self-organizing maps; association rule learning, e.g., the Apriori algorithm, the Eclat algorithm, and the FPgrowth algorithm; hierarchical clustering, e.g., shortest distance clustering and concept clustering; cluster analysis, e.g., the K-means algorithm, fuzzy clustering, DBSCAN, and the OPTICS algorithm; and outlier detection, e.g., the local outlier factor method.Semi-supervised learning concepts may include generative models, sparse separation, graph-based methods, and co-training. Reinforcement learning concepts may include temporal difference learning, Q-learning, learning automata, and SARSA. Deep learning concepts may include deep belief networks, deep Boltzmann machines, deep convolutional neural networks, deep recurrent neural networks, and hierarchical temporal memory. A computer system may be configured to perform the methods described herein. The system includes a central computer server programmed to perform the methods described herein. The server includes a central processing unit (CPU, or "processor"), which may be a single-core processor, a multi-core processor, or multiple processors for parallel processing. The server also includes memory (e.g., random access memory, read-only memory, flash memory); electronic storage (e.g., hard disk); communication interfaces (e.g., network adapters) for communicating with one or more other systems; and peripheral devices, which may include cache, other memory, data storage, and / or electronic display adapters. The memory, storage, interfaces, and peripherals are connected to the processor via a communication bus (solid lines), such as a motherboard. The storage device may be a data storage device for storing data. The server is operably coupled to a computer network ("network") utilizing a communications interface. The network may be the Internet, an intranet and / or an extranet, an intranet and / or an extranet connected to the Internet, a telecommunications network, or a data network. The network in some cases may utilize the server to implement a peer-to-peer network, whereby devices coupled to the server may operate as either clients or servers.

[0163] The storage device can store files, such as subject reports, and / or communications regarding data about an individual, or any aspect of data relevant to the present disclosure.

[0164] The computer server can communicate over a network with one or more remote computer systems, which can be, for example, personal computers, laptops, tablets, phones, smartphones, or personal digital assistants.

[0165] In some applications, the computer system includes a single server. In other situations, the system includes multiple servers connected to each other via an intranet, an extranet, and / or the Internet.

[0166] The server can be configured to store measurement data or databases as provided herein, patient information from the subject, such as medical history, family history, demographic data, etc., and / or other clinical or personal information of potential relevance to a particular application. Such information can be stored on a storage device or server, and such data can be transmitted over a network.

[0167] The methods described herein can be performed by machine (or computer processor) executable code (or software) stored in an electronic storage location on a server, such as, for example, on a memory or electronic storage device. During use, the code can be executed by the processor. In some cases, the code can be read from storage and stored in memory for easy access by the processor. In some situations, electronic storage can be omitted, and the machine-executable instructions are stored in memory. Alternatively, the code can be executed on a second computer system.

[0168] Aspects of the systems and methods provided herein, such as the server, can be implemented with programming. Various aspects of the technology can be generally considered "products" or "articles of manufacture" in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such memory (e.g., read-only memory, random-access memory, flash memory), or a hard disk. "Storage" type media can include any or all of the tangible memory of a computer, processor, or the like, or its associated modules, such as various semiconductor memories, tape drives, disk drives, and the like, that can provide non-transitory storage at any time for software programming. All or portions of the software can sometimes be linked via the Internet or various other telecommunications networks. Such communication can enable, for example, loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, over wired and optical telephone networks, and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, or the like, may also be considered media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" may refer to any medium that participates in providing instructions to a processor for execution.

[0169] The computer systems described herein may include computer executable code for performing any of the algorithms or methods based on the algorithms described herein. In some applications, the algorithms described herein will use storage devices comprised of at least one database.

[0170] Data related to the present disclosure can be transmitted over a network or connection for receipt and / or review by a recipient. A recipient can be, but is not limited to, the subject to whom the report pertains; or their caregiver, such as a healthcare provider, administrator, other healthcare professional, or other caretaker; or the person or company that performs and / or orders the analysis. A recipient can also be a local or remote system (e.g., a server or other system in a "cloud computing" architecture) for filtering such reports. In one embodiment, the computer-readable medium comprises a medium suitable for transmitting the results of an analysis of a biological sample using the methods described herein.

[0171] Aspects of the systems and methods provided herein can be implemented in programming. Various aspects of this technology can be generally considered as an "article of manufacture" or "article of manufacture" in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, or the like, or its associated modules, such as various semiconductor memories, tape drives, disk drives, and the like, that can provide non-transitory storage at any time for software programming. All or portions of the software can sometimes be linked via the Internet or various other telecommunications networks. Such communication can enable, for example, loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, over wired and optical telephone networks, and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, or the like, may also be considered media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0172] Thus, machine-readable media such as computer-executable code may take many forms, including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any of the storage devices in any computer, such as those shown in the figures that may be used to implement databases, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, such as the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links which transport such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution. Classification of protein coronas using machine learning

[0173] A method for determining a set of proteins associated with a disease or disorder and / or condition involves analyzing the coronas of at least two samples. This determination, analysis, or statistical classification is performed by methods known in the art, including, but not limited to, a wide range of supervised and unsupervised data analysis, machine learning, deep learning, and clustering techniques, such as hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), random forests, logistic regression, decision trees, support vector machines (SVM), k-nearest neighbors, naive Bayes, linear regression, polynomial regression, SVM for regression, k-means clustering, and hidden Markov models, among others. In other words, the proteins in the coronas of each sample are compared / analyzed with each other using statistical significance to determine what patterns are common between the individual coronas and determine a set of proteins associated with the disease or disorder or condition.

[0174] Generally, machine learning algorithms are used to build models that accurately assign class labels to examples based on input features that describe the examples. In some cases, it may be advantageous to utilize machine learning and / or deep learning techniques in the methods described herein. For example, machine learning can be used to associate protein coronas with various disease states (e.g., disease-free, precursors of disease, early or late stages of disease, etc.). For example, in some cases, one or more machine learning algorithms are utilized in conjunction with the methods of the present invention to analyze the detected and obtained data for each set of protein coronas and proteins derived therefrom. For example, in one embodiment, machine learning can be coupled with the sensor arrays described herein to determine whether a subject is in a pre-cancer stage, has cancer, or has and does not have cancer, as well as to distinguish between types of cancer. Numbered Embodiments

[0175] The following embodiments describe non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is considered dependent or related to any preceding or following numbered embodiment, regardless of the order in which they are listed. 1. A method for identifying proteins in a sample, comprising incubating a panel comprising multiple particle types with the sample to form multiple protein coronas, digesting the multiple protein coronas to generate proteomic data, and identifying proteins in the sample by quantifying the proteomic data. 2. The method of embodiment 1, wherein the sample is from a subject. 3. The method of embodiment 2, further comprising determining a protein profile of the sample from the identifying step and associating the protein profile with a biological state of the subject. 4. The method of embodiment 1, further comprising determining a biological state of a sample from the subject by digesting multiple protein coronas to generate proteomic data, determining a protein profile of the multiple protein coronas, and associating the protein profile with the biological state, wherein the panel comprises at least two different particle types. 5. The method of embodiment 4, wherein the associating is performed by a trained classifier. 6. The method of any one of embodiments 1-5, wherein the panel comprises at least 3 different particle types, at least 4 different particle types, at least 5 different particle types, at least 6 different particle types, at least 7 different particle types, at least 8 different particle types, at least 9 different particle types, at least 10 different particle types, at least 11 different particle types, at least 12 different particle types, at least 13 different particle types, at least 14 different particle types, at least 15 different particle types, or at least 20 different particle types. 7. The method of any one of embodiments 1-6, wherein the panel comprises at least 4 different particle types. 8. The method of any one of embodiments 1-7, wherein at least one particle type of the panel has a different physical characteristic than a second particle type of the panel. 9. The method of embodiment 8, wherein the physical characteristic is size, polydispersity index, surface charge, or morphology.10. The method of any one of embodiments 1-9, wherein the size of at least one particle type of the plurality of particle types in the panel is between 10 nm and 500 nm. 11. The method of any one of embodiments 1-10, wherein the polydispersity index of at least one particle type of the plurality of particle types in the panel is between 0.01 and 0.25. 12. The method of any one of embodiments 1-11, wherein the morphology of at least one particle type of the plurality of particle types comprises a spherical, colloidal, square, rod, wire, cone, pyramidal, or ellipsoidal shape. 13. The method of embodiments 1-12, wherein the surface charge of at least one particle type of the plurality of particle types comprises a positive surface charge. 14. The method of any one of embodiments 1-12, wherein the surface charge of at least one particle type of the plurality of particle types comprises a negative surface charge. 15. The method of any one of embodiments 1-12, wherein the surface charge of at least one particle type of the plurality of particle types comprises a neutral surface charge. 16. The method of any one of embodiments 1-15, wherein at least one particle type of the plurality of particle types has a different chemical characteristic than a second particle type of the panel. 17. The method of embodiment 16, wherein the chemical characteristic is a surface functional chemical group. 18. The method of embodiment 17, wherein the functional chemical group is an amine or a carboxylate. 19. The method of any one of embodiments 1-18, wherein at least one particle type of the plurality of particle types is made of a polymer, a lipid, or a material comprising a metal, silica, a protein, a nucleic acid, a small molecule, or a large molecule. 20. The method of embodiment 19, wherein the polymer comprises polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), polystyrene, or a copolymer of two or more polymers.21. Lipids include dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidyl Dioleoylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans 20. The method of embodiment 19, comprising PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, or cholesterol.22. The method of embodiment 19, wherein the metal comprises gold, silver, copper, nickel, cobalt, palladium, platinum, iridium, osmium, rhodium, ruthenium, rhenium, vanadium, chromium, manganese, niobium, molybdenum, tungsten, tantalum, iron, or cadmium, titanium, or gold. 23. The method of any one of embodiments 1-22, wherein at least one particle type of the plurality of particle types is surface-functionalized with a polymer comprising polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or a polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), polystyrene, or a copolymer of two or more polymers, polyethylene glycol. 24. The method of any one of embodiments 3-23, wherein the protein profile is associated with a biological state with at least 70% accuracy, at least 75% accuracy, at least 80% accuracy, at least 85% accuracy, at least 90% accuracy, at least 92% accuracy, at least 95% accuracy, at least 96% accuracy, at least 97% accuracy, at least 98% accuracy, at least 99% accuracy, or 100% accuracy. 25. The method of any one of embodiments 3-24, wherein the protein profile is associated with a biological state with at least 70% sensitivity, at least 75% sensitivity, at least 80% sensitivity, at least 85% sensitivity, at least 90% sensitivity, at least 92% sensitivity, at least 95% sensitivity, at least 96% sensitivity, at least 97% sensitivity, at least 98% sensitivity, at least 99% sensitivity, or 100% sensitivity.26. The method of any one of embodiments 3-25, wherein the protein profile is associated with the biological state with at least 70% specificity, at least 75% specificity, at least 80% specificity, at least 85% specificity, at least 90% specificity, at least 92% specificity, at least 95% specificity, at least 96% specificity, at least 97% specificity, at least 98% specificity, at least 99% specificity, or 100% specificity. 27. The method of any one of embodiments 1-26, wherein at least 100 unique proteins, at least 200 unique proteins, at least 300 unique proteins, at least 400 unique proteins, at least 500 unique proteins, at least 600 unique proteins, at least 700 unique proteins, at least 800 unique proteins, at least 900 unique proteins, at least 1000 unique proteins, at least 1100 unique proteins, at least 1200 unique proteins, at least 1300 unique proteins, at least 1400 unique proteins, at least 1500 unique proteins, at least 1600 unique proteins, at least 1700 unique proteins, at least 1800 unique proteins, at least 1900 unique proteins, or at least 2000 unique proteins are identified. 28. The method of any one of embodiments 1-27, wherein at least one particle type of the plurality of particle types comprises superparamagnetic iron oxide nanoparticles. 29. The method of any one of embodiments 1-28, wherein the sample is a body fluid. 30. The method of embodiment 29, wherein the body fluid comprises plasma, serum, CSF, urine, tears, or saliva. 31. A method for selecting a panel for protein corona analysis, comprising selecting a plurality of particle types having at least three different physicochemical properties. 32. The method of embodiment 31, wherein the different physicochemical properties are selected from the group consisting of surface charge, surface chemical structure, size, and morphology. 33. The method of embodiment 32, wherein the different physicochemical properties comprise surface charge. 34. A composition comprising a panel of particles, the panel comprising a plurality of particle types, the plurality of particle types having at least three different physicochemical properties.35. The composition of embodiment 35, wherein the different physicochemical properties are selected from the group consisting of surface charge, surface chemical structure, size, and morphology. 36. The composition of embodiment 36, wherein the different physicochemical properties comprise surface charge. 37. A system comprising a panel of any one of embodiments 1 to 34. 38. A system comprising a panel, wherein the panel comprises a plurality of particle types. 39. The system of embodiment 38, wherein the plurality of particle types have at least three different physicochemical properties. 40. Embodiment 3, wherein the panel comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different particle types. 8. The system of embodiment 38, wherein the plurality of particle types are capable of adsorbing a plurality of proteins from the sample to form a plurality of protein coronas. 42. The system of embodiment 41, wherein the plurality of protein coronas are digested to determine a protein profile. 43. The system of embodiment 42, wherein the protein profile is associated with a biological state using a trained classifier. [Example]

[0176] The following examples are included to further illustrate some aspects of the disclosure, but should not be used to limit the scope of the invention. Example 1 Synthesis of SPNMPs or superparamagnetic iron oxide nanoparticles (SPIONs)

[0177] SPMNPs or SPIONs are described in the literature (Angew. Chem. Int. Ed. 2009, 48, 5875 - 5879 & Langmuir 2012, 28, 3271 - 3278) It was synthesized by the solvothermal reaction at 200 °C by the reduction of FeCl with ethylene glycol (EG) in the presence of sodium acetate (NaOAc) as an alkali source and trisodium citrate (NaCit) as an electrostatic stabilizer. Excess EG acted as both the solvent and the reducing agent.

[0178] Starting materials: Iron(III) chloride hexahydrate (FeCl3·6H2O), MW 270.30, CAS No. 10025-77-1; Sodium acetate (NaOAc), MW 82.03, CAS No. 127-09-3; Trisodium citrate dihydrate (Na3Cit·2H2O), MW 294.10, CA# 6132-04-3; Ethylene glycol (EG), MW 62.07, CAS No. 107-21-1.

[0179] Procedure for Fe3O4 nanoparticles via solvothermal reaction:

[0180] (1) Typically, FeCl3 (0.65 g, 4.0 mmol) and sodium citrate (0.20 g, 0.68 mmol) were first dissolved in EG (20 mL), and then sodium acetate (1.20 g, 14.6 mmol) was added with stirring. The mixture was vigorously stirred at 160 °C for 30 minutes in a Teflon-lined stainless steel autoclave (50 mL capacity) and then sealed. (2) The autoclave was heated to 200 °C, maintained for 12 hours, and then allowed to cool to room temperature. (3) The black product was isolated with a magnet and washed >5 times with DI water. (4) The final Fe3O4 nanoparticle product was dried in vacuo or freeze-dried at 60 °C for 12 hours to form a black powder. The Fe3O4@SiO2 core / shell was prepared by a modified Stöber method by following the method in the literature (J. Am. Chem. Soc. 2008, 130, 28-29 & J. Mater. Chem. B, 2013, 1, 4684-4691).

[0181] Starting materials: Fe3O4 nanoparticles synthesized by solvothermal reaction; tetraethyl orthosilicate (TEOS), MW 208.33, CAS number 78-10-4; ammonia solution 25%; cetrimonium bromide (CTAB), MW 364.45, CAS number 57-09-0; (3-aminopropyl)triethoxysilane (APTES), MW 221.37, CAS number 919-30-2.

[0182] Procedure for Fe3O4@SiO2 core / shell nanoparticles:

[0183] (1) Superparamagnetic Fe3O4 NPs were synthesized according to a previously reported solvothermal reaction (Angew. Chem. Int. Ed. 2009, 48, 5875–5879 & Langmuir 2012, 28, 3271–3278). The resulting Fe3O4 nanoparticles were then used to prepare highly aminated superparamagnetic mesoporous composite nanoparticles via a two-step coating procedure. (2) Briefly, 0.08 g of Fe3O4 nanoparticles were uniformly dispersed in a mixture of ethanol (50 mL), DI water (1 mL), and concentrated aqueous ammonia solution (1.7 mL, 25 wt%), followed by the addition of TEOS (140 μL). After stirring at 40 °C for 6 h, amorphous silica-coated superparamagnetic nanoparticles (denoted as Fe3O4@SiO2) were obtained and washed five times with water. (3) Then, by using CTAB as a template, the Fe3O4@SiO2 nanoparticles were coated with a highly aminated mesoporous silica shell via a base-catalyzed sol-gel silica reaction. Typically, the Fe3O4@SiO2 nanoparticles (5 mg) prepared above were dispersed in a mixed solution containing CTAB (0.08 g), ethyl acetate (0.7 mL), DI water (113 mL), and concentrated ammonia (2.42 mL, 25 wt%). TEOS (0.18 mL) and APTES (0.22 mL) were added to the dispersion using a stirring speed of 300 rpm. After 3 h of reaction at room temperature, the product was collected with a magnet and repeatedly washed with water and ethanol, respectively.

[0184] (4) To remove the pore-generating template (CTAB), the as-synthesized material was transferred to ethanol (60 mL) with continuous stirring at 60 °C for 3 h. The surfactant extraction step was repeated twice to ensure the removal of CTAB. The template-removed product was washed twice with ethanol to obtain sandwich-structured, highly aminated superparamagnetic mesoporous composite nanoparticles (FeO@SiO@mSiO-NH).

[0185] Fe3O4@polymer composite nanoparticles were synthesized by seeded emulsion polymerization (Langmuir 2012, 28, 3271 - 3278) in the absence of surfactants.

[0186] Fe3O4 nanoparticles synthesized by solvothermal reaction.

[0187] Starting materials: Fe3O4 nanoparticles synthesized by solvothermal reaction; tetraethyl orthosilicate (TEOS), MW 208.33, CAS No. 78-10-4; 3-(trimethoxysilyl)propyl methacrylate (MPS), MW 248.35, CAS No. 2530-85-0; ammonia solution 25%; divinylbenzene (DVB), MW 130.19, CAS No. 1321-74-0; styrene (St), MW 104.15, CAS No. 100-42-5; methacrylic acid (MAA), MW 86.09, CAS No. 79-41-4; ammonium persulfate (APS), MW 228.20, CAS No. 7727-54-0.

[0188] Procedure for Fe3O4@polymer core / shell nanoparticles:

[0189] (1) SPIONs were synthesized according to a previously reported solvothermal reaction (Angew. Chem. Int. Ed. 2009, 48, 5875–5879 & Langmuir 2012, 28, 3271–3278). (2) Fe3O4@MPS was prepared by a modified Stöber method. Typically, 1 g of Fe3O4 nanoparticles was homogeneously dispersed in a mixture of ethanol (50 mL), DI water (2 mL), and concentrated aqueous ammonia (2 mL, 25 wt%), followed by the addition of TEOS (200 μL) and MPS (2 mL). After stirring at 70 °C for 24 h, MPS-coated superparamagnetic NPs were obtained, washed five times with water, freeze-dried to a dark brown powder, and stored at −20 °C. (3) Fe3O4@Polymer nanoparticles were synthesized by seeded emulsion polymerization in the absence of surfactants. Typically, 100 mg of Fe3O4@MPS was uniformly dispersed in 125 mL of DI water. After bubbling with N2 for 30 min, 2 mL of St, 0.2 mL of DVB, and 0.4 mL of MAA were added to the Fe3O4@MPS suspension. After adding 0.2 g of NaOH and 5 mL of an aqueous solution containing 40 mg of APS, the resulting mixture was heated to 75 °C overnight. (4) After cooling, Fe3O4@P(St-co-MAA) was obtained, washed five times with water, and freeze-dried to a dark brown powder. Example 2 Building particle-type panels

[0190] This example describes the construction of a panel of particle types. A particle library containing approximately 170 total particle types was constructed. Biomolecular coronas for each particle type were generated by incubating each particle with a biological sample. Proteomic data from the protein coronas was analyzed for each particle type, including qualitative analysis based on electrophoresis and quantitative analysis based on mass spectrometry data and heat maps. The panel was constructed by selecting specific particle types based on particle and corona characteristics. Particle types for the particle panel were selected based on the breadth of coverage they had for proteins in plasma samples. Figure 5 illustrates the process for generating proteomic data and the process for panel selection. As shown in Figure 6, particle characteristics include size / geometry, charge, surface functional groups, and magnetic force, among other properties. Each of these properties can be assayed by various tests during the complete characterization of a particle type before adding it to a panel. As shown in Figure 7, dynamic light scattering was used to characterize the size distribution of two particle types: SP-002 (phenol-formaldehyde coated particles) and SP-010 (carboxylate, PAA coated particles). As shown in Figure 8, TEM was used to characterize the size and morphology of two particle types, including SP-002 (phenol-formaldehyde coated particles) (Figure 8A) and SP-339 (polystyrene carboxyl particles) (Figure 8B). As shown in Figure 9, XPS was used to analyze the chemical groups present on the surface of various particle types, including SP-333 (carboxylate), SP-339 (polystyrene carboxylate), SP-356 (silica amino), SP-374 (silica silanol), HX-20 (silica coated), HX-42 (silica coated, amine), and HX-74 (PDMPAPMA coated (dimethylamine)). Example 3 Synthesis and characterization of iron oxide NPs with distinct surface chemistries

[0191] This example describes the synthesis and characterization of iron oxide NPs with distinct surface chemistries. To address the need for robust particles that can withstand, but are easily prepared without, repeated centrifugation or membrane filtration to separate the particle protein corona from free plasma proteins and to wash loosely bound proteins off the particles, superparamagnetic iron oxide NPs (SPIONs) were developed for protein corona formation (Figure 23, top). The iron oxide particle core facilitated rapid separation of the particles from the plasma solution in <30 seconds using a magnet (Figure 4). This significantly reduced the time required for extraction of the particle protein corona for analysis by LC-MS / MS. Furthermore, SPIONs were robustly modified with various surface chemistries that facilitated the generation of distinct patterns of protein corona for broader interrogation of the proteome.

[0192] Three differently surface-functionalized SPIONs (SP-003, SP-007, and SP-011) were synthesized (Figure 28). A thin layer of silica was coated on SP-003 using tetraethyl orthosilicate (TEOS) by a modified Stöber method. To synthesize the poly(dimethylaminopropyl methacrylamide) (PDMAPMA)-coated SPION (SP-007) and poly(ethylene glycol) (PEG)-coated SPION (SP-011), the iron oxide particle cores were first modified with vinyl groups by a modified Stöber method using TEOS and 3-(trimethoxysilyl)propyl methacrylate. The vinyl-functionalized SPIONs were then surface-modified with N-[3-(dimethylamino)propyl]methacrylamide and poly(ethylene glycol) methyl ether methacrylate to prepare SP-007 and SP-011, respectively.

[0193] We characterized the three SPIONs using various techniques, including scanning electron microscopy (SEM), dynamic light scattering (DLS), transmission electron microscopy (TEM), high-resolution TEM (HRTEM), and X-ray photoelectron spectroscopy (XPS), to evaluate the size, morphology, and surface properties of the SPIONs (Figure 23). DLS measurements indicated that SP-003, SP-007, and SP-011 had average sizes of approximately 233 nm, 283 nm, and 238 nm, respectively. This was consistent with SEM measurements, which showed that all three SPIONs had spherical and hemispherical morphologies with sizes ranging from 200 nm to 300 nm. The surface charge of the SPIONs was assessed by zeta potential (ζ) analysis, which showed ζ potential values ​​of -36.9 mV, +25.8 mV, and -0.4 mV for SP-003, SP-007, and SP-011, respectively, at pH 7.4 (Tables 2–4). [Table 2] [Table 3] [Table 4]

[0194] This indicated that SP-003, SP-007, and SP-011 had negative, positive, and neutral surfaces, consistent with the charge of the coating functional groups used to modify the surface of each particle, as shown in the schematic diagram in Figure 23. HRTEM was used to evaluate the coating thickness. For SP-003, a complete amorphous shell was observed around the iron oxide core with a thickness of more than 10 nm (Figure 23, top fifth column). For SP-007 and SP-011, relatively thin (<10 nm) amorphous features were observed on the particle surface (arrows in Figure 23, middle and bottom fifth columns). In addition, XPS was performed for surface analysis, which, together with the HRTEM images, confirmed the successful coating of the particles with the respective functional groups. Example 4 Detecting proteins in panels and associating protein profiles with cancer

[0195] This example describes the detection of proteins in a panel and the association of protein profiles with cancer. The particle panel includes three different cross-reactive liposomes with varying surface charges: anionic (DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol))), cationic (DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)-DOPE (dioleoylphosphatidylethanolamine)), and neutral (dioleoylphosphatidylcholine with cholesterol (DOPC)).

[0196] Figure 17A shows a schematic diagram of the present process, which includes sample collection from healthy and cancer patients, isolation of plasma from the samples, incubation with uncoated liposomes to form a protein corona, and enrichment of selected plasma proteins. Protein corona formation can vary based on the physicochemical properties of the particles. Figure 17B shows the corona analysis signals for a three-particle panel. Plasma was collected from 45 subjects (eight subjects each with five cancers, including glioblastoma, lung cancer, meningioma, myeloma, and pancreatic cancer, and five healthy controls). Corona analysis was performed for each particle in the three-particle panel. Random forest models were constructed in each of 1000 rounds of cross-validation. There is strong evidence of a robust corona analysis signal. Initial exploratory analysis was performed using PCA. Figures 18A and 18B show that early-stage cancers can be separated up to eight years before symptom onset. The Golestan cohort enrolled 50,000 healthy subjects between 2004 and 2008. As shown in Figure 18A, stored plasma from the registry was tested. Eight years after enrollment, approximately 1,000 patients had developed cancer. Figure 18B shows the classification of stored plasma. Corona analysis of stored plasma from the date of enrollment correctly classified cancer in 15 of the 15 subjects tested (5 patients for each of the 3 cancer types). Figure 19 shows robust classification of 5 cancer types with an overall accuracy of 95% using 3-particle type corona analysis. The data show that adding particle type diversity improves performance. Three different liposomes with negative, neutral, and positive net surface charges (pH 7.4) were used. Example 5 Rapid and deep proteome analysis with the Corona Analysis workflow

[0197] This example illustrates rapid and deep proteome analysis using a corona analysis workflow. To evaluate the multiparticle protein corona analysis platform (Figure 22B) for plasma proteome analysis, SPIONs were tested using combined pooled plasma samples from eight colorectal cancer (CRC) subjects. Each of these three particle types was first incubated with the plasma sample for approximately 1 hour at approximately 37°C for protein corona formation, followed by magnet-based purification of the particles from unbound proteins (three cycles at 6 minutes per cycle). The particle-bound proteins were then dissolved, digested, purified, and eluted. These steps combined took approximately 2-4 hours, and then analyzed by MS. Notably, this preparation workflow required only approximately 4-6 hours in total for one batch of 96 corona samples.

[0198] After MS analysis and data processing, the resulting MS2 peptide-spectrum matches (PSMs) were used to identify proteins present in each particle-type corona. In parallel, proteins were also detected directly from raw plasma samples, without particle corona formation. Identified proteins from the samples were compared to a compiled database of MS-measured or estimated plasma protein concentrations, and the depth and extent of coverage by the particle corona or plasma was investigated by plotting the measured proteins against the database values ​​of reported protein concentrations (Figure 24). First, 1,255 proteins from the database, covering nearly 11 orders of magnitude, were plotted from most abundant to least abundant. Database matches for each of the experimentally evaluated samples (raw plasma vs. SP-003 / SP-007 / SP-011 particle coronas) were similarly plotted. As can be seen in Figure 24, the dynamic range of the measured plasma proteome, as defined by the range of concentrations for database match proteins, was twice as large for particle coronas (e.g., 40 mg / mL to 0.54 ng / mL for SP-007) as for raw plasma (40 mg / mL to 1.2 ng / mL), and there was a 10-fold increase in the number of low-abundance proteins present at less than 100 ng / mL (842 for particles and 84 for raw plasma). Only 12 proteins in the database were annotated at concentrations lower than the least abundant protein detected on particles. In addition, the total number of unique proteins for each of the particle-type coronas (approximately 1,000) was greater (>2-fold) than that observed for raw plasma (<500), as clearly demonstrated in Table 5. [Table 5]

[0199] Additionally, the proportion of proteins not previously observed by comparison with literature MS compilations was greater for particles (61-64%) compared to raw plasma (45%). In other words, more proteins not annotated with previous MS concentrations in public databases were identified in the particle corona than were observed in raw plasma. Plots of particle protein identifications overlapping with databases confirm that different particle types select for different subsets of plasma proteins. This may be due to the different surface properties of the three SPION particle types, which largely determine the protein composition of the corona.

[0200] To evaluate the ability of particles to compress the measured dynamic range, we compared the measured and identified protein feature intensities with previously reported values ​​for the same protein concentrations. First, we selected the peptide features obtained for each protein (as presented in Figure 24) using the maximum MS-determined intensity of all possible features for the protein (by extracting monoisotopic peak values ​​using the OpenMS MS data processing tool), and then modeled their intensities against previously reported abundance levels for these same proteins (Figure 25). Comparing the slope of the regression model with the intensity span of the measured data, we found that the particle corona contains more low-abundance (measured or reported) proteins than plasma does, similar to Figure 24. The dynamic range of these measurements was compressed (the slope of the regression model was reduced) for particle measurements compared to plasma measurements. This is consistent with previous observations that particles can effectively compress the measured dynamic range for the resulting corona abundance compared to the original dynamic range in plasma, likely due to a combination of the protein's absolute concentration, its binding affinity for the particle, and its interactions with neighboring proteins. All of the above results indicate that the multiparticulate protein corona strategy facilitated the identification of a wide range of plasma proteins, especially those with low abundance that are difficult to rapidly detect using conventional proteomic techniques.

[0201] To assess the robustness of protein identification using the particle corona MS assay, full assay triplicates were performed using a three-particle type panel to generate individual protein corona samples from the same pooled CRC plasma sample. For each combination of particle types, ranging from any one to two whole groups to three single groups, the number of unique proteins listed per combination is shown in Table 6. [Table 6]

[0202] Protein counts in the "only one" column were obtained by using each of the three replicates independently and then taking the mean and standard deviation for all of the combined counts. As can be seen from the table, more proteins were found as the number of particle types in the particle panel increased, with >1,500 unique proteins found in the three particle type group (65 of which are FDA-cleared / approved biomarkers, as listed in Table 7 below). Protein counts in the "any one" replicate column were obtained using the union of the particle type repeat protein lists. Protein counts in the "all three" replicate column were obtained using the intersection of the particle type repeat protein lists. As a further measure of the overlap of particle repeats of the identified proteins, the Jaccard coefficient, a metric of inter-set similarity, was calculated for each pairwise comparison. The values ​​for SP-003, SP-007, and SP-011 were 0.74 ± 0.018, 0.65 ± 0.078, and 0.76 ± 0.019 (mean ± sd), respectively. Enumeration of protein abundance in a given MS sample depends on the stochastic nature of MS2 data collection and may result in an undercount of proteins that are represented within or commonly shared between samples. PSM mapping to shared MS1 features represents one approach that can mitigate this issue and will be developed for future analyses. [Table 7-1] [Table 7-2] Table 7-3

[0203] Dynamic Range. The three-particle panel was evaluated for its ability to assay proteins in samples across a wide dynamic range of protein concentrations. Feature intensities corresponding to proteins identified by mass spectrometry were compared to values ​​determined by other assays for the same protein at the same concentration. After mass spectrometry analysis and data processing, MS2 peptide spectral matching (PSM) was used to identify peptides and associated proteins present in the corona of distinct particle types within the particle panel. In parallel, direct detection of peptides in plasma samples without the three-particle panel for corona analysis using the Proteograph workflow was also performed. Monoisotopic peak values ​​were extracted and determined using the OpenMS MS data processing tool, and the resulting peptide feature with the maximum MS-determined intensity of all observed features was selected for each protein. The MS-determined intensity was then modeled against comparable reported abundance levels for the same protein. Figure 25 shows the correlation between the maximum intensity of proteins in distinct coronas from distinct nanoparticle types for each particle type within the three-particle panel for plasma proteins and the concentration of the same protein determined using other models. As indicated by the slope of the regression model and the intensity span of the measured data, the particle corona had more low-abundance protein hits than did plasma. Additionally, the dynamic range of these measurements was compressed for particle measurements compared to plasma measurements, as indicated by the reduced slope of the regression model, thus indicating that particles effectively compressed the measurement dynamic range of protein abundance in the corona compared to plasma. This may be due to a combination of absolute protein concentration, protein binding affinity to particles, and protein interactions with neighboring proteins. These results indicate that the method disclosed herein, using a multi-particle type panel for enrichment of proteins in distinct coronas corresponding to distinct particle types, facilitated the identification of a wide range of plasma proteins, particularly those with low abundances that are difficult to rapidly detect using conventional proteomic techniques. Example 6 Coronavirus Analysis Assay Accuracy

[0204] This example describes the accuracy of the corona analysis assay. Precision, a measure of assay repeatability and reproducibility, was assessed by comparing multiple measurements under the same conditions and determining the variability between individual measurements. To investigate the reproducibility of the particle-protein corona analysis Proteograph workflow, peptide MS feature intensities were extracted and compared from three complete assay replicates for each of the three particle types. The raw MS files from each replicate were converted to mzML format, a standard compatible MS file format, using the msconvert.exe utility from the openMS suite of programs. Additionally, MS1 features were extracted from the raw data by overlapping dwell times and mz values ​​using the openMS processing pipeline, and aligned into clusters. Clusters containing features from each of the three replicates were selected and filtered based on the quality scores of the clustering algorithm to remove the bottom decile (90% of the feature clusters were retained for subsequent accuracy analysis). A total of 2,744, 2,785, and 3,209 clustered feature clusters were used for accuracy analysis for SP-003, SP-007, and SP-011 nanoparticles, respectively. The distribution of log-transformed raw intensities for each of the replicates for these feature sets is plotted in Figure 26, with values ​​for each particle type presented in the labeled panel. As shown in Figure 26, the feature data for each particle type was highly reproducible, and this reproducibility was consistent across both high- and low-intensity features.

[0205] To assess performance more quantitatively than visual inspection of raw data, we estimated the overall precision of particle coronas after quantile-normalizing the feature intensities of the groups. This normalization method was based on the assumption that all distributions being compared should be identical, so the intensities were adjusted for each distribution being compared. This assumption is reasonable given the reproducibility of the physical characteristics of the particle type itself and of the physical characteristics from other analyses of these particles (e.g., X-ray photoelectron spectroscopy, transmission electron microscopy, and other analytical methods). Using the normalized values, we estimated the standard deviation and determined the coefficient of variation (CV) using appropriate transformations of the logarithmically processed data. For each particle, the median CV (percent of quantile-normalized CV, or QNCV%) is shown in Table 8. This result demonstrates that the protein MS feature intensities measured by the particles have sufficient precision across the thousands of MS feature intensities observed to detect relatively small differences in a reasonably small-scale study. For example, assuming a 25% CV, the power to detect a two-fold change using Bonferroni-corrected significance was approximately 100%.

[0206] The overall precision of the particle corona was estimated by normalizing the group feature intensities using quantile normalization, which makes the a priori assumption that all distributions being compared should be identical and adjusts the intensities for each distribution being compared appropriately. The normalized values ​​were used to estimate the standard deviation, and the coefficient of variation (CV) was determined using an appropriate transformation of the logarithmic data. For each particle type, the median CV (percent of quantile-normalized CV, i.e., QNCV%) is shown in Table 8. A low coefficient of variation (CV) indicates a high degree of assay precision. [Table 8]

[0207] The results demonstrated that particle-measured protein MS feature intensities have sufficient precision across the thousands of MS feature intensities observed to detect relatively small differences in a reasonably small-scale study. Example 7 Accuracy of Coronavirus Analysis Assays

[0208] This example describes the accuracy of the corona analysis assay. The accuracy of the method should be robust enough to detect true intergroup differences in samples in biomarker discovery and validation studies. The accuracy of the corona analysis assay was determined by comparing the results with those obtained by other methods. To evaluate the accuracy of the corona analysis assay, a spike recovery study was performed using SP-007 nanoparticles. C-reactive protein (CRP) was selected for analysis based on its endogenous level measurements. Using endogenous plasma levels of CRP determined by enzyme-linked immunosorbent assay (ELISA), known amounts of purified protein (see "Methods") were spiked to testable multiples of its endogenous level. Post-spiking CRP levels were determined experimentally by ELISA and were 4.11, 7.10, 11.5, 22.0, and 215.0 μg / mL for the 1x (unspiked), 2x, 5x, 10x, and 100x samples, respectively. Extracted MS1 feature intensities were plotted against CRP concentration for the four indicated CRP tryptic peptides detected by MS using SP-007 particles (Figure 14A). Figure 30 also shows the accuracy of CRP protein measurement with SP-007 particles in spike recovery experiments of four different peptides. MS1 feature intensities could not be detected for two of the peptides at an unspiked 1x concentration of CRP. The line fitted using the spike intensities of a given feature was a linear model.

[0209] Fitting a regression model to all four of the CRP tryptic peptides yielded a slope of 0.9 (95% CI 0.81-0.98) for the response of coronavirus MS signal intensity to ELISA plasma levels, close to the slope of 1 considered to be perfect analytical performance. In contrast, a similar regression model fitted to 1,308 other (non-spiked) MS features identified in at least four of five plasma samples, where no sample-to-sample differences in signal from the relevant MS features should be observed, had a slope of -0.086 (95% CI -0.1 to -0.068). These results demonstrate that the particle type's ability to accurately represent differences between samples makes it a useful tool for quantifying potential markers in comparative studies. If protein levels in a sample change due to any factor, the method disclosed herein will detect similar changes in the levels of proteins bound to the particle types in the particle panel, a critical characteristic for the particle types to be effective in any given assay. Furthermore, the response of peptide features of spiked proteins also suggests that with proper calibration, the particle-protein corona method could be used to determine absolute analyte levels rather than just relative quantitation. Example 8 Proteomic analysis of NSCLC samples and healthy controls

[0210] This example describes the proteome analysis of NSCLC samples and healthy controls. To demonstrate the potential utility of the corona analysis platform, we evaluated the platform's capabilities using a single particle type, SP-007, and serum samples from 56 subjects (28 with stage IV NSCLC and 28 age- and sex-matched controls) to observe differences between groups. The selected subject samples represented a fairly balanced study for identifying MS features that may differ between groups. Subject age and gender characteristics are summarized in Table 6, and all data regarding subject annotation, including disease status and comorbidities, are summarized in Table 9. [Table 9]

[0211] After collection and filtering of MS1 features followed by log2 transformation of their intensities, the dataset was median scaled without considering class.

[0212] Figure 29 shows the normalized intensity distributions for all 56 subject datasets. All 56 sample MS raw data files from this NSCLC versus control study were processed with the OpenMS pipeline script to extract MS1 features and their intensities, and cluster them into feature groups based on mz and RT value overlap within a specified tolerance range. Only feature groups that 1) had at least 50% presence of the feature in the group from at least one of the comparison arms and 2) had a feature group cluster quality above the first quartile were retained. Retained features were median normalized without regard to class, and these features were used for subsequent univariate analysis comparisons. Inspection of the distribution revealed no outliers, and all datasets were retained for univariate analysis.

[0213] Upon inspection, no outlier datasets were apparent. Univariate comparisons of feature group strengths between classes were performed with nonparametric Wilcoxon tests (two-tailed). The p-values ​​obtained for the comparisons were corrected for multiple testing using the Benjamini-Hochberg method. As summarized in Figure 27, a total of seven feature groups demonstrated statistical significance using an adjusted p-value cutoff of 0.05.

[0214] All five of the proteins identified as differentially abundant between NSCLC patients and controls have been previously implicated in cancer, if not actually NSCLC itself. PON1, or paraoxanase-1, has been implicated as a risk factor in lung cancer. The pattern is complex, including the involvement of a relatively common small allelic variant (Q192R). At the protein level, PON1 is modestly decreased in lung adenocarcinoma. SAA1 is an acute-phase protein shown to be overexpressed in NSCLC in MS-related studies, and the identified peptide was found to be 5.4-fold increased in affected subjects. The matrisome factor tenascin-C (TENA) has been shown to be increased in primary lung tumors and associated lymph node metastases compared with normal tissue, and this study found that the associated MS feature was increased 2-fold. Neural cell adhesion molecule 1 (NCAM1) serves as a marker for diagnosing pulmonary neuroendocrine tumors. FIBA ​​peptides were identified by MS analysis with increased levels that correlated with the progression of lung cancer progression. Of particular note are two unknown features, group 2 and group 7, that show differences between control and affected subjects. Group 2 was present in 54 of 56 controls and had a small 33% decrease in affected subjects. In contrast, group 7 was only found in diseased subjects (14 of 28 members of this class). These results demonstrate the potential utility of particle coronas to help identify known and unknown markers for different disease states. Example 9 Particle panels for assaying proteins in samples

[0215] This example illustrates a 10-particle type particle panel for assaying proteins in a sample. This particle panel, shown in Table 10, contains 10 distinct particle types that vary in size, charge, and polymer coating. All particle types in this particle panel are superparamagnetic. The panel shown below was used to assay proteins in a sample. [Table 10]

[0216] Protein Coverage of the V1 Panel. To assess the total protein group coverage across multiple samples in a clinical sample set, plasma samples from 16 individuals were evaluated using the sample preparation, MS data collection, and MS data analysis methods described herein for a panel of 10 distinct particle types, shown in Table 10 and referred to as the V1 panel. A combination of small cell lung cancer (NSCLC) patients and healthy individuals (n = 8 for each) was used to obtain a diverse set of proteins and protein groups present in both healthy and cancer cells for analysis and identification using the methods described herein. A total of 2,009 protein groups were efficiently identified at a 1% FDR (protein and peptide) rate. For comparison, the previously referenced published study detected 4,500 protein groups across 16 individual plasma samples in a complex workflow involving over 70 steps and over 30 MS fractions per sample, likely taking weeks to complete. Example 10 10-particle particle panel for protein assays

[0217] This example illustrates the development of a 10-particle particle panel for the method described herein for assaying proteins using biomolecular corona analysis.

[0218] Particle Screen: Biomolecular coronas from 43 particle types with distinct physicochemical properties, screened in a manner similar to the 3-particle type particle panel disclosed herein, to demonstrate that the corona analysis platform can expand its coverage by adding guide particles. [Table 11-1] [Table 11-2]

[0219] Forty-three particle types were evaluated using six conditions as described in the Methods section, and the optimal conditions were used in secondary analysis to select the best combination based on the total number of identified proteins. To demonstrate platform validation across biological samples, a 43-particle type screen was performed using a plasma pool of healthy and lung cancer patients, different from the CRC pool used for the 3-particle type particle panel. Pooled samples were used to increase protein diversity. Stringent conditions were used to identify potential proteins for panel selection and optimization. For maximum likelihood evaluation, proteins had to demonstrate at least one peptide-spectrum match (PSM; 1% false positive rate (FDR)) in each of three complete assay replicates to be counted as "identified." The panel with the greatest number of individual unique Uniprot identifiers was selected for the 10-particle type particle panel.

[0220] Protein Coverage of a 10-Particle Particle Panel. The data disclosed herein confirm that the particle panels provided can be used to determine changes in proteome content across many biological samples. The particle panels disclosed herein provide a highly accurate and precise method that takes an unbiased approach without the need for specific ligands for known proteins. Therefore, these panels are particularly well suited for biomarker discovery. The breadth and depth of plasma protein coverage using the 10-particle panel were investigated. Using a database (n=5,304) of MS-derived plasma protein intensities (one that correlates closely with concentration), the coverage of the 10-particle panel was compared to the full database and also to the coverage obtained by simple MS evaluation of plasma (direct MS analysis of the same plasma without particle-based sample extraction). Figure 36 shows the matching and coverage of a particle panel of 10 distinct particle types against a 5,304 plasma protein database of MS intensities. Ranked intensities for database proteins are shown in the top panel ("Database"); intensities for proteins from the simple plasma MS assessment are shown in the second panel ("Plasma"); and intensities for the optimal 10-particle panel are shown in the remaining panels. The plasma protein intensity database is from Keshishian et al. (2015). Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields: Novel Candidates for Early Myocardial Injury. Molecular & Cellular Proteomics, 14(9), 2375-2393. The results shown in Figure 36 confirm and extend the results shown for the particle panel of the three distinct particle types used in the precision experiment shown in Figure 26. The particle panel of 10 distinct particle types identified 1,598 proteins compared to 268 proteins for simple plasma. Furthermore, each individual particle type detected substantially more proteins than direct analysis of simple plasma by MS.Unlike MS analysis of simple plasma, the particle panel of 10 distinct particle types matched the entire range of plasma protein concentrations. In other words, while proteins identified from simple plasma samples were biased toward more intense proteins (i.e., more abundant proteins), proteins identified from the particle panel of 10 distinct particle types spanned the dynamic range of concentrations in the database by more than eight orders of magnitude. Only 21 proteins in the database had an intensity lower than the lowest matched protein from the particle panel of 10 distinct particle types. As demonstrated in Figure 36, the particle panel of 10 distinct particle types demonstrated high precision, accuracy, and broad coverage across a wide range of protein concentrations in plasma, enabling parallel, large-scale, unbiased proteome analysis across a large number of biomolecules, potentially matching the cost and speed currently available for genomic data collection.

[0221] Precision of a Particle Panel Containing 10 Distinct Particle Types. This example describes the reproducibility of particle coronas for a particle panel containing 10 distinct nanoparticle types. Particles were analyzed to determine the coefficient of variation (CV) for each feature group between replicate runs for each particle type in a particle panel containing 10 distinct nanoparticle types. A low CV indicated high precision and reproducibility between replicate runs. Data were processed using the software program OpenMS, and feature groups with measured precursor features from each of the three replicates were retained. Statistical outliers were eliminated by removing the bottom 5% of the data based on the quality score of the clustering algorithm. Group feature intensities were median-normalized to estimate the overall precision of the corona for each particle type. Normalization was performed so that the overall median intensity for each injection remained the same, and intensities were adjusted to account for intensity shifts for each of the distributions being compared, e.g., intensity shifts due to overall differences in instrument response. Differences in instrument response can occur in various analytical methods, including X-ray photoelectron spectroscopy, high-resolution transmission electron microscopy, and other analytical methods. The normalized coefficient of variation (CV) for each feature was then evaluated for each particle type in a particle panel containing 10 distinct nanoparticle types. Table 12 shows the optimized panel of 10 distinct particle types. [Table 12]

[0222] Table 13 shows the median quantile-normalized CV percent (QNCV%) values ​​for the accuracy assessment of the protein corona-based Proteograph workflow for features, peptides, and proteins for plasma and a particle panel containing 10 distinct particle types. A 1% peptide and 1% protein false positive rate (FDR) was applied. Data were processed using MaxLFQ analysis software, applying the condition that each protein group had at least one peptide ratio count and detection in all replicates, reducing the number of groups used in the accuracy analysis. Table 13 shows the median CV, including the quantile-normalized CV percent, or QNCV%, for each particle type in the particle panel containing 10 distinct nanoparticle types. Similar analysis was performed at the peptide and protein levels using MaxQuant to align distinguishable feature groups with features, peptides, and proteins (Table 13). Because peptides can contain multiple features and proteins can contain multiple peptides, the number of distinguishable feature groups decreases from features to peptides to proteins. This nanoparticle panel detected 1,184 protein groups with a 1% false positive rate (FDR). [Table 13]

[0223] Coefficients of variation (CV) were investigated independently at the feature, peptide, and protein levels. Analysis of feature, peptide, and protein CVs provides a comprehensive view of assay precision. The OpenMS and MaxQuant software engines were used for feature, peptide, and protein matching. MaxQuant was used for protein clustering with FDR. OpenMS was used to perform peptide-spectrum matching (PSM) using the X!Tandem matching tool. MaxQuant was configured to use the Andromeda algorithm. Peptide and protein CVs were used to assess the platform's precision for use with biological variables. The mean CV decreased with increasing peptide size; therefore, the mean CV was lower for peptides than for proteins. Peptides maintained similar CVs to plasma, but particles had higher feature, peptide, and protein occurrence rates than plasma. Specifically, the number of proteins on particles of any given particle type was higher than plasma (mean: 218% more, range: 133%–296% more) while maintaining comparable CVs (21.1% vs. 17.1% for particles and plasma, respectively). Furthermore, the particle-type panel identified 1,184 proteins, whereas plasma alone identified only 162 proteins.

[0224] Accuracy of a particle panel containing 10 distinct particle types. The accuracy of a particle panel containing 10 distinct particle types for detecting real-world group differences in samples in biomarker discovery and validation studies was assessed. Accuracy was determined by measuring spike recovery in the presence of nanoparticle type SP-007 and C-reactive protein (CRP). Spike recovery data were further measured in the presence of one of three additional polypeptides (S100A8 / 9 and angiogenin) in combination with each of three particle types (SP-006, SP-339, and SP-374). Known amounts of each polypeptide were spiked in at different concentrations in increments of 10 (e.g., 1x, 2x, 5x, 10x, and 100x). The levels of each polypeptide were measured by ELISA. Derived peptide and protein intensities were plotted against ELISA protein concentration. Peptide intensities were derived using the OpenMS MS1 / MS2 pipeline to find clustered feature groups with target protein MS2 IDs assigned to at least one feature within the cluster. Only clusters with a representation of the highest spike level in at least one replicate were used for analysis. Protein intensities were derived using MaxQuant software. Intensity values ​​for each protein were summarized, and the data were scaled so that the maximum concentration was 2. MS datasets were run three times for each spike concentration (e.g., 1x, 2x, 5x, 10x, and 100x) to obtain 15 individual protein or peptide measurements. Not all peptides were detected in all particle types or particle type replicates. The results of the MS datasets are shown in Figures 31-34. Figure 31 shows the accuracy of peptide feature measurements for angiogenin in a spike recovery experiment. Figure 32 shows the accuracy of peptide feature measurements for S10A8 in a spike recovery experiment. Figure 33 shows the accuracy of peptide feature measurements for S10A9 in a spike recovery experiment. Figure 34 shows the accuracy of peptide feature measurements for CRP in a spike recovery experiment. The fitted lines are linear fits to the spike intensities of each feature.

[0225] Figures 31-34 show the results of three spike recovery experiments to determine the accuracy of angiogenin, S10A8, S10A9, and CRP peptide feature measurements, respectively. Data are presented for peptides (mean r 2 is 0.81) and protein (average r 2 The results demonstrate a high degree of correlation between the individual measurements for each protein (r = 0.97). The average value of the slope across all proteins is 1.06. Table 14 shows the r for each comparison. 2 Correlation is shown, and the average r for each protein 2 Correlations are also shown. Only two of the 20 peptides showed no correlation between the ELISA assays on the two different particle types; in this case, one peptide existed in two charge states. These anomalies decreased with increasing peptide size, and therefore the frequency of anomalies was lower for peptides than for proteins. The two peptides that showed correlation in the ELISA on the two different particles here also showed a high degree of correlation with the ELISA on the other particle types. A problematic peptide may co-elute with another peptide that masks its signal, for example, by removing its charge.

[0226] Table 14 provides a summary of the regression fits for protein intensity as measured by corona analysis or ELISA. Values ​​are shown for individual particle types, averaged across four replicates per particle type. Protein concentrations, as measured by corona analysis, were consistent across the various conditions and particle types. As shown in Table 14, protein measurements were consistent across the various conditions and particle types, with a high r 2 The correlation was good, as shown by the values ​​(mean 0.97, range between individual particles 0.92-1.0; averaged range between particles 0.94-0.99). This consistent behavior among the four proteins as measured by ELISA illustrates the accuracy of the corona analysis assay. [Table 14]

[0227] Comparison with Other Platforms: The methods disclosed herein, which use multi-particle panels to enrich for proteins in distinct coronas corresponding to each protein type in the panel (e.g., corona analysis using the Proteograph workflow), provide broad, unbiased coverage of protein identification in the proteome. Other methods that attempt to achieve broad coverage of the proteome require multiple fractionation steps, complex workflows, and are slow compared to the methods presented herein. Other methods lack the breadth and unbiasedness of the methods disclosed herein, and are compared here with the methods disclosed herein for assaying proteins.

[0228] Geyer et al. (Cell Systems 2016) used a high-throughput shotgun proteomic approach. This resulted in an average of 284 protein groups per assay and 321 protein groups across all replicates. The evaluation utilized a slower multi-day protocol with fractionation that yielded approximately 1,000 protein groups. Replicate experiments were not performed, likely due to prohibitive cost and time requirements, so variance could not be determined.

[0229] Geyer used a short run to generate 321 protein groups and determined the CV of each protein. The 321 groups assessed by Geyer et al. and the 1,184 protein groups identified by the 10-particle panel contained 88 protein groups common to the two models. The identification of 88 common protein groups is unexpectedly sophisticated because protein groups can contain multiple related proteins that can be combined differently based on the peptides detected.

[0230] For a group of 88 common proteins, we analyzed the data from Geyer et al. and determined a median CV of 12.1%. In contrast, the same group of 88 common proteins had a lower CV of only 7.2% when analyzed by Proteograph. Thus, the present method of corona analysis using a multi-particle panel and Proteograph workflow provides improved precision over the method of Geyer et al. In addition, Geyer et al.'s assessment showed an assay accuracy of 0.99 for four proteins, r 2 Similarly, the proteographic assay showed an r 2 showed.

[0231] Geyer et al. further evaluated the number of protein groups with a CV of <20%, a cutoff commonly used for in vitro diagnostic assays. The particle panel method detected 761 protein groups with a CV of <20%, which was 3.7 times higher than the number identified by Geyer et al. Further evaluation by Dr. Mann (Niu et al., 2019) identified 272 protein groups with a CV of <20%, which was 2.8 times lower than the number identified by the multiparticulate panel and methods for using it disclosed herein.

[0232] Bruderer et al. assessed protein group CV using data generated by the Biognosys platform (Bruderer et al., 2019). This assessment was based on 4 65 proteins were identified, and of these 465 proteins, there was a median CV of 5.2%, with 404 of these proteins having a CV < 20%. In contrast, the best 465 proteins from the 1,184 proteins identified using the methods disclosed herein had a median CV of 4.7%, with 761 of the 1,184 proteins identified by Proteography having a CV < 20%.

[0233] Compared to the assessments of Geyer et al., Niu et al., and Bruderer et al., the present particle panels provided improved CVs for comparable numbers of proteins as well as the number of proteins meeting the CV threshold compared to other identification methods. The methods disclosed herein further have reduced bias compared to other methods, such as targeted mass spectrometry and other analyte-specific reagents (e.g., Olink). Such approaches introduce bias during the protein panel selection process by measuring a small number of preselected proteins. As a result, these approaches experience lower CVs and higher r for proteins in their panels compared to proteins identified by proteography. 2 and is limited to the detection of proteins in a panel. Example 11 Materials and methods for particle synthesis

[0234] This example describes materials and methods for particle synthesis.

[0235] Materials. Iron(III) chloride hexahydrate (ACS), sodium acetate (anhydrous ACS), ethylene glycol, ammonium hydroxide 28–30%, ammonium persulfate (APS) (≥98%, Pro-Pure, proteome grade), ethanol (reagent alcohol ACS), and methanol (≥99.8% ACS) were purchased from VWR. N,N'-methylenebisacrylamide (99%) was purchased from EMD Millipore. Trisodium citrate dihydrate (ACS reagent, ≥99.0%), tetraethyl orthosilicate (TEOS) (reagent grade, 98%), 3-(trimethoxysilyl)propyl methacrylate (MPS) (98%), and poly(ethylene glycol) methyl ether methacrylate (OEGMA, average Mn 500, containing 100 ppm MEHQ as an inhibitor and 200 ppm BHT as an inhibitor) were purchased from Sigma-Aldrich. 4,4'-Azobis(4-cyanovaleric acid) (ACVA, 98%, containing approximately 18% water) and divinylbenzene (DVB, 80%, a mixture of isomers) were purchased from Alfa Aesar and purified to remove inhibitors by passage through a short silica column. N-(3-dimethylaminopropyl)methacrylamide (DMAPMA) was purchased from TCI and purified to remove inhibitors by passage through a short silica column. An ELISA kit for measuring human C-reactive protein (CRP) was purchased from R&D Systems (Minneapolis, MN). Human CRP protein purified from human serum was from Sigma-Aldrich.

[0236] Synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) based on SP-003, SP-007, and SP-011. The iron oxide core was synthesized by solvothermal reaction (Figures 28A-E, top (Figure 28A)) (Liu, J., et al. Highly water-dispersible biocompatible nanoparticles). magnetite particles with low cytotoxicity stabilized by citrate groups. Angew Chem Int Ed Engl 48, 5875-5879 (2009);Xu, S., et al. Toward designer magnetite / polystyrene colloidal composite microspheres with controllable nanostructures and desirable surface functionalities. Langmuir 28, 3271-3278 (2012)). Typically, approximately 26.4 g of iron(III) chloride hexahydrate was dissolved in approximately 220 mL of ethylene glycol at approximately 160°C for approximately 10 minutes with stirring. Approximately 8.5 g of trisodium citrate dihydrate and approximately 29.6 g of anhydrous sodium acetate were then added and mixed for approximately 15 minutes at 160°C until complete dissolution. The solution was then sealed in a Teflon-lined stainless steel autoclave (300 mL capacity) and heated to approximately 200°C for approximately 12 hours. After cooling to room temperature, the black paramagnetic product was isolated with a magnet and washed 3-5 times with DI water. The final product was freeze-dried to a black powder for further use.

[0237] Silica-coated iron oxide nanoparticles (SP-003) were prepared by a modified Stöber method as previously reported (Figure 28B) (Deng, Y., Qi, D., Deng, C., Zhang, X. & Zhao, D. Superparamagnetic high-magnetization microspheres with an Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins. J Am Chem Soc 130, 28-29 (2008);Teng, ZG, et al. Superparamagnetic high-magnetization composite spheres with highly aminated ordered mesoporous silica shell for biomedical applications. J Mater Chem B 1, 4684-4691 (2013)). Typically, about 1 g The SPIONs were homogeneously dispersed in a mixture of ethanol (approximately 400 mL), DI water (approximately 10 mL), and concentrated aqueous ammonia (approximately 10 mL, 28-30 wt%), followed by the addition of TEOS (approximately 2 mL). After stirring at approximately 70 °C for approximately 6 hours, amorphous silica-coated SPIONs (designated Fe3O4@SiO2) were obtained. These were washed three times with methanol and three more times with water, and the final product was freeze-dried to a powder.

[0238] To prepare SP-007 (PDMAPMA-modified SPION) and SP-011 (PEG-modified SPION), vinyl-functionalized SPIONs (denoted as Fe3O4@MPS) were first prepared by a modified Stöber method previously reported (Figure 28C) (Crutchfield, CA, Thomas, SN, Sokoll, LJ & Chan, DW Advances in mass spectrometry-based clinical biomarker discovery. Clin Proteomics 13, 1 (2016)). Briefly, with the aid of vortexing (or sonication), Approximately 1 g of SPIONs was homogeneously dispersed in a mixture of ethanol (approximately 400 mL), DI water (approximately 10 mL), and concentrated aqueous ammonia solution (approximately 10 mL, 28–30 wt%), followed by the addition of TEOS (approximately 2 mL). After stirring at 70 °C for approximately 6 h, approximately 2 mL of 3-(trimethoxysilyl)propyl methacrylate was added to the reaction mixture and stirred overnight at approximately 70 °C. Vinyl-functionalized SPIONs were obtained and washed three times with methanol and three more times with water. The final product was freeze-dried to a powder. Next, for the synthesis of poly(dimethylaminopropylmethacrylamide) (PDMAPMA)-coated SPIONs (denoted as Fe3O4@PDMAPMA; SP-007 in Figure 28D), approximately 100 mg of Fe3O4@MPS was homogeneously dispersed in approximately 125 mL of DI water. After bubbling with N2 for approximately 30 minutes, approximately 2 g of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) and approximately 0.2 g of divinylbenzene (DVB) were added to the Fe3O4@MPS suspension under N2 protection. The resulting mixture was heated to approximately 75 °C, and then approximately 40 mg of ammonium persulfate (APS) in approximately 5 mL of DI water was added and stirred at approximately 75 °C overnight. After cooling, the Fe3O4@PDMAPMA was isolated with a magnet and washed with water 3–5 times. The final product was freeze-dried to a dark brown powder. For the synthesis of poly(ethylene glycol) (PEG)-coated SPIONs (denoted as Fe3O4@PEGOMA; SP-011 in Figure 28E), approximately 100 mg of Fe3O4@MPS was uniformly dispersed in approximately 125 mL of DI water. After bubbling with N2 for approximately 30 minutes, approximately 2 g of poly(ethylene glycol) methyl ether methacrylate (OEGMA, average Mn 500) and approximately 50 mg of N,N'-methylenebisacrylamide (MBA) were added to the Fe3O4@MPS suspension under N2 protection. The resulting mixture was heated to approximately 75 °C, and then approximately 50 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA) in approximately 5 mL of ethanol was added and stirred at approximately 75 °C overnight. After cooling, the Fe3O4@POEGMA was isolated with a magnet and washed with water 3 to 5 times. The final product was freeze-dried to a dark brown powder. Example 12 Patient samples

[0239] This example describes the patient samples used in this disclosure. A set of eight colorectal cancer (CRC) plasma samples and eight age- and sex-matched controls were purchased from BioIVT (Westbury, NY). A set of 28 non-small cell lung cancer (NSCLC) serum samples and 28 age- and sex-matched controls were also purchased from BioIVT. Detailed information on the CRC / NSCLC patient samples and controls is shown in Tables 15 and 16. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 16] Example 13 Characterization of the physicochemical properties of particle types.

[0240] This example describes the characterization of particle physicochemical properties by various techniques. Dynamic light scattering (DLS) and zeta potential were performed on a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Particles were suspended in water at 10 mg / mL with approximately 10 minutes of bath sonication prior to testing. Samples were then diluted to approximately 0.02 wt.% in their respective buffers for both DLS and zeta potential measurements. DLS was performed in water at approximately 25°C in disposable polystyrene semi-microcuvettes (VWR, Randor, PA, USA) with a temperature equilibration time of approximately 1 minute and consisted of an average from three approximately 1-minute runs using a 633 nm laser in 173° backscattering mode. DLS results were analyzed using the cumulant method. Zeta potential was measured in 5% pH 7.4 PBS (Gibco, PN 10010-023, USA) at approximately 25°C in a disposable collapsible capillary cell (Malvern Instruments, PN DTS1070) with an equilibration time of approximately 1 minute. Three measurements were performed with an automated measurement time of 1 minute between measurements, with a minimum of 10 runs and a maximum of 100 runs. Zeta potential was determined from electrophoretic mobility using the Smoluchowski model.

[0241] Scanning electron microscopy (SEM) was performed using an FEI Helios 600 Dual-Beam FIB-SEM. Aqueous dispersions of particles were prepared from weighed particle powder by redispersing in DI water with approximately 10 minutes of sonication to a concentration of approximately 10 mg / mL. The sample was then diluted 4-fold with methanol (from Fisher) to form a water / methanol dispersion, which was used directly for electron microscopy. SEM substrates were prepared by drop-casting approximately 6 μL of particle sample onto a Si wafer (from Ted Pella), and the droplet was then thoroughly dried in a vacuum desiccator for approximately 24 hours before measurement.

[0242] A Titan 80-300 transmission electron microscope (TEM) was used for both low- and high-resolution TEM measurements at an accelerating voltage of 300 kV. TEM grids were prepared by drop-casting approximately 2 μL of particle dispersion in a water-ethanol mixture (25-75 v / v%) at a final concentration of approximately 0.25 mg / mL and allowed to dry in a vacuum desiccator for approximately 24 h before TEM analysis. All measurements were performed on lace-hole TEM grids from Ted Pella.

[0243] X-ray photoelectron spectroscopy (XPS) was performed using a PHI VersaProbe and a ThermoScientific ESCALAB 250e III. XPS analysis was performed using finely divided powders, which were kept sealed and stored dry before measurement. The materials were placed on carbon tape to provide a uniform surface for analysis. A pale Al K-alpha X-ray source (50 W and 15 kV) was used with a 200 μm 2 A pass energy of 140 eV was used for the scanned area, and all binding energies were referenced to the C-C peak at 284.8 eV. Both survey and high-resolution scans were performed to assess the elements of interest in detail. The atomic concentration of each element was determined from the integrated intensity of the element's photoemission signature, corrected by the relative atomic sensitivity factor, by averaging results from two different locations on the sample. In some cases, four or more locations were averaged to assess uniformity. Example 14 Protein corona preparation and proteome analysis

[0244] This example describes protein corona preparation and proteomic analysis. Plasma and serum samples were diluted in TE buffer (10 mM Tris, 1 mM HCl) containing 0.05% CHAPS. The particles were diluted 1:5 with dilution buffer (SP-003, 5 mg / ml; SP-007, 2.5 mg / ml; SP-011, 10 mg / ml). The particle powder was reconstituted by sonication in DI water for approximately 10 minutes, followed by vortexing for approximately 2-3 seconds. To create the protein corona, approximately 100 μL of particle suspension (SP-003, 5 mg / ml; SP-007, 2.5 mg / ml; SP-011, 10 mg / ml) was mixed with approximately 100 μL of diluted biological sample in a microtiter plate. The plate was sealed and incubated at 37°C for approximately 1 hour with shaking at 300 rpm. After incubation, the plate was placed on a magnet for approximately 5 minutes to pellet and settle the nanoparticles. Unbound protein in the supernatant was removed with a pipette. The protein corona was further washed three times with approximately 200 μL of dilution buffer while magnetically separating. The five additional assay conditions evaluated for the 10-particle particle panel screen were identical to those described above, with one exception: First, a low particle concentration was evaluated that was 50% of the original particle concentration (2.5–15 mg / ml for each particle, depending on the expected peptide yield). For the second and third assay variants, both low and high particle concentrations were performed using undiluted raw plasma rather than diluting the particles with buffer. For the fourth and fifth assay variants, both low and high particle concentrations were performed using pH 5 citrate buffer for both dilution and rinsing.

[0245] To digest the proteins bound to the nanoparticles, a trypsin digestion kit (iST 96X, PreOmics, Germany) was used according to the provided protocol. Briefly, approximately 50 μL of lysis buffer was added to each well and heated to approximately 95°C for approximately 10 minutes with agitation. After the plate was cooled to room temperature, trypsin digestion buffer was added, and the plate was incubated at approximately 37°C for approximately 3 hours with shaking. The digestion process was stopped with stop buffer. The supernatant was separated from the nanoparticles using a magnetic collector and further purified using a peptide cleanup cartridge included in the kit. Peptides were eluted twice with approximately 75 μL of elution buffer and combined. Peptide concentrations were measured using a quantitative colorimetric peptide assay kit from Thermo Fisher Scientific (Waltham, MA).

[0246] The peptide eluate was then lyophilized and reconstituted with 0.1% TFA. A 2 μg aliquot from each sample was analyzed by nano LC-MS / MS using a Waters NanoAcquity HPLC system interfaced to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer from Thermo Fisher Scientific. Peptides were loaded onto the trapping column and eluted at 350 nL / min using a 75 μm analytical column. Both columns were packed with Luna C18 resin from Phenomenex (Torrance, CA). The mass spectrometer was operated in data-dependent mode, with MS and MS / MS performed at 60,000 and 15,000 FWHM resolutions on the Orbitrap, respectively. The instrument was run in 3-second cycles for MS and MS / MS. Example 15 Mass spectrometry data analysis

[0247] This example describes a mass spectrometry data analysis method. Acquired MS data files were processed using a suite of tools from OpenMS. These tools include modules and pipeline scripts for converting vendor-provided instrument-supplied raw files to mzML files, for MS1 feature identification and intensity extraction, for MS dataset runtime alignment and feature clustering, and for MS2 spectral database matching with the X!Tandem search engine. The precursor ion and fragment ion matching tolerances during the spectrum-database search were set to 10 and 30 ppm, respectively. Default settings were enabled for fixed carbamidomethyl (C) modifications and variable acetyl (N-terminal) and oxidative (M) modifications. The UniProtKB / Swiss-Prot protein sequence database (accepted January 27, 2019) was used for the search, and peptide spectral matches (PSMs) were scored using a standard reverse sequence decoy database strategy at 1% FDR. Using PSMs, a protein list for each particle type iteration was compiled, using a single PSM as sufficient evidence to add the protein to the enumerated protein list for a given particle type iteration. In addition, PSMs that matched more than one protein added all of the potential proteins to the enumerated protein list for a given particle type iteration. This threshold for protein enumeration is permissive and can include false positives (higher sensitivity, lower specificity), while more stringent tests requiring two or more peptides (including at least one unique peptide) suffer from the opposite problem of false negatives (lower sensitivity, higher specificity). For quantitative analysis of known peptides, a custom R script was used to assign MS2 PSMs to MS1 feature sets based on positional overlap with a tolerance of 1 Da and 30 s for mz and dwell time, respectively. In the unlikely event that more than one PSM was initially located on an MS1 ​​feature within a pre-specified tolerance, we used the PSM that was closest to the MS1 feature (within the MS dataset) or closest to the center of the MS1 feature cluster (between the MS datasets).It should be noted that not all MS2s are assigned to MS1 feature groups, and not all MS1 feature group clusters have assigned MS2s, and research continues in this area to improve mapping and subsequent peptide feature identification. Example 16 Protein group identification

[0248] This example describes a method for protein group identification by mass spectrometry. For protein group-level analysis, MS data at the protein group level were analyzed as follows: MS raw files were processed with MaxQuant (v. 1.6.7 (49) and Andromeda (50)), and MS / MS spectra were searched against the UniProtKB human FASTA database (UP000005640, 74,349 forward entries; version from August 2019) using standard settings. Enzyme digestion specificity was set to trypsin, allowing cleavage at the N-terminal side of proline and up to two false cleavages. The minimum peptide length was set to 7 amino acids, and the maximum peptide mass was set to 4,600 Da. Methionine oxidation and protein N-terminal acetylation were set as variable modifications, and carbamidomethylation of cysteine ​​was set as a fixed modification. MaxQuant improves precursor ion mass accuracy through a time-dependent recalibration algorithm, defining individual mass tolerances for each peptide. The initial maximum precursor mass tolerance allowed was 20 ppm during the initial search and 4.5 ppm for the main search. The MS / MS mass tolerance was set to 20 ppm. For the analysis, a 1% false positive rate (FDR) cutoff was applied at the peptide and protein levels (all protein groups are reported with their corresponding q values ​​in the proteinGroups.txt table). "Matches between runs" was disabled. The number of identifications was counted based on protein intensity (only proteins with q values ​​lower than 1% were counted), requiring at least one razor peptide. MaxLFQ-normalized protein intensities (requiring a peptide ratio count of at least 1) were reported in the raw output, and these intensities were used only for CV accuracy analysis. Peptides that could be distinguished were sorted into their own protein groups, while proteins that could not be distinguished based on unique peptides were combined into protein groups. Proteins were further filtered against the list of common contaminants included in MaxQuant. Proteins identified solely by site modifications were strictly excluded from the analysis. Example 17 Spike recovery rate

[0249] This example describes a method for C-reactive protein (CRP) spike recovery experiments. Baseline concentrations of CRP in pooled healthy plasma samples were measured using the ELISA kit described above ("Materials") according to the manufacturer's suggested protocol. Stock solutions and appropriate dilutions of CRP were prepared and spiked into identical pooled plasma samples to achieve final concentrations that were 2x, 5x, 10x, and 100x the baseline endogenous concentration of CRP. The volume added to the pooled plasma was 10% of the total sample volume. Spiked controls were created by adding the same volume of buffer to the pooled plasma samples. The concentrations of the spiked samples were measured again by ELISA to confirm the CRP levels at each spike level. These samples were used to evaluate particle corona measurement accuracy as described above in "Results." Example 18 Proteomic analysis of NSCLC samples and healthy controls

[0250] This example describes proteomic analysis of NSCLC samples and healthy controls. Serum samples from 56 subjects, 28 with stage IV NSCLC and 28 age- and sex-matched controls, were commercially purchased and evaluated for SP-007 nanoparticle formation (see above for sample collection and corona formation and processing). MS spectral data for each corona was collected as described, and raw data was processed as described above (MS data analysis). 19,214 feature groups were identified and extracted across the 56 subject samples, with group sizes ranging from 1 (singleton features in only one sample, n = 6,249, i.e., 0.29% of the data) to 56 (features present in all samples, n = 450, i.e., 12% of the data). The clustering algorithm calculates a "group_quality" metric, which relates to the spatial uniformity of feature grouping with groups across the dataset. We then partitioned by group size, eliminating the bottom quartile of groups from consideration due to the skewed nature of the distribution of low quality scores, resulting in 15,967 remaining groups. As an additional filter before analysis, only groups with features present in at least 50% of at least one of the classes, diseased or control, were brought forward, resulting in a set of 2,507 feature groups remaining for analysis.

[0251] Peptide and protein identities were assigned to feature sets as follows: MS2 PSM and MS1 feature sets were assigned together as described above (MS data analysis). Using this approach, 25% of the original 19,249 feature sets were associated with peptide sequences. Univariate statistical comparisons between sets were performed for all feature sets with and without assigned peptide sequences. Example 19 statistical analysis

[0252] This example describes the statistical analysis of the data disclosed herein. Statistical analysis and visualization were performed using R (v3.5.2) with appropriate packages (R: A Language and Environment for Statistical Computing. The R Foundation for Statistical Computing). Computing, Vienna, Austria.URL https: / / www.R-project.org / ).

[0253] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is to be understood that alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for identifying a protein in a sample, comprising: incubating the particle panel with the sample to form a plurality of distinct biomolecular coronas corresponding to distinct particle types of the particle panel; magnetically isolating the particle panel from unbound proteins in the sample to enrich for proteins in the plurality of distinct biomolecular coronas; and assaying said plurality of distinct biomolecular coronas to identify enriched proteins. A method comprising: (Item 2) Item 10. The method according to item 1, wherein the assaying step can identify 1 to 20,000 protein groups. (Item 3) 3. The method according to any one of items 1 to 2, wherein the assaying step can identify 1,000 to 10,000 protein groups. (Item 4) 4. The method according to any one of items 1 to 3, wherein the assaying step can identify 1,000 to 5,000 protein groups. (Item 5) 5. The method according to any one of items 1 to 4, wherein the assaying step can identify 1,200 to 2,200 protein groups. (Item 6) 6. The method according to any one of items 2 to 5, wherein the group of proteins comprises a peptide sequence having a minimum length of 7 amino acid residues. (Item 7) 7. The method according to any one of items 1 to 6, wherein the assaying step is capable of identifying 1,000 to 10,000 proteins. (Item 8) 8. The method according to any one of items 1 to 7, wherein the assaying step can identify 1,800 to 5,000 proteins. (Item 9) 9. The method according to any one of items 1 to 8, wherein the sample comprises a plurality of samples. (Item 10) 10. The method of claim 9, wherein the plurality of samples comprises at least two or more spatially separated samples. (Item 11) Item 11. The method of item 10, wherein the incubating step comprises simultaneously contacting the at least two or more spatially separated samples with the particle panel. (Item 12) 12. The method of any one of items 10-11, wherein the magnetically isolating step comprises simultaneously magnetically isolating the particle panel from unbound proteins in the at least two or more spatially separated samples of the plurality of samples. (Item 13) 13. The method of any one of items 10 to 12, wherein the assaying step comprises assaying the plurality of distinct biomolecular coronas to simultaneously identify the at least two or more spatially separated proteins in the sample. (Item 14) 14. The method of any one of items 1 to 13, further comprising repeating the method of any one of items 1 to 7, wherein when repeated, the incubating, isolating and assaying steps result in a quantile normalized coefficient of variation (QNCV) percent of 20% or less, as determined by comparing peptide mass spectrometry signatures from at least three complete assay replicates for each particle type in the particle panel. (Item 15) 14. The method of any one of items 1 to 13, wherein the incubating, isolating and assaying steps, when repeated, result in a quantile normalized coefficient of variation (QNCV) percent of 10% or less, as determined by comparing peptide mass spectrometry signatures from at least three complete assay replicates for each particle type in the particle panel. (Item 16) 16. The method of any one of items 1 to 15, wherein the assaying step is capable of identifying proteins over a dynamic range of at least 7, at least 8, at least 9, or at least 10. (Item 17) 17. The method of any one of items 1 to 16, further comprising washing the particle panel at least once or at least twice after magnetically isolating the particle panel from the unbound protein. (Item 18) 18. The method of any one of items 1 to 17, further comprising, after the assaying step, lysing the proteins in the plurality of distinct biomolecular coronas. (Item 19) 20. The method of claim 18, further comprising digesting the proteins in the plurality of distinct biomolecular coronas to produce digested peptides. (Item 20) 20. The method of claim 19, further comprising purifying the digested peptides. (Item 21) 21. The method of any one of items 1 to 20, wherein the assaying step comprises identifying proteins in the sample using mass spectrometry. (Item 22) 22. The method of any one of items 1 to 21, wherein the assaying step is carried out within about 2 to about 4 hours. (Item 23) 23. The method according to any one of items 1 to 22, which is carried out within about 1 to about 20 hours. (Item 24) 24. The method according to any one of items 1 to 23, which is carried out within about 2 to about 10 hours. (Item 25) 25. The method according to any one of items 1 to 24, wherein the method is carried out within about 4 to about 6 hours. (Item 26) 26. The method of any one of items 1 to 25, wherein the isolating step takes about 30 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, or about 2 minutes or less. (Item 27) 27. The method of any one of items 3 to 26, wherein the plurality of samples comprises at least 10 spatially separated samples, at least 50 spatially separated samples, at least 100 spatially separated samples, at least 150 spatially separated samples, at least 200 spatially separated samples, at least 250 spatially separated samples, or at least 300 spatially separated samples. (Item 28) 28. The method of claim 27, wherein the plurality of samples comprises at least 96 samples. (Item 29) 29. The method of any one of items 1 to 28, wherein the particle panel comprises at least two distinct particle types, at least three distinct particle types, at least four distinct particle types, at least five distinct particle types, at least six distinct particle types, at least seven distinct particle types, at least eight distinct particle types, at least nine distinct particle types, at least ten distinct particle types, at least eleven distinct particle types, at least 12 distinct particle types, at least thirteen distinct particle types, at least fourteen distinct particle types, at least fifteen distinct particle types, at least twenty distinct particle types, at least twenty-five particle types, or at least thirty distinct particle types. (Item 30) 30. The method of claim 29, wherein the particle panel comprises at least 10 distinct particle types. (Item 31) 31. The method according to any one of items 10 to 30, wherein the at least two spatially separated samples differ in at least one physicochemical property. (Item 32) 32. The method of any one of items 1 to 31, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, wherein the first distinct type of particle and the second distinct type of particle share at least one physicochemical property but differ in at least one physicochemical property, and thus the first distinct type of particle and the second distinct type of particle are different. (Item 33) 33. The method of any one of items 1 to 32, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, wherein the first distinct type of particle and the second distinct type of particle share at least two physicochemical properties but differ in at least two physicochemical properties, and thus the first distinct type of particle and the second distinct type of particle are different. (Item 34) 34. The method of any one of items 1 to 33, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, wherein the first distinct type of particle and the second distinct type of particle share at least one physicochemical property but differ in at least two physicochemical properties, and thus the first distinct type of particle and the second distinct type of particle are different. (Item 35) 35. The method of any one of items 1 to 34, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, wherein the first distinct type of particle and the second distinct type of particle share at least two physicochemical properties but differ in at least one physicochemical property, and thus the first distinct type of particle and the second distinct type of particle are different. (Item 36) 36. The method according to any one of items 31 to 35, wherein the physicochemical property comprises size, charge, core material, shell material, porosity, or surface hydrophobicity. (Item 37) 37. The method of claim 36, wherein the size is a diameter or radius measured by dynamic light scattering, SEM, TEM, or any combination thereof. (Item 38) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle comprising a carboxylate material, the first distinct particles being microparticles, and the second distinct type of particle being nanoparticles. (Item 39) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type having surface charges of 0 mV and -50 mV, and the first distinct particle type having a diameter less than 200 nm and the second distinct particle type having a diameter greater than 200 nm. (Item 40) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle having a diameter of 100 to 400 nm, the first distinct type of particle having a positive surface charge, and the second distinct type of particle having a neutral surface charge. (Item 41) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle being nanoparticles, the first distinct type of particle having a surface charge less than −20 mV, and the second distinct type of particle having a surface charge greater than −20 mV. (Item 42) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle being microparticles, the first distinct type of particle having a negative surface charge, and the second distinct type of particle having a positive surface charge. (Item 43) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a subset of nanoparticles that are negatively charged, each particle of the subset differing in at least one surface chemical group. (Item 44) 38. The method of any one of items 1 to 37, wherein the particle panel comprises a first distinct type of particle, a second particle, and a third distinct type of particle, wherein the first distinct type of particle, the second distinct type of particle, and the third distinct type of particle comprise an iron oxide core, a polymer shell, and are less than about 500 nm in diameter, wherein the first distinct type of particle has a negative charge, the second distinct type of particle has a positive charge, and the third distinct type of particle has a neutral charge, and wherein the diameters are average diameters measured by dynamic light scattering. (Item 45) 45. The method of claim 44, wherein the first separate type of particles comprises a silica coating, the second separate type of particles comprises poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), and the third separate type of particles comprises a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating. (Item 46) 46. ​​The method of any one of items 1 to 45, wherein at least one distinct particle type of the particle panel is a nanoparticle. (Item 47) 47. The method of any one of items 1 to 46, wherein at least one distinct particle type of the particle panel is a microparticle. (Item 48) 48. The method of any one of items 1 to 47, wherein at least one distinct particle type of the particle panel is superparamagnetic iron oxide particles. (Item 49) 49. The method of any one of items 1 to 48, wherein each particle of the particle panel comprises an iron oxide material. (Item 50) 50. The method of any one of items 1 to 49, wherein at least one distinct particle type of the particle panel has an iron oxide core. (Item 51) 50. The method of any one of items 1 to 49, wherein at least one distinct particle type of the particle panel has iron oxide crystals embedded in a polystyrene core. (Item 52) 48. The method of any one of items 1 to 47, wherein each of the distinct particle types of the particle panel is a superparamagnetic iron oxide particle. (Item 53) 53. The method of any one of items 1 to 52, wherein each distinct particle type of the particle panel comprises an iron oxide core. (Item 54) 53. The method of any one of items 1 to 52, wherein each one distinct particle type of the particle panel has iron oxide crystals embedded in a polystyrene core. (Item 55) 56. The method of any one of items 1 to 55, wherein at least one distinct particle type of the particle panel comprises a carboxylated polymer, an aminated polymer, a zwitterionic polymer, or any combination thereof. (Item 56) 57. The method of any one of items 1 to 56, wherein at least one particle type of the particle panel comprises an iron oxide core with a silica shell coating. (Item 57) 57. The method of any one of items 1 to 56, wherein at least one particle type of the particle panel comprises an iron oxide core having a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating. (Item 58) 57. The method of any one of items 1 to 56, wherein at least one particle type of the particle panel comprises an iron oxide core having a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating. (Item 59) 59. The method of any one of items 1 to 58, wherein at least one distinct particle type of the particle panel has a negative surface charge. (Item 60) 59. The method of any one of items 1 to 58, wherein at least one distinct particle type of the particle panel has a positive surface charge. (Item 61) 59. The method of any one of items 1 to 58, wherein at least one distinct particle type of the particle panel has a neutral surface charge. (Item 62) 62. The method of any one of items 1 to 61, wherein the particle panel comprises one or more distinct particle types selected from Table 10. (Item 63) 63. The method of any one of items 1 to 62, wherein the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 10. (Item 64) 64. The method of any one of items 1 to 63, wherein the particle panel comprises one or more distinct particle types selected from Table 12. (Item 65) 65. The method of any one of items 1 to 64, wherein the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 12. (Item 66) 67. A composition comprising three or more distinct magnetic particle types that differ in two or more physicochemical properties, wherein a subset of the three or more distinct magnetic particle types share a physicochemical property among the two or more physicochemical properties, and wherein such particle types of the subset bind to different proteins. 67. The composition of claim 66, wherein the three or more distinct magnetic particle types adsorb proteins from a sample over a dynamic range of at least 7, at least 8, at least 9, or at least 10. (Item 68) 68. The composition of any one of items 66-67, wherein the three or more distinct magnetic particle types are capable of adsorbing a group of 1 to 20,000 proteins from a sample. (Item 69) 69. The composition of any one of items 66 to 68, wherein the three or more distinct magnetic particle types are capable of adsorbing 1,000 to 10,000 protein groups from a sample. (Item 70) 70. The composition of any one of items 66 to 69, wherein the three or more distinct magnetic particle types are capable of adsorbing 1,000 to 5,000 protein groups from a sample. (Item 71) 71. The composition of any one of items 66 to 70, wherein the three or more distinct magnetic particle types are capable of adsorbing 1,200 to 2,200 protein groups from a sample. (Item 72) 72. The composition according to any one of items 69 to 71, wherein the group of proteins comprises a peptide sequence having a minimum length of 7 amino acid residues. (Item 73) 73. The composition of any one of items 66 to 72, wherein the three or more distinct magnetic particle types are capable of adsorbing 1 to 20,000 proteins from a sample. (Item 74) 74. The composition of any one of items 66 to 73, wherein the three or more distinct magnetic particle types are capable of adsorbing 1,000 to 10,000 proteins from a sample. (Item 75) 75. The composition of any one of items 66 to 74, wherein the three or more distinct magnetic particle types are capable of adsorbing 1,800 to 5,000 proteins from a sample. (Item 76) 76. The composition of any one of items 66 to 75, comprising at least 4 distinct magnetic particle types, at least 5 distinct magnetic particle types, at least 6 distinct magnetic particle types, at least 7 distinct magnetic particle types, at least 8 distinct magnetic particle types, at least 9 distinct magnetic particle types, at least 10 distinct magnetic particle types, at least 11 distinct magnetic particle types, at least 12 distinct magnetic particle types, at least 13 distinct magnetic particle types, at least 14 distinct magnetic particle types, at least 15 distinct magnetic particle types, at least 20 distinct magnetic particle types, or at least 30 distinct magnetic particle types. (Item 77) 77. The composition according to item 76, comprising at least 10 distinct magnetic particle types. (Item 78) 78. The composition according to any one of items 66 to 77, comprising a first distinct type of particle and a second distinct type of particle, wherein said first distinct type of particle and said second distinct type of particle share at least two physicochemical properties but differ in terms of at least two physicochemical properties, and thus said first distinct type of particle and said second distinct type of particle are different. (Item 79) 79. The composition according to any one of items 66 to 78, comprising a first distinct type of particle and a second distinct type of particle, wherein said first distinct type of particle and said second distinct type of particle share at least two physicochemical properties but differ in at least one physicochemical property, and thus said first distinct type of particle and said second distinct type of particle are different. (Item 80) 80. The composition according to any one of items 66 to 79, wherein the physicochemical property comprises size, charge, core material, shell material, porosity, or surface hydrophobicity. (Item 81) 81. The composition of claim 80, wherein the size is a diameter or radius measured by dynamic light scattering, SEM, TEM, or any combination thereof. (Item 82) 82. The composition of any one of items 66 to 81, comprising a first distinct type of particles and a second distinct type of particles, wherein the first distinct type of particles and the second distinct type of particles comprise a carboxylate material, the first distinct particles being microparticles and the second distinct type of particles being nanoparticles. (Item 83) 82. The composition of any one of items 66 to 81, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle having surface charges of 0 mV and -50 mV, and the first distinct type of particle has a diameter less than 200 nm and the second distinct type of particle has a diameter greater than 200 nm. (Item 84) 82. The composition of any one of items 66 to 81, wherein the particle panel comprises a first distinct particle type and a second distinct particle type, the first distinct particle type and the second distinct particle type having diameters of 100 to 400 nm, the first distinct particle type having a positive surface charge, and the second distinct particle type having a neutral surface charge. (Item 85) 82. The composition of any one of items 66 to 81, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle being nanoparticles, the first distinct type of particle having a surface charge less than −20 mV, and the second distinct type of particle having a surface charge greater than −20 mV. (Item 86) 82. The composition of any one of items 66 to 81, wherein the particle panel comprises a first distinct type of particle and a second distinct type of particle, the first distinct type of particle and the second distinct type of particle being microparticles, the first distinct type of particle having a negative surface charge, and the second distinct type of particle having a positive surface charge. (Item 87) Item 88. The composition of any one of items 66 to 81, comprising a subset of negatively charged nanoparticles, each particle type of the subset differing in at least one surface chemical group. 82. The composition of any one of items 66 to 81, comprising a first distinct type of particles, a second distinct type of particles, and a third distinct type of particles, wherein the first distinct type of particles, the second distinct type of particles, and the third distinct type of particles comprise an iron oxide core, a polymer shell, and are less than about 500 nm in diameter, the first distinct type of particles have a negative charge, the second distinct type of particles have a positive charge, and the third distinct type of particles have a neutral charge, and the diameters are average diameters measured by dynamic light scattering. (Item 89) 89. The composition of claim 88, wherein the first separate type of particles comprises a silica coating, the second separate type of particles comprises poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), and the third separate type of particles comprises a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating. (Item 90) 89. The composition of any one of items 66 to 89, wherein the three or more distinct magnetic particle types comprise nanoparticles. (Item 91) 91. The composition of any one of items 66 to 90, wherein the three or more distinct magnetic particle types comprise microparticles. (Item 92) 92. The composition of any one of items 66 to 91, wherein at least one distinct particle type of the three or more distinct magnetic particle types is superparamagnetic iron oxide particles. (Item 93) 93. The composition of any one of items 66 to 92, wherein at least one distinct particle type of the three or more distinct magnetic particle types comprises an iron oxide material. (Item 94) 94. The composition of any one of items 66 to 93, wherein at least one distinct particle type of the three or more distinct magnetic particle types has an iron oxide core. (Item 95) 95. The composition of any one of items 66 to 94, wherein at least one distinct particle type of the three or more distinct magnetic particle types has iron oxide crystals embedded in a polystyrene core. (Item 96) 96. The composition of any one of items 66 to 95, wherein each distinct particle type of the three or more distinct magnetic particle types is a superparamagnetic iron oxide particle. (Item 97) 97. The composition of any one of items 66 to 96, wherein each distinct particle type of the three or more distinct magnetic particle types comprises an iron oxide core. (Item 98) 98. The composition of any one of items 66 to 97, wherein each one distinct particle type of the three or more distinct magnetic particle types has iron oxide crystals embedded in a polystyrene core. (Item 99) 99. The composition of any one of items 66 to 98, wherein at least one particle type of the three or more distinct magnetic particle types comprises a polymer coating. (Item 100) 99. The composition of any one of items 66-99, wherein the three or more distinct magnetic particle types comprise carboxylated polymers, aminated polymers, zwitterionic polymers, or any combination thereof. (Item 101) 101. The composition of any one of items 66 to 100, wherein at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core with a silica shell coating. (Item 102) 101. The composition of any one of items 66 to 100, wherein at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core having a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating. (Item 103) 101. The composition of any one of items 66 to 100, wherein at least one particle type of the three or more distinct magnetic particle types comprises an iron oxide core having a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating. (Item 104) 104. The composition of any one of items 66 to 103, wherein at least one particle type of the three or more distinct magnetic particle types has a negative surface charge. (Item 105) 104. The composition of any one of items 66 to 103, wherein at least one particle type of the three or more distinct magnetic particle types has a positive surface charge. (Item 106) 104. The composition according to any one of items 66 to 103, wherein at least one particle type of the three or more distinct magnetic particle types has a neutral surface charge. (Item 107) 107. The composition of any one of items 66 to 106, wherein the three or more distinct magnetic particle types comprise one or more particle types of Table 10. (Item 108) 108. The composition of any one of items 66 to 107, wherein the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 10. (Item 109) 109. The composition of any one of items 66 to 108, wherein the three or more distinct magnetic particle types comprise one or more particle types of Table 12. (Item 110) 109. The composition of any one of items 66 to 109, wherein the particle panel comprises two or more distinct particle types, three or more distinct particle types, four or more distinct particle types, five or more distinct particle types, six or more distinct particle types, seven or more distinct particle types, eight or more distinct particle types, nine or more distinct particle types, or all ten distinct particle types selected from Table 12. (Item 111) 111. The method according to any one of items 1 to 64 or the composition according to any one of items 67 to 110, wherein the sample is a biological sample. (Item 112) 112. The method of claim 111, wherein the biological sample is plasma, serum, CSF, urine, tears, cell lysate, tissue lysate, cell homogenate, tissue homogenate, nipple aspirate, fecal sample, synovial fluid, and whole blood, or saliva. (Item 113) 111. The method according to any one of items 1 to 64 or the composition according to any one of items 67 to 110, wherein the sample is a non-biological sample. (Item 114) Item 114. The method of item 113, wherein the non-biological sample is a water, milk, solvent, or homogenate sample.

Claims

1. 1. A high-throughput method for identifying proteins in a biological sample, comprising: a. incubating magnetic microparticles with the biological sample to form a protein corona containing proteins, the magnetic microparticles comprising (i) silica, (ii) carboxylic acid functional groups, (iii) a negative surface charge with a zeta potential of −30 mV to −10 mV in the absence of the biological sample, and (iv) a heterogeneous size distribution of 1 μm to 10 μm in diameter, the magnetic microparticles configured to form a protein corona with at least 200 unique protein groups that can be identified using mass spectrometry; b. magnetically isolating the magnetic microparticles from unbound proteins in the biological sample by applying an external magnetic field to concentrate proteins in the protein corona; c. washing the concentrated proteins and the magnetic microparticles after magnetically isolating the magnetic microparticles; d. Incubating the concentrated proteins in the protein corona by adding a denaturing reagent to the protein corona and the magnetic microparticles; e. digesting the concentrated proteins in the protein corona by adding a digestion reagent to the protein corona and the magnetic microparticles to generate digested peptides; f. purifying the digested peptides; A method comprising:

2. 2. The method of claim 1, wherein the step of purifying the digested peptides comprises binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides.

3. 10. The method of claim 1, further comprising using a stopping reagent to stop the digestion reagent.

4. The method of claim 1 , wherein the digestion reagent comprises trypsin.

5. The method of claim 4 , wherein the digestion reagent further comprises LysC.

6. 10. The method of claim 1, wherein the magnetic microparticles are configured to form a protein corona having at least 500 unique protein groups that can be identified using mass spectrometry.

7. The method of claim 1 , wherein the magnetic microparticles comprise a polydispersity index greater than 0.

5.

8. 10. The method of claim 1, further comprising assaying the purified digested peptides using mass spectrometry to identify the unique protein group from the enriched proteins.

9. 9. The method of claim 8, wherein the assaying step produces a quantile normalized coefficient of variation (QNCV) percent of 20% or less, as determined by comparing peptide mass spectrometric features from at least three complete assay replicates.

10. 10. The method of claim 1, wherein the magnetic microparticles exhibit a negative surface charge with a zeta potential of -30 mV to -10 mV when the zeta potential is measured at a concentration of 0.02 wt% in a solution containing 5% pH 7.4 PBS at about 25°C in a disposable folded capillary cell with an equilibration time of about 1 minute.

11. The method of claim 1 , wherein the washing of the concentrated protein and the magnetic microparticles is performed at least twice.

12. 10. The method of claim 1, wherein the biological sample comprises plasma or serum.

13. The method of claim 1 , wherein the magnetic microparticles are incubated with the biological sample at a basic pH.

14. 10. The method of claim 1, wherein (a) through (f) are carried out for about 4 to 6 hours.

15. A high-throughput method for preparing a method for identifying proteins in a biological sample, comprising: a. incubating magnetic particles with the biological sample to form a protein corona comprising proteins, wherein the magnetic particles have a negative surface charge with a zeta potential of -30 mV to -10 mV in the absence of a biological sample, and the magnetic particles are configured to form a protein corona having at least 400 unique protein groups that can be identified using mass spectrometry; b. magnetically isolating the magnetic particles from unbound proteins in the biological sample by applying an external magnetic field to concentrate proteins in the protein corona; c. washing the concentrated proteins and the magnetic particles at least two times after magnetically isolating the magnetic particles; d. Incubating the concentrated proteins in the protein corona by adding a denaturing reagent to the protein corona and the magnetic particles; e. digesting the concentrated proteins in the protein corona by adding a digestion reagent to the protein corona and the magnetic particles to generate digested peptides; f. purifying the digested peptides; Including, the step of purifying the digested peptides comprises binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides, and the biological sample is plasma or serum; i. incubating second magnetic particles with the biological sample to form a second protein corona, wherein the second magnetic particles comprise a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating; ii. magnetically isolating the second magnetic particles from unbound proteins in the biological sample by applying an external magnetic field to concentrate a second protein in the second protein corona; iii. washing the second concentrated protein and the second magnetic particles after magnetically isolating the second magnetic particles; iv. incubating the second concentrated protein in the second protein corona by adding a denaturing reagent to the second protein corona and the second magnetic particles; v. digesting the second enriched proteins in the second protein corona by adding a digestion reagent to the second protein corona and the second magnetic particles to produce second digested peptides; vi. purifying the second digested peptide; The method further comprises:

16. A high-throughput method for preparing a method for identifying proteins in a biological sample, comprising: a. incubating magnetic particles with the biological sample to form a protein corona comprising proteins, wherein the magnetic particles have a negative surface charge with a zeta potential of -30 mV to -10 mV in the absence of a biological sample, and the magnetic particles are configured to form a protein corona having at least 400 unique protein groups that can be identified using mass spectrometry; b. magnetically isolating the magnetic particles from unbound proteins in the biological sample by applying an external magnetic field to concentrate proteins in the protein corona; c. washing the concentrated proteins and the magnetic particles at least two times after magnetically isolating the magnetic particles; d. Incubating the concentrated proteins in the protein corona by adding a denaturing reagent to the protein corona and the magnetic particles; e. digesting the concentrated proteins in the protein corona by adding a digestion reagent to the protein corona and the magnetic particles to generate digested peptides; f. purifying the digested peptides; Including, the step of purifying the digested peptides comprises binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides, and the biological sample is plasma or serum; The method, wherein the magnetic particles comprise a first type of particle and a second type of particle, the first type of particle being nanoparticles and the second type of particle being microparticles.

17. 17. The method of claim 16, wherein the first type of grain comprises an iron oxide core.

18. The method of claim 17 , wherein the second type of grain comprises a non-magnetic core.

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