US20210215709A1 - Compositions, methods and systems for protein corona analysis from biofluids and uses thereof - Google Patents

Compositions, methods and systems for protein corona analysis from biofluids and uses thereof Download PDF

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US20210215709A1
US20210215709A1 US17/216,510 US202117216510A US2021215709A1 US 20210215709 A1 US20210215709 A1 US 20210215709A1 US 202117216510 A US202117216510 A US 202117216510A US 2021215709 A1 US2021215709 A1 US 2021215709A1
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proteins
sample
particle
protein
corona
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Xiaoyan Zhao
William Manning
John Blume
Lyndal HESTERBERG
Gregory Troiano
Michael Figa
Hope Liou
Shadi Roshdiferdosi
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Seer Inc
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Seer Inc
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Assigned to SEER, INC. reassignment SEER, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HESTERBERG, Lyndal, ROSHDIFERDOSI, Shadi, FIGA, MICHAEL, LIOU, Hope, MANNING, WILLIAM, ZHAO, XIAOYAN, BLUME, JOHN, TROIANO, GREGORY
Priority to US17/823,924 priority patent/US20230076807A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • Biofluids contain a wide variety of proteins whose presence and concentrations may be indicative of subject health status. High abundance proteins and other proteins may overshadow the signal of an assay. Protein coronas can form on particles when they are exposed to biological fluids containing proteins. Most work on protein coronas has been performed on fluid from the blood stream (plasma or serum). The interaction of diverse biofluids with particles is largely unknown.
  • Fractionation may be used to reduce the signal of high abundance proteins when assaying a biological fluid.
  • various fractionation methods exist, which facilitate the isolation and extraction of proteins. These methods allow for different profiles of proteins in assays to be detected.
  • analysis of more diverse proteomics remains desired.
  • the present disclosure provides a method of serially interrogating a sample, the method comprising: a) contacting the sample to a first particle type and incubating the first particle type with the sample to form a first biomolecule corona, wherein the first biomolecule corona forms upon incubation of the first particle type with the sample and wherein the first biomolecule corona comprises a protein; b) separating the first particle type comprising the first biomolecule corona from the sample; c) contacting the sample to a second particle type and incubating the second particle type with the sample to form a second biomolecule corona, wherein the second biomolecule corona forms upon incubation of the second particle type with the sample, wherein the second biomolecule corona comprises a protein, and further wherein (i) the first particle type and the second particle type are a same particle type or (ii) the first particle type and the second particle type are different; d) separating the second particle type comprising the second biomolecule corona from the sample; e
  • the dynamic range of the plurality of proteins in the first biomolecule corona is compressed relative to a dynamic range of a plurality of proteins in the sample, as measured by a total protein analysis method.
  • the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio of: a) a signal produced by a higher abundance protein of the plurality of proteins in the first biomolecule corona; and b) a signal produced by a lower abundance protein of the plurality of proteins in the first biomolecule corona.
  • the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the first biomolecule corona.
  • the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio of a top decile of proteins to a bottom decile of proteins in the plurality of proteins in the first biomolecule corona. In some aspects, the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio comprising a span of the interquartile range of proteins in the plurality of proteins in the first biomolecule corona. In some aspects, the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio comprising a slope of fitted data in a plot of all concentrations of proteins in the plurality of proteins in the first biomolecule corona versus known concentrations of the same proteins in the sample.
  • the known concentrations of the same proteins in the sample are obtained from a database.
  • the dynamic range of a plurality of proteins in the sample is a second ratio of: a) a signal produced by a higher abundance protein of the plurality of proteins in the sample, as measured by a total protein analysis method; and b) a signal produced by a lower abundance protein of the plurality of proteins in the sample, as measured by a total protein analysis method.
  • the dynamic range of the plurality of proteins in the sample, as measured by a total protein analysis method is a second ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the sample.
  • the dynamic range of the plurality of proteins in the sample, as measured by a total protein analysis method is a second ratio of a top decile of proteins to a bottom decile of proteins in the plurality of proteins in the sample.
  • the dynamic range of the plurality of proteins in the sample is a second ratio comprising a span of the interquartile range of proteins in the plurality of proteins in the sample.
  • the dynamic range of the plurality of proteins in the sample is a second ratio comprising a slope of fitted data in a plot of all concentrations of proteins in the plurality of proteins in the sample versus known concentrations of the same proteins in the sample.
  • the known concentrations of the same proteins in the sample are obtained from a database.
  • the compressing the dynamic range comprises a decreased first ratio relative to the second ratio.
  • the decreased first ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, or at least 10-fold less than the second ratio.
  • the method further comprises: determining the composition and concentration of a plurality of proteins in the second biomolecule corona. In some aspects, the determining the composition and concentration of the plurality of proteins in the first biomolecule corona or determining the composition and concentration of the plurality of proteins in the second biomolecule corona comprises performing mass spectrometry. In some aspects, the method further comprising repeating step a), b), and e) with one or more additional particle types to form one or more additional biomolecule coronas. In some aspects, the one or more additional particle types are the same as the first particle type or the second particle type. In some aspects, the one or more additional particle types are different from the first particle type, the second particle type, or a combination thereof.
  • the one or more additional biomolecule coronas comprise proteins from the sample.
  • the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises at least 100 distinct proteins.
  • the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises at least 200 distinct proteins, at least 300 distinct proteins, at least 400 distinct proteins, at least 500 distinct proteins, at least 1000 distinct proteins, at least 2000 distinct proteins, or at least 5000 distinct proteins.
  • the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises a different composition of proteins, a different concentration of a subset of proteins, or a combination thereof.
  • a dynamic range of a plurality of proteins in the one or more additional biomolecule coronas is compressed relative to a dynamic range of a plurality of proteins in the sample, as measured by a total protein analysis method.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of: a) a signal produced by a higher abundance protein of the plurality of proteins in the one or more additional biomolecule coronas; and b) a signal produced by a lower abundance protein of the plurality of proteins in the one or more additional biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the one or more biomolecule coronas. In some aspects, the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of a top decile of proteins to a bottom decile of proteins in the plurality of proteins in the one or more biomolecule coronas. In some aspects, the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio comprising a span of the interquartile range of proteins in the plurality of proteins in the one or more biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio comprising a slope of fitted data in a plot of all concentrations of proteins in the plurality of proteins in the one or more biomolecule coronas versus known concentrations of the same proteins in the sample.
  • the known concentrations of the same proteins in the sample are obtained from a database.
  • the compressing the dynamic range comprises a decreased additional ratio relative to the second ratio.
  • the decreased first ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, or at least 10-fold less than the second ratio.
  • the total protein analysis method comprises direct quantification of the plurality of proteins in the sample by mass spectrometry, gel electrophoresis, or liquid chromatography.
  • the sample comprises a sample volume of no more than about 1000 ⁇ L, no more than about 900 ⁇ L, no more than about 800 ⁇ L, no more than about 700 ⁇ L, no more than about 600 ⁇ L, no more than about 500 ⁇ L, no more than about 400 ⁇ L, no more than about 300 ⁇ L, no more than about 200 ⁇ L, no more than about 100 ⁇ L, no more than about 50 ⁇ L, no more than about 20 ⁇ L, no more than about 10 ⁇ L, no more than about 5 ⁇ L, no more than about 2 ⁇ L, or no more than about 1 ⁇ L.
  • the sample comprises a biological sample.
  • the biological sample comprises a biofluid.
  • the biofluid is selected from the group consisting of plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, and cell culture samples.
  • the biofluid is plasma or serum.
  • the biofluid is cerebrospinal fluid.
  • the first particle type, the second particle type, the one or more additional particle types, or any combination thereof are selected from TABLE 1.
  • the method further comprises removing high abundance proteins from the sample by depleting or fractioning the sample prior to step a).
  • the one or more additional biomolecule coronas comprises a protein.
  • the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises a lipid, a nucleic acid, a polysaccharide, or any combination thereof.
  • the first particle type, the second particle type, or the one or more additional particle types differ by at least one physicochemical properties.
  • the first particle type and the second particle type differ by at least one physicochemical property and wherein the first biomolecule corona comprises a distinct but overlapping set of proteins relative to the second biomolecule corona.
  • the composition of the plurality of proteins and the concentration of a protein of the plurality of proteins is determined based on the distinct but overlapping set of proteins in the first biomolecule corona and the second biomolecule corona.
  • the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, or any combination thereof.
  • the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is a superparamagnetic particle.
  • the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is selected from TABLE 1.
  • the first particle type and the second particle type are the same particle type and wherein at least 70%, at least 80%, at least 90%, or at least 95% of proteins assayed in the first biomolecule corona and the second biomolecule corona are the same.
  • the first particle type and the second particle type are different and wherein no more than 70%, no more than 80%, no more than 90%, or no more than 95% of proteins assayed in the first biomolecule corona and the second biomolecule corona are the same.
  • the present disclosure provides a high throughput method of analyzing a plurality of biofluids, the method comprising: a) contacting a first biofluid and a second biofluid of the plurality of biofluids with a particle type; b) in a first volume, separating the particle type from a first biofluid of the plurality of biofluids and determining the composition and concentration of a plurality of proteins in a first biomolecule corona corresponding to the particle type; and c) in a second volume, separating the particle type from a second biofluid of the plurality of biofluids and determining the composition and concentration of a plurality of proteins in a second biomolecule corona corresponding to the particle type, wherein the first biofluid and the second biofluid are different biofluids.
  • a biofluid of the plurality of biofluids is selected from the group consisting of plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, and cell culture samples.
  • the biofluid is plasma or serum.
  • the biofluid is cerebrospinal fluid.
  • a biomolecule corona forms upon contacting the particle type with the sample and wherein the first biomolecule corona comprises a protein.
  • the biomolecule corona comprises a lipid, a nucleic acid, a polysaccharide, or any combination thereof.
  • the method further comprises removing high abundance proteins from the sample by depleting or fractioning the sample prior to step a).
  • the particle type is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, or any combination thereof.
  • the particle type is a superparamagnetic particle.
  • the particle type is selected from TABLE 1.
  • the separating comprises magnetic separation, filtration, gravitational separation, or centrifugation.
  • the contacting occurs in vitro.
  • the method further comprises identifying a biological state of the biofluid processing the composition and concentration of the plurality of proteins in the biomolecule corona using a trained algorithm to identify the biological state.
  • the biofluid is isolated from a subject.
  • the subject is a human.
  • the subject is a non-human animal.
  • the subject has a condition.
  • the condition is a disease state.
  • the disease state is cancer.
  • the cancer is prostate cancer, colorectal cancer, lung cancer, or breast cancer.
  • the disease state is Alzheimer's disease.
  • the separating comprises magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density or gradient-based centrifugation, gravitational separation, or any combination thereof.
  • the present disclosure provides a method of analyzing a biofluid, the method comprising: a) contacting a biofluid in a biofluid collection tube with a particle type, wherein the blood collection tube comprises a stabilization reagent, wherein the stabilization reagent stabilizes cell-free nucleic acid molecules in the biofluid; and b) incubating the biofluid and the particle type in the biofluid collection tube to permit binding of proteins of the biofluid to the particle type, thereby forming a biomolecule corona comprising proteins bound to the particle type, wherein the biomolecule corona comprises a population of proteins that is also detected when the particle type is incubated in the biofluid when stored in EDTA and permitted to form a control biomolecule corona; and wherein the biofluid and the particle type are incubated in the biofluid collection tube at from about 20° C. to about 35° C. and wherein 50% or more of the population of proteins of the biomolecule
  • 60% or more, 70% or more, 80% or more, or 90% or more of the population of proteins of the biomolecule corona is also detectable in the control biomolecule corona.
  • at least a subset of the population of proteins is detected at a higher abundance than when the particle type is incubated in the biofluid when stored in EDTA.
  • at least 5% of the population of proteins is detected at a higher abundance than when the particle type is incubated in the biofluid when stored in EDTA.
  • At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the population of proteins is detected at a higher abundance than when the particle type is incubated in the biofluid when stored in EDTA.
  • the method further comprises assaying the proteins in the biomolecule corona at a lower limit of detection than when the particle type is incubated in the biofluid when stored in EDTA.
  • the incubating comprises incubating at room temperature.
  • the incubating comprises incubating for up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, or up to 3 days.
  • the incubating comprises incubating for up to 14 days.
  • the biomolecule corona comprises a unique protein that is absent from the control biomolecule corona.
  • the biomolecule corona comprises at least 100 distinct proteins, at least 200 distinct proteins, at least 300 distinct proteins, at least 400 distinct proteins, at least 500 distinct proteins, at least 1000 distinct proteins, at least 2000 distinct proteins, or at least 5000 distinct proteins.
  • the particle type binds 100 or more distinct proteins.
  • the biofluid comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, or cell culture samples.
  • the biofluid is plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, a fluid from swabbing, a bronchial aspirant, a fluidized solid, a fine needle aspiration sample, a tissue homogenate, or a cell culture sample.
  • the method further comprises separating the particle type from the biofluid. In some aspects, the separating comprises magnetic separation, filtration, gravitational separation, or centrifugation. In some aspects, the method further comprises determining the composition and concentration of proteins in the biomolecule corona. In some aspects, the method further comprises determining the composition and concentration of proteins in the control biomolecule corona. In some aspects, the determining the composition and concentration of proteins comprises mass spectrometry, gel electrophoresis, or liquid chromatography.
  • the stabilization reagent comprises a metabolic inhibitor, a protease inhibitor, a phosphatase inhibitor, a nuclease inhibitor, a preservative agent, or a combination thereof.
  • the metabolic inhibitor comprises glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodium fluoride, or K2C2O4.
  • the protease inhibitor comprises antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, EDTA, pepstatin, VdLPFFVdL, 1,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, or egg white ovostatin egg white cystatin.
  • PMSF phenylmethylsulfonyl fluoride
  • APMSF phenylmethylsulfonyl fluoride
  • TLCK phenylmethylsulfonyl fluoride
  • TPCK TPCK
  • leupeptin soybean tryp
  • the phosphatase inhibitor comprises calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, or sodium orthovanadate.
  • the nuclease inhibitor comprises diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide, vanadyl-ribonucleoside complexes, macaloid, ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl) phosphene hydrochloride, or a divalent cation such as Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, or Cu+2.
  • ATA aurintricarboxylic acid
  • ATA aurintricarboxylic acid
  • ATA aurintricarboxylic acid
  • formamide vanadyl-ribonu
  • the preservative agent comprises diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, or quaternary adamantine.
  • the biofluid collection tube is a blood collection tube.
  • the particle type is a nanoparticle or a microparticle.
  • the particle type is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, and any combination thereof.
  • the particle type is selected from TABLE 1.
  • the method further comprises contacting the biofluid with one or more additional particle types, incubating the biofluid with the one or more additional particle types to permit binding of proteins of the biofluid to the one or more additional particle type, thereby forming one or more additional biomolecule coronas comprising proteins bound to the one or more additional particle types.
  • the one or more additional particle types binds a population of proteins that is distinct but overlapping with the population of proteins in the biomolecule proteins of the particle type.
  • the contacting the biofluid with the one or more additional particle types occurs after the contacting the biofluid with the particle type.
  • the present disclosure analyzes the protein corona from the interaction of nanoparticles and diverse fluids such as CSF, urine, tear and saliva.
  • Various biofluids including CSF, urine, tear and saliva are used to incubate with nanoparticles.
  • the resulted protein coronas were separated and analyzed with mass spectrometry.
  • the present disclosure provides how proteins from diverse biofluids will interact with nanoparticles.
  • the proteins (or biomarkers) identified from these protein corona may be used in the discovery of advanced diagnostic tools as well as therapeutic targets.
  • the present disclosure provides a method of analyzing of a plurality of biofluids, the method comprising: (a) separately contacting each biofluid of the plurality of biofluids with a nanoparticle; (b) isolating the first nanoparticle and analyzing a first protein corona corresponding to a first biofluid of the plurality of biofluids; and (c) isolating the second nanoparticle and analyzing a second protein corona corresponding to a second biofluid of the plurality of biofluids, wherein step (b) and step (c) occur simultaneously.
  • the method further comprises contacting the plurality of biofluids with a third nanoparticle. In some embodiments, the method further comprises contacting the plurality of biofluids with a fourth nanoparticle. In some embodiments, the method further comprises contacting the plurality of biofluids with a fifth nanoparticle. In some embodiments, the first nanoparticle and the second nanoparticle are the same.
  • the method further comprises contacting the plurality of biofluids with up to 10 different nanoparticles.
  • the nanoparticle is made of a material selected from a lipid, a polymer, a metal, or any combination thereof.
  • the polymer comprises PS, PLA, PGA, PLGA, or PVP.
  • the lipid comprises EPC, DOPC, DOPG, or DPPG.
  • the metal comprises iron oxide, gold, or silver.
  • the nanoparticle comprises a positive surface charge. In some embodiments, the nanoparticle comprises a negative surface charge. In some embodiments, the nanoparticle comprises a neutral surface charge.
  • the nanoparticle comprises a size of 1-400 nm.
  • the biofluid comprises plasma, serum, CSF, urine, tear, or saliva.
  • the method further comprises comparing the first protein corona to a first control protein corona identified by contacting the first biofluid with the first nanoparticle. In some embodiments, the method further comprises comparing the second protein corona to a second control protein corona identified by contacting the second biofluid with the second nanoparticle.
  • the method further comprises correlating the first protein corona to a first biological state. In some embodiments, the method further comprises correlating the second protein corona to a second biological state.
  • the present disclosure provides methods and systems for analyzing diverse proteomics.
  • abundant proteins are first removed by fractionation or by nanoparticle depletion, thereby generating a depleted sample.
  • the depleted sample is then incubated with a nanoparticle, which leads to the formation of a protein corona with high affinity low abundant proteins.
  • the methods and systems disclosed herein provided a diverse protein corona profile, which may be used in the discovery of advanced diagnostic tools as well as therapeutic targets.
  • the present disclosure provides a method of identifying a biological state of a sample of a subject, the method comprising: incubating the sample with a depletion nanoparticle, thereby generating a depleted sample; incubating the depleted sample with a first nanoparticle to generate a protein corona; generating protein data corresponding to a protein in the protein corona associated with the first nanoparticle; and processing the protein data using a trained algorithm to identify the biological state of the subject.
  • incubating the sample with the depletion nanoparticle comprises the incubating the sample with the depletion nanoparticle for at least about 30 minutes and extracting the depletion nanoparticle from the sample. In some embodiments, incubating the sample with the depletion nanoparticle for at least about 30 min and extracting the depletion nanoparticle from the sample removes at least about 80% of high abundance proteins from the sample.
  • the method further comprises analyzing a protein corona associated with the depletion nanoparticle.
  • incubating the sample with the depletion nanoparticle comprises incubating the sample with the depletion nanoparticle for at least about 60 minutes and extracting the depletion nanoparticle from the sample at least two times. In some embodiments, incubating the sample with the depletion nanoparticle for at least about 60 minutes and removing the depletion nanoparticle from the sample at least two times removes 99% of high abundance proteins.
  • the depletion nanoparticle comprises at least one different physicochemical property from the first nanoparticle.
  • the different physicochemical property may comprise size, surface charge, or a chemical moiety.
  • processing the protein data comprises analyzing the protein corona, analyzing the sample prior to depletion, or analyzing the depleted sample.
  • the present disclosure provides a method of identifying a biological state of a protein sample from a subject, the method comprising: fractionating the protein sample, thereby generating a fractionated sample comprising a subset of proteins in the protein sample; incubating the depleted sample with a first nanoparticle to generate a protein corona; generating protein data corresponding to a protein of the protein corona associated with the first nanoparticle; and processing the protein data using a trained algorithm to identify the biological state of the subject, wherein fractionating the sample comprises separating high abundance proteins with a method selected from the group consisting of Cohn's method, column chromatograph, ion-exchange chromatography, affinity chromatography, size exclusion chromatography, centrifugation, filtration, ultrafiltration and cryo-precipitation.
  • processing the protein data comprises analyzing the protein corona, analyzing the protein sample prior to fractionation, or analyzing the fractionated sample.
  • a concentration of the first nanoparticle in the depleted sample is from 1-50 mg/mL.
  • generating protein data corresponding to the protein in the protein corona associated with the first nanoparticle comprises determining a concentration of each unique protein in the protein corona and correlating the concentration of each unique protein in the protein corona to the biological state.
  • processing the protein data using a trained algorithm to identify the biological state of the subject comprises associating the biological state with a biomolecule fingerprint.
  • the sample is plasma or serum.
  • the present disclosure provides a method for assaying a biological sample, comprising (a) obtaining a biological sample comprising a plurality of biomolecules, which plurality of biomolecules comprises protein; (b) enriching said biological sample based on one or more features selected from the group consisting of: size, charge, hydrophobicity, structure and affinity, to yield a processed biological sample; (c) generating at least a first subset of biomolecules and a second subset of biomolecules from said processed biological sample of (b) with the aid of one or more nanoparticles, wherein said first subset of biomolecules associates with said one or more nanoparticles and said second subset of biomolecules does not associate with said one or more nanoparticles; and (d) separately conducting one or more assays on at least said first subset of biomolecules or said second subset of biomolecules.
  • the method further comprises (e) isolating said first subset of biomolecules from said second subset of biomolecules.
  • the one or more assays comprise an assay selected from the group consisting of: mass spectrometry and enzyme-linked immunosorbent assay (ELISA). In some embodiments, the one or more assays comprise mass spectrometry.
  • enriching said biological sample comprises fractionation to yield said processed biological sample.
  • the fractionation comprises separating high abundance proteins with a method selected from the group consisting of Cohn's method, ion-exchange chromatography, affinity chromatography, size exclusion chromatography, centrifugation, filtration, ultrafiltration and cryo-precipitation.
  • enriching said biological sample to yield said processed biological sample comprises depleting one or more proteins.
  • one or more depletion nanoparticles are used to yield said processed biological sample.
  • the one or more depletion nanoparticles of (b) are different from said one or more nanoparticles of (c).
  • the one or more assays are conducted on said first subset, which assay is ELISA.
  • the biological sample is a biofluid selected from the group consisting of: plasma, serum, urine, cerebrospinal fluid, tears, saliva, whole blood, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, pericrevicular fluid, semen, prostatic fluid, sputum, synovial fluid, fecal matter, trabecular fluid, bronchial lavage, fluid from swabbings, and bronchial aspirants.
  • a biofluid selected from the group consisting of: plasma, serum, urine, cerebrospinal fluid, tears, saliva, whole blood, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, pericrevicular fluid, semen, prostatic fluid, sputum, synovial fluid
  • the one or more assays yields proteomic data, and further comprising processing said proteomic data to predict a biological state.
  • processing said proteomic data comprises use of a trained algorithm, which trained algorithm is trained using an independent set of biological samples.
  • the first subset of biomolecules associates with said one or more nanoparticles upon incubation with said one or more nanoparticles to form protein coronas.
  • the biological sample is depleted prior to (a).
  • the first subset of biomolecules and said second subset of biomolecules comprise protein.
  • FIG. 1 shows the total number of proteins identified in different cerebral spinal fluid (“CSF”) samples.
  • CSF cerebral spinal fluid
  • FIG. 2A and FIG. 2B show heatmap clustering of CSF protein corona and plasma/serum protein corona demonstrated good separation of the two types of samples such that separate profiles for CSF and plasma/serum were observed.
  • Four samples of serum or plasma protein corona were clustered on the left four nodes of the heatmap. All CSF protein coronas were clustered on the right side of the heatmap.
  • FIG. 3 shows the spectral counts associated with CSF protein corona and plasma protein corona.
  • the proteins shown have known association with neurological disease.
  • Spectral counts for plasma protein corona fall along the x-axis for many proteins listed (shown in hollow red circles).
  • Spectral counts for CSF plasma protein corona are shown with solid black dots.
  • FIG. 4 shows the total number of proteins identified in CSF protein corona derived from different particles. A pooled CSF sample was used for all particles. More than 1000 unique proteins were identified with all particles.
  • FIG. 5 shows the total number of proteins identified in different urine samples.
  • a set of 10 individual urine samples (U1 to U10) and 1 control plasma sample (PC) were interacted with P39 particles to create 11 protein profiles from mass spectrometry analysis.
  • the total number of identified proteins in urine was greater than the total number of proteins identified in the plasma sample.
  • FIG. 6A and FIG. 6B show the proteins identified from multiplexed measurement of protein coronas derived from particles P39, HX42 and HX56.
  • a pooled plasma sample that was diluted 5 ⁇ with TE buffer was used in this experiment for particles and particle combinations.
  • FIG. 6A shows the total number of proteins identified with different conditions. With single particle protein corona (P39, HX42 and HX56), 234, 303 and 353 unique proteins can be identified for the three particles, respectively.
  • FIG. 6B shows the overlap of proteins between particle run separately (“P39+HX42+HX56”), particle mixing (“P39+HX42+HX56 NP Mixing”), and post-digestion mixing (“P39+HX42+HX56 Post Digestion Mixing”).
  • FIG. 7A shows the broad range of the proteome in biofluids. This shows that broad interrogation is difficult and slow. Proteins are ranked based on concentration of identified proteins from plasma or serum extracted from the Plasma Proteome Project as described in Geyer, et al. Mol Syst Biol 13, 942-15 (2017), which is incorporated herein by reference in its entirety.
  • FIG. 7B shows the particle biosensors of the present disclosure (listed on the X-axis label and in a legend to the right of FIG. 7B ) are able to interrogate the proteome with depth, breadth, and efficiency. Each point represents a protein identified in a corona of the corresponding particle and the plasma concentration of that protein as found in literature.
  • Proteins can be identified at a wide range of concentrations in the protein corona of a number of different particle types.
  • the particle biosensors rapidly interrogate the proteome across its wide dynamic range (e.g., by assaying proteins present at concentrations at about 0.001 ug/ml up to almost 10 5 ⁇ g/ml).
  • FIG. 8A - FIG. 8G illustrate various workflows for depletion methods disclosed herein.
  • FIG. 8A illustrates a workflow for serial interrogation of a sample with particles. The sample may be repeatedly interrogated using the same or different particle types.
  • FIG. 8B illustrates a workflow comprising serial interrogation of a sample with a first particle type and a second particle type. The corona of each particle type are analyzed following interrogation.
  • FIG. 8C illustrates a workflow comprising serial interrogation of a sample with a first particle type, a second particle type, and a third particle type. The corona of each particle type are analyzed following interrogation.
  • FIG. 8A illustrates a workflow for serial interrogation of a sample with particles. The sample may be repeatedly interrogated using the same or different particle types.
  • FIG. 8B illustrates a workflow comprising serial interrogation of a sample with a first particle type and a second particle type. The corona of each particle type are analyzed following
  • FIG. 8D illustrates a workflow comprising serial interrogation of a sample with a first particle type, a second particle type, a third particle type, and a fourth particle type.
  • the corona of each particle type are analyzed following interrogation.
  • FIG. 8E illustrates a workflow comprising serial interrogation of a sample with a particle type a first time and a second time. The corona of the particle type are analyzed following each interrogation.
  • FIG. 8F illustrates a workflow comprising serial interrogation of a sample with a particle type a first time, a second time, and a third time. The corona of the particle type are analyzed following each interrogation.
  • FIG. 8G illustrates a workflow comprising serial interrogation of a sample with a particle type a first time, a second time, a third time, and a fourth time. The corona of the particle type are analyzed following each interrogation.
  • FIG. 9 shows a gel of a plasma sample that has been serially interrogated with particles.
  • the sample was interrogated with P39 particles, the particles were extracted, and the resulting corona were run on the gel (P39 Corona 1).
  • the supernatant was interrogated with P39 particles a second time and a third time, the particles were extracted each time, and the resulting corona were run on the gel (P39 Corona 2 and P39 Corona 3).
  • the supernatant was then interrogated with HX-42 particles, the particles were extracted, and the resulting corona were run on the gel (P39 and HX-42 Corona 4).
  • the sample was interrogated with HX-42 particles, the particles were extracted, and the resulting corona were run on the gel (HX-42 Corona 1).
  • the supernatant was interrogated with HX-42 particles a second time and a third time, the particles were extracted each time, and the resulting corona were run on the gel (HX-42 Corona 2 and HX-42 Corona 3).
  • the supernatant was then interrogated with P39 particles, the particles were extracted, and the resulting corona were run on the gel (HX-42 and P39 Corona 4).
  • Separated particles were treated with 8M urea to release proteins from the particles to be run on the gel. Plasma denatured with 8M Urea (HP1 Urea) was run as a control.
  • FIG. 10 shows that protein corona derived from undepleted and depleted plasma had different proteins bound to particles based on gel image.
  • Undepleted or depleted plasma samples were interrogated with P39 particles, and the protein corona were assayed.
  • Red arrows indicate proteins (exemplified by bands) which differ in protein coronas derived from undepleted vs. depleted plasma samples.
  • Particles were treated with 8M urea to release proteins from the particles to be run on the gel.
  • FIG. 11 shows corona analysis experimental results from a single synovial fluid (SF) sample assayed with nine different particles. Specific particle treatments are indicated on the x-axis.
  • a corona analysis (“Proteograph”) assay was also run with synovial fluid that had not been contacted to particles (“neat SF”) as a control. The number of unique proteins identified by all nine particles was also plotted (“All NP”).
  • FIG. 12 shows corona analysis experimental results for two types of samples assayed with particles.
  • Fine needle aspiration (FNA) samples were prepared using 1 mL syringes with 25 G needles. FNA samples were lysed with Mammalian Protein Extraction Reagent (M-PER) and buffer exchanged into TE buffer before corona analysis. Due to the small mass provided by FNA, a single particle (P-039) was used to assess the FNA-corona.
  • Tissue samples were also lysed with M-PER and buffer exchanged to TE buffer before corona analysis. 100 ⁇ L of tissue samples were incubated with 100 ⁇ L of particles to form tissue-particle corona. Peptides were run on an Orbitrap Lumos instrument with 1 hour liquid chromatography (LC) gradient. Corona analysis was also performed on a neat tissue sample that had not been contacted to a particle (“Tissue neat”) as a control.
  • M-PER Mammalian Protein Extraction Reagent
  • FIG. 13 shows corona analysis experimental results from a matched pair of plasma and serum samples assayed with particles.
  • Plasma and serum in 5 ⁇ dilutions (“Plasma 5 ⁇ ” and “Serum 5 ⁇ ,” respectively) and undiluted plasma and serum (“Undiluted Plasma” and “Undiluted Serum,” respectively) were tested. Bars for each particle treatment correspond to, from left to right, Plasma 5 ⁇ , Plasma neat, Serum 5 ⁇ , Serum Neat, and Depleted plasma. Additionally, an aliquot of the plasma sample was deeply depleted with IgY14 and Supermix columns and tested with neat and diluted samples. Each sample was assayed against each of seven particles in separate wells. A set of samples without particles (“No NP”) were also tested as controls.
  • No NP samples without particles
  • FIG. 14 shows results of experiments comparing blood samples collected in EDTA blood collection tubes (“EDTA”) to samples collected in Streck blood collection tubes (“STRECK”). Streck sample treatment was designed to simulate overnight shipping of a sample at ambient temperature. Analysis of recovered peptide mass was performed on blood samples collected from three patients (#1, #2, and #3). Each sample was assayed with particles (“P39 CORONA”) and without particles (“NEAT PLASMA”). Bar height indicates the mean protein mass recovered in each sample. No difference in peptide yield was observed between EDTA and STRECK plasma corona.
  • EDTA EDTA blood collection tubes
  • STRECK Streck blood collection tubes
  • FIG. 15 shows mean number of proteins identified by corona analysis experiments performed on the samples assayed in FIG. 14 . Bar height indicates the mean number of proteins identified in each sample. No difference in number of identified proteins was observed between EDTA and STRECK plasma corona.
  • FIG. 16 shows mean number of peptides detected by corona analysis experiments performed on the samples assayed in FIG. 14 - FIG. 15 . Bar height indicates the mean number of peptides detected in each sample. No difference in number of identified peptides was observed between EDTA and STRECK plasma corona.
  • FIG. 17 shows mean number of features identified by corona analysis experiments performed on the samples assayed in FIG. 14 - FIG. 16 . Bar height indicates the mean number of features identified in each sample. No difference in number of features was observed between EDTA and STRECK plasma corona.
  • FIG. 18 shows a summary of the experimental results presented in FIG. 14 - FIG. 17 .
  • Circles represent the proteins identified in each sample under the indicated condition. Overlapping regions of the circles indicate proteins that were identified in both the corresponding EDTA sample and Streck sample. Non-overlapping regions of the circles indicate proteins that were identified in only the EDTA sample or only the Streck sample assayed under corresponding conditions. Circles positioned toward the left in each pair represent EDTA samples, and circles positioned toward the right in each pair represent Streck samples.
  • FIG. 19 shows numbers of individual proteins of selected protein types identified by corona analysis in each patient sample assayed in FIG. 14 - FIG. 18 .
  • Numbers of proteins of the select protein types identified in each of the three patient samples in only Streck samples assayed with particles (“Streck”) or only EDTA samples assayed with particles (“EDTA”) are plotted.
  • the number of proteins of selected protein type identified in each of the three patient samples in both Streck samples assayed with particles and EDTA samples assayed with particles (“Overlap”) are also plotted.
  • FIG. 20 shows a distribution of protein group intensity for each sample assayed in FIG. 14 - FIG. 19 .
  • FIG. 21 shows the percent coefficient of variation (% CV) of protein group intensities for each sample assayed in FIG. 14 - FIG. 20 .
  • FIG. 22A and FIG. 22B show the number of common and unique proteins identified in each sample assayed in the presence of particles in FIG. 14 - FIG. 21 . Numbers in overlapping regions of each circle indicate proteins that were identified in each sample represented by an overlapping circle.
  • FIG. 22A shows the number of common and unique proteins identified in each patient sample collected in EDTA blood collection tubes
  • FIG. 22B shows the number of common and unique proteins identified in each patient sample collected in Streck blood collection tubes.
  • FIG. 23 shows the coefficient of variation (CV) of protein group intensities, as plotted in FIG. 21 , for each sample assayed in FIG. 14 - FIG. 22 .
  • FIG. 24 shows plots of the intensities of common protein groups identified in samples collected in both Streck blood collection tubes and EDTA blood collection tubes in FIG. 14 - FIG. 23 .
  • Protein group intensities of Streck samples are plotted on the x-axes.
  • Protein group intensities of EDTA samples are plotted on the y-axes.
  • R 2 values (“Rsq”) of the linear fits are provided in the lower right corner of each plot.
  • FIG. 25A and FIG. 25B show numbers of protein groups identified in samples collected in both Streck blood collection tubes and EDTA blood collection tubes in FIG. 14 - FIG. 24 .
  • FIG. 25A shows the number of protein groups containing at least one plasma protein identified in each sample.
  • FIG. 25B shows the number of protein groups identified in each sample that do not contain at least one plasma protein identified in each sample. Classification of proteins as plasma proteins was based on the set of 5,304 plasma proteins identified by Keshishian et al. ( Mol. Cell Proteomics 14, 2375-2393 (2015)), which is incorporated herein by reference in its entirety.
  • FIG. 26 shows the overlap of proteins identified experimentally in biological samples collected into an EDTA sample collection tube (“EDTA”) or a Streck sample collection tube (“Streck”).
  • EDTA EDTA sample collection tube
  • Streck Streck sample collection tube
  • FIG. 27 shows mean protein group intensities of proteins identified in samples collected in EDTA sample collection tubes and samples collected in Streck sample collection tubes. Protein group intensities were plotted for proteins present in the Carr plasma proteome database and identified in both the analyzed in the samples collected in EDTA sample collection tubes (y-axis) and the samples collected in Streck sample collection tubes (x-axis).
  • FIG. 28A and FIG. 28B show Spearman ( FIG. 28A ) and Pearson ( FIG. 28B ) correlation coefficients between the 115 protein groups identified in FIG. 27 .
  • FIG. 29 shows a computer system that is programmed or otherwise configured to implement methods provided herein.
  • FIG. 30A - FIG. 30C show correlation of three protein concentrations as measured by enzyme-linked immunosorbent assay (ELISA) between plasma samples collected in EDTA blood collection tubes (x-axis) and plasma samples collected in Streck sample collection tubes (y-axis).
  • FIG. 30A shows the correlation of C-reactive protein (CRP).
  • FIG. 30B shows the correlation of a Calgranulin A and B heterodimer (“S100”).
  • FIG. 30C shows the correlation of MUC16/CA125 (“CA125”).
  • FIG. 31 shows the percent coefficient of variation (% CV) of CRP (left two box and whiskers), S100 (middle two box and whiskers), and CA125 (right two box and whiskers) of protein concentrations detected by ELISA in plasma samples collected in either EDTA blood collection tubes or Streck blood collection tubes.
  • FIG. 32 shows the matched pair difference in protein concentration detected by ELISA in samples collected in either EDTA blood collection tubes or Streck blood collection tubes for CRP (left plot), S100 (middle plot), and CA125 (right plot).
  • FIG. 33 illustrates examples of methods of processing biological sample collected in EDTA sample collection tubes (left) or sample collection tube comprising a nucleic acid stabilizing agent (right).
  • FIG. 34 shows correlation of the maximum intensities of particle corona proteins and plasma proteins to the published concentration of the same proteins.
  • the blue plotted lines are linear regression models to the data and the shaded regions represent the standard error of the model fit.
  • the dynamic range of the samples assayed with particles (“S-003,” “S-007,” and “S-011”) exhibited a compressed dynamic range as compared to the plasma sample not assayed with particles (“Plasma”), as shown by the decrease in slopes of the linear fits.
  • the slopes of each plot are 0.47, 0.19, 0.22, and 0.18 for, plasma without particles, plasma with S-003 particles, plasma with S-007 particles, and plasma with S-011 particles, respectively.
  • FIG. 35 shows the dynamic range compression of a protein corona analysis assay with mass spectrometry as compared to mass spectrometry without particle corona formation.
  • Protein intensities of common proteins identified in particle corona in the plasma samples assayed in FIG. 34 (“Nanoparticle MS ln Intensity”) are plotted against the protein intensity identified by mass spectrometry of plasma without particles (“Plasma MS ln Intensity”).
  • the lightest dotted line shows a slope of 1, indicating the dynamic range of mass spectrometry without particles.
  • the slopes of the linear fits to the protein intensity was 0.12, 0.36, and 0.093 for S-003, S-007, and S-011 particles, respectively.
  • the grayed area indicates the standard error region of the regression fit.
  • compositions of particles that may be incubated with various biological samples.
  • Various particle types can be incubated with a wide range of biological samples to analyze the proteins present in said biological sample based on binding to particle surface to form protein coronas.
  • a single particle type may be used to assay the proteins in a particular biological sample or multiple particle types can be used together to assay the proteins in the biological sample.
  • a protein corona analysis may be performed on a biological sample (e.g., a biofluid) by contacting the biological sample with a plurality of particles, incubating the biological sample with the plurality of particles to form a protein corona, separating the particles from the biological sample, and analyzing the protein corona to determine the composition of the protein corona.
  • analyzing the protein corona is performed using mass spectrometry. Interrogation of a sample with a plurality of particles followed by analysis of the protein corona formed on the plurality of particles may be referred to herein as “protein corona analysis.”
  • a biological sample may be interrogated with one or more particle types.
  • the protein corona of each particle type may be analyzed separately.
  • the protein corona of one or more particle types may be analyzed in combination.
  • a biological sample may be a biofluid sample such as cerebral spinal fluid (CSF), synovial fluid (SF), urine, plasma, serum, tears, crevicular fluid, semen, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, sweat or saliva.
  • CSF cerebral spinal fluid
  • SF synovial fluid
  • urine plasma
  • serum tears
  • crevicular fluid semen
  • whole blood milk
  • milk nipple aspirate
  • ductal lavage vaginal fluid
  • nasal fluid nasal fluid
  • ear fluid gastric fluid
  • pancreatic fluid pancreatic fluid
  • trabecular fluid trabecular fluid
  • lung lavage prostatic fluid
  • sputum sputum
  • a biofluid may be a fluidized solid, for example a tissue homogenate, or a fluid extracted from a biological sample.
  • a biological sample may be, for example, a tissue sample or a fine needle aspiration (FNA) sample.
  • a biological sample may be a cell culture sample.
  • a biofluid is a fluidized biological sample.
  • a biofluid may be a fluidized cell culture extract.
  • depletion can comprise using one or more particles to deplete a biological sample (e.g., a biofluid) of highly abundant proteins.
  • Depletion of a sample with particles may include one or more rounds of depletion of the sample with a one or more particle types (e.g., a particle with a unique material composition).
  • plasma depletion kits, columns, chromatography, centrifugation, or other systems may be used to deplete a biological sample of highly abundant proteins. Any of these methods of depletion can be followed up by serial interrogation of the sample using the various particle types disclosed herein and corona analysis of said various particle types, as disclosed herein.
  • FIG. 8A illustrates a workflow for serial interrogation of a sample with particles.
  • a sample e.g., a biological sample
  • the sample may be repeatedly interrogated using the same or different particle types.
  • the sample may be depleted or fractioned prior to serial interrogation.
  • Interrogation of the sample with the same particle type or different particle types may be repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.
  • any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more particle types may be the same particle type.
  • any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more particle types may be different particle types.
  • a sample may be interrogated with a particle type by incubating the sample with the particles to form biomolecule corona on the particles.
  • the particles comprising the biomolecule corona may be separated from the sample (e.g., using magnetic separation, centrifugation, filtration, or gravitational separation).
  • the biomolecule corona on the separated particles may be analyzed (e.g., by mass spectrometry, gel electrophoresis, chromatography, spectroscopy, or immunoassays). Protein corona analysis of the biomolecule corona may compress the dynamic range of the analysis compared to a total protein analysis method.
  • FIG. 8B illustrates a workflow comprising serial interrogation of a sample with a first particle type and a second particle type.
  • the first particle type may be a particle selected from TABLE 1.
  • the second particle type may be a particle selected from TABLE 1.
  • the corona of each particle type are analyzed following interrogation.
  • the sample is depleted or fractioned prior to interrogation.
  • FIG. 8C illustrates a workflow comprising serial interrogation of a sample with a first particle type, a second particle type, and a third particle type.
  • the first particle type may be a particle selected from TABLE 1.
  • the second particle type may be a particle selected from TABLE 1.
  • the third particle type may be a particle selected from TABLE 1.
  • the corona of each particle type are analyzed following interrogation.
  • the sample is depleted or fractioned prior to interrogation.
  • FIG. 8D illustrates a workflow comprising serial interrogation of a sample with a first particle type, a second particle type, a third particle type, and a fourth particle type.
  • the first particle type may be a particle selected from TABLE 1.
  • the second particle type may be a particle selected from TABLE 1.
  • the third particle type may be a particle selected from TABLE 1.
  • the fourth particle type may be a particle selected from TABLE 1.
  • the corona of each particle type are analyzed following interrogation.
  • the sample is depleted or fractioned prior to interrogation.
  • FIG. 8E illustrates a workflow comprising serial interrogation of a sample with a particle type a first time and a second time.
  • the particle type may be a particle selected from TABLE 1.
  • the corona of the particle type are analyzed following each interrogation.
  • the sample is depleted or fractioned prior to interrogation.
  • FIG. 8F illustrates a workflow comprising serial interrogation of a sample with a particle type a first time, a second time, and a third time.
  • the particle type may be a particle selected from TABLE 1.
  • the corona of the particle type are analyzed following each interrogation.
  • the sample is depleted or fractioned prior to interrogation.
  • FIG. 8G illustrates a workflow comprising serial interrogation of a sample with a particle type a first time, a second time, a third time, and a fourth time.
  • the particle type may be a particle selected from TABLE 1.
  • the corona of the particle type are analyzed following each interrogation.
  • serial interrogation of a sample may be performed any number of times with any number or types of particle types.
  • Serial interrogation may be performed with a combination of same particle types and different particle types in any order.
  • the present disclosure also provides methods and compositions for fractioning a biological sample, such as a biofluid.
  • a biofluid may be separated into fractions by sequentially contacting the fluid or a supernatant of the fluid to different types of particles.
  • a biofluid may be contacted with a first particle type, and the first particle type may be separated from the biological sample (e.g., by centrifugation or magnetic separation) along with any biological molecules (e.g., proteins) bound to the first particle type.
  • the separated first particle type and bound biological molecules may constitute a first fraction.
  • the remaining biofluid (e.g., a supernatant) may be contacted with a second particle type, and the second particle type may be separated from the biological sample along with any biological molecules bound to the second particle type. This process may be repeated with additional particle types, thereby fractioning the biofluid.
  • a biological sample may be fractionated using a chromatography, centrifugation, precipitation, electrophoresis, or any other method of biochemical separation. After fractioning a sample, the sample may be assayed for proteins using the methods of serial interrogation with various particle types, as disclosed herein.
  • the particles, compositions, and methods disclosed herein are compatible with various sample collection and stabilization methods and compositions.
  • the protein corona analysis methods provided herein may be performed on a stabilized biological sample.
  • a stabilized biological sample may comprise a biological sample (e.g., a biofluid) that has been treated with a stabilizing reagent (e.g., a preservative).
  • a biological sample may be treated with a liquid preservative that maintains cellular antigen expression.
  • the stabilizing reagent may be contained within a sample collection container (e.g., a blood collection tube).
  • the stabilized biological sample may be analyzed by protein corona analysis using any of the particles, compositions, or methods disclosed herein.
  • the stabilized biological sample may be stored unrefrigerated prior to protein corona analysis.
  • particle types consistent with the methods disclosed herein can be made from various materials.
  • particle materials consistent with the present disclosure include metals, polymers, magnetic materials, and lipids.
  • Magnetic particles may be iron oxide particles.
  • metal materials include any one of or any combination of 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. Pat. No. 7,749,299.
  • a particle may be a superparamagnetic iron oxide nanoparticle (SPION).
  • polymers include any one of or any combination of polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, or polyamines, a polyalkylene glycol (e.g., polyethylene glycol (PEG)), a polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), or a copolymer of two or more polymers, such as a copolymer of a polyalkylene glycol (e.g., PEG) and a polyester (e.g., PLGA).
  • the polymer is e.g.
  • particles can be made of any one of or any combination of dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidyl
  • a particle of the present disclosure may be synthesized, or a particle of the present disclosure may be purchased from a commercial vendor.
  • particles consistent with the present disclosure may be purchased from commercial vendors including Sigma-Aldrich, Life Technologies, Fisher Biosciences, nanoComposix, Nanopartz, Spherotech, and other commercial vendors.
  • a particle of the present disclosure may be purchased from a commercial vendor and further modified, coated, or functionalized.
  • a particle of the present disclosure may be a nanoparticle.
  • a nanoparticle of the present disclosure may be from about 10 nm to about 1000 nm in diameter.
  • the nanoparticles 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, from 10 nm to 50 nm, from 50 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 250 nm, from 250 nm to 300 nm, from 300 nm to 350 nm, from 350 nm to 400 nm, from 400 nm to 450 nm, from 450 nm to 500 nm, from 500 nm to 550 nm, from 550 nm to 600 nm, from 600 nm to 650 nm, from 650 nm to 700 nm, from 700 nm to 750 nm
  • a particle of the present disclosure may be a microparticle.
  • a microparticle may be a particle that is from about 1 ⁇ m to about 1000 ⁇ m in diameter.
  • the microparticles disclosed here can be at least 1 ⁇ m, at least 10 ⁇ m, at least 100 ⁇ m, at least 200 ⁇ m, at least 300 ⁇ m, at least 400 ⁇ m, at least 500 ⁇ m, at least 600 ⁇ m, at least 700 ⁇ m, at least 800 ⁇ m, at least 900 ⁇ m, from 10 ⁇ m to 50 ⁇ m, from 50 ⁇ m to 100 ⁇ m, from 100 ⁇ m to 150 ⁇ m, from 150 ⁇ m to 200 ⁇ m, from 200 ⁇ m to 250 ⁇ m, from 250 ⁇ m to 300 ⁇ m, from 300 ⁇ m to 350 ⁇ m, from 350 ⁇ m to 400 ⁇ m, from 400 ⁇ m to 450 ⁇ m, from 450 ⁇ m to 500 ⁇ m, from 500 ⁇ m to
  • a particle of the present disclosure may be contacted with a biological sample (e.g., a biofluid) to form a biomolecule corona.
  • the particle and biomolecule corona may be separated from the biological sample, for example by centrifugation, magnetic separation, filtration, or gravitational separation.
  • the particle types and biomolecule corona may be separated from the biological sample using a number of separation techniques.
  • separation techniques include comprises magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density or gradient-based centrifugation, gravitational separation, or any combination thereof.
  • a protein corona analysis may be performed on the separated particle and biomolecule corona.
  • a protein corona analysis may comprise identifying one or more proteins in the biomolecule corona, for example by mass spectrometry.
  • a single particle type e.g., a particle of a type listed in TABLE 1
  • a plurality of particle types e.g., a plurality of the particle types provided in TABLE 1 may be contacted to a biological sample.
  • the plurality of particle types may be combined and contacted to the biological sample in a single sample volume.
  • the plurality of particle types may be sequentially contacted to a biological sample and separated from the biological sample prior to contacting a subsequent particle type to the biological sample.
  • Protein corona analysis of the biomolecule corona may compress the dynamic range of the analysis compared to a total protein analysis method.
  • the particles of the present disclosure may be used to serially interrogate a sample by incubating a first particle type with the sample to form a biomolecule corona on the first particle type, separating the first particle type, incubating a second particle type with the sample to form a biomolecule corona on the second particle type, separating the second particle type, and repeating the interrogating (by incubation with the sample) and the separating for any number of particle types.
  • the biomolecule corona on each particle type used for serial interrogation of a sample may be analyzed by protein corona analysis.
  • the biomolecule content of the supernatant may be analyzed following serial interrogation with one or more particle types.
  • a particle type of the present disclosure can be used to serially interrogate a sample followed by corona analysis of proteins in the protein corona formed upon incubation of the particle type with the sample.
  • Serial interrogation may be performed with two particle types in a round-by-round fashion.
  • Serial interrogation may also include subsequent interrogation with additional particle times.
  • a particle of the present disclosure may be used to deplete a sample prior to the above described method of serial interrogation.
  • a particle type may be contacted to a sample to form biomolecule corona on a surface of the particle type, and the particle may be separated from the sample, thereby depleting the sample.
  • This strategy may be used to deplete one or more proteins (e.g., one or more high abundance proteins) from a sample.
  • the biomolecule content of the supernatant of a depleted sample may be analyzed.
  • the supernatant of the depleted sample may be used in any of the protein corona analysis methods disclosed herein.
  • An example of a particle type of the present disclosure may be a carboxylate (Citrate) superparamagnetic iron oxide nanoparticle (SPION), a phenol-formaldehyde coated SPION, a silica-coated SPION, a polystyrene coated SPION, a carboxylated poly(styrene-co-methacrylic acid) coated SPION, a N-(3-Trimethoxysilylpropyl)diethylenetriamine coated SPION, a poly(N-(3-(dimethylamino)propyl) methacrylamide) (PDMAPMA)-coated SPION, a 1,2,4,5-Benzenetetracarboxylic acid coated SPION, a poly(Vinylbenzyltrimethylammonium chloride) (PVBTMAC) coated SPION, a carboxylate, PAA coated SPION, a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-coated
  • samples may be assayed in accordance with the methods and compositions of this disclosure.
  • the samples disclosed herein may be analyzed by protein corona analysis after serially interrogating the sample with various particle types.
  • a sample may be fractioned prior to protein corona analysis.
  • a sample may be depleted prior to protein corona analysis.
  • a method of this disclosure may comprise contacting a sample with one or more particle types and performing a protein corona analysis on the sample.
  • a sample may be a biological sample.
  • a biological sample may be a biofluid sample such as cerebrospinal fluid (CSF), synovial fluid (SF), urine, plasma, serum, tear, crevicular fluid, semen, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, sweat or saliva.
  • a biofluid may be a fluidized solid, for example a tissue homogenate, or a fluid extracted from a biological sample.
  • a biological sample may be, for example, a tissue sample or a fine needle aspiration (FNA) sample.
  • a biological sample may be a cell culture sample.
  • a sample that may be used in the methods disclosed herein can either include cells grow in cell culture or can include acellular material taken from cell cultures.
  • a biofluid is a fluidized biological sample.
  • a biofluid may be a fluidized cell culture extract.
  • a sample may be extracted from a fluid sample, or a sample may be extracted from a solid sample.
  • a sample may comprise gaseous molecules extracted from a fluidized solid (e.g., a volatile organic compound).
  • the protein corona analysis methods described herein may comprise assaying proteins in a sample of the present disclosure across a wide dynamic range.
  • the dynamic range of proteins assayed in a sample may be a range of measured signals of protein abundances as measured by an assay method (e.g., mass spectrometry, chromatography, gel electrophoresis, spectroscopy, or immunoassays) for the proteins contained within a sample.
  • an assay capable of detecting proteins across a wide dynamic range may be capable of detecting proteins of very low abundance to proteins of very high abundance.
  • the dynamic range of an assay may be directly related to the slope of assay signal intensity as a function of protein abundance.
  • an assay with a low dynamic range may have a low (but positive) slope of the assay signal intensity as a function of protein abundance, e.g., the ratio of the signal detected for a high abundance protein to the ratio of the signal detected for a low abundance protein may be lower for an assay with a low dynamic range than an assay with a high dynamic range.
  • the protein corona analysis methods described herein may compress the dynamic range of an assay.
  • the dynamic range of an assay may be compressed relative to another assay if the slope of the assay signal intensity as a function of protein abundance is lower than that of the other assay.
  • a plasma sample assayed using protein corona analysis with mass spectrometry may have a compressed dynamic range compared to a plasma sample assayed using mass spectrometry alone, directly on the sample or compared to provided abundance values for plasma proteins in databases (e.g., the database provided in Keshishian et al., Mol. Cell Proteomics 14, 2375-2393 (2015), also referred to herein as the “Carr database”), as shown in FIG. 34 and FIG. 35 .
  • the compressed dynamic range may enable the detection of more low abundance proteins in the plasma sample using protein corona analysis with mass spectrometry than using mass spectrometry alone.
  • Compression of a dynamic range of an assay may enable the detection of low abundance proteins using the methods disclosed herein (e.g., serial interrogation with a particle followed by an assay for quantitating protein abundance such as mass spectrometry).
  • an assay e.g., mass spectrometry
  • an assay may be capable of detecting a dynamic range of 3 orders of magnitude.
  • the assay e.g., mass spectrometry
  • the assay may detect proteins B, C, D, and E.
  • proteins A, B, C, D, and E may have different affinities for the particle surface and may adsorb to the surface of the particle to form the biomolecule corona at different abundancies than present in the sample.
  • proteins A, B, C, D, and E may be present in the biomolecule corona at abundancies of 1 ng/mL, 231 ng/mL, 463 ng/mL, 694 ng/mL, and 926 ng/mL, respectively.
  • using the particles disclosed herein in methods of interrogating a sample results in compressing the dynamic range to 2 orders of magnitude and the resulting assay (e.g., mass spectrometry) can detect all five proteins.
  • a protein corona analysis may be performed in parallel on multiple biological samples by performing each step of a protein corona analysis assay on multiple biological samples at the same time.
  • a protein corona analysis may be performed in parallel on multiple biological samples by performing a first step of a protein corona analysis assay on the multiple biological samples simultaneously, performing a second step of a protein corona analysis assay on the multiple biological samples simultaneously, and performing a third step of a protein corona analysis assay on the multiple biological samples simultaneously.
  • Simultaneous performance of each step of a protein corona analysis on the multiple biological samples may be repeated until all steps of a protein corona analysis assay have been performed on the multiple biological samples.
  • the multiple biological samples may be obtained from the same subject. These samples may be portions of a sample obtained from a subject (e.g., multiple aliquots of a plasma sample). These samples may be different biofluids collected from the same subject.
  • a protein corona analysis may be performed in parallel on multiple biological samples by sequentially performing a first step of a protein corona analysis assay on each sample of a plurality of biological samples, sequentially performing a second step of a protein corona analysis assay on each sample of a plurality of biological samples, and sequentially performing a third step of a protein corona analysis assay on each sample of a plurality of biological samples. Sequential performance of each step of a protein corona analysis may be performed on each sample of a plurality of biological samples may be repeated until all steps of a protein corona analysis assay have been performed on each biological sample of the plurality of biological samples.
  • a protein of interest may be enriched in a biomolecule corona relative to the untreated sample (e.g., a sample that is not assayed using particles).
  • a level of enrichment may be the percent increase or fold increase in concentration of the protein of interest relative to the total protein concentration in the biomolecule corona as compared to the untreated sample.
  • a protein of interest may be enriched in a biomolecule corona by increasing the concentration of the protein of interest in the biomolecule corona as compared to the sample that has not been contacted to a particle.
  • a protein of interest may be enriched by decreasing the concentration of a high abundance protein in the biomolecule corona as compared to the sample that has not been contacted to a particle.
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a biological sample (e.g., a biofluid).
  • a protein corona analysis may identify at least about 500 low abundance proteins in a biological sample in no more than about 8 hours from first contacting the biological sample with a particle.
  • a protein corona analysis may identify at least about 1000 low abundance proteins in a biological sample in no more than about 8 hours from first contacting the biological sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a biological sample in no more than about 4 hours from first contacting the biological sample with a particle.
  • a protein corona analysis may identify at least about 1000 low abundance proteins in a biological sample in no more than about 4 hours from first contacting the biological sample with a particle.
  • a biological sample may be collected in a sample collection tube comprising a stabilizing reagent.
  • the stabilizing reagent may stabilize nucleated cells in a biofluid sample or other biomolecules in the biofluid sample, such as proteins or nucleic acids (e.g., RNA or DNA, including cell free RNA or DNA).
  • a sample collection tube may comprise a metabolic inhibitor, a protease inhibitor, a phosphatase inhibitor, a nuclease inhibitor, a preservative agent, a polyamide, or a combination thereof.
  • a metabolic inhibitor may comprise glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodium fluoride, or K 2 C 2 O 4 .
  • a protease inhibitor may comprise antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, EDTA, pepstatin, VdLPFFVdL, 1,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, or egg white ovostatin egg white cystatin.
  • PMSF phenylmethylsulfonyl fluoride
  • APMSF phenylmethylsulfonyl fluoride
  • TLCK phenylmethylsulfonyl fluoride
  • TPCK TPCK
  • leupeptin soybean try
  • a phosphatase inhibitor may comprise calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, or sodium orthovanadate.
  • a nuclease inhibitor may comprise diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide, vanadyl-ribonucleoside complexes, macaloid, ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl) phosphene hydrochloride, or a divalent cation such as Mg +2 , Mn +2 , Zn +2 , Fe +2 , Ca +2 , or Cu +2 .
  • a preservative agent may comprise diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, or quaternary adamantine.
  • a sample collection tube may comprise stabilizing agents disclosed in U.S. Pat. Nos. 8,304,187, 8,586,306, 9,657,227, 9,926,590, 10,144,955, and 10,294,513, and U.S. application Ser. No. 16/377,344, each of which is incorporated herein by reference in its entirety, and other protein separation techniques.
  • Cerebrospinal Fluid (CSF) Sample The present disclosure provides methods of assaying for proteins in a cerebrospinal fluid sample using one or more of the particles disclosed herein (e.g., and one or more of the particles disclosed in TABLE 1).
  • the relatively low abundance of proteins in CSF may confound protein detection using traditional means (e.g., ELISA, immunofluorescence, gel electrophoresis, or chromatography).
  • the methods and compositions disclosed herein may be well suited for detecting proteins in samples with low protein concentration.
  • the protein corona analysis assays disclosed herein may be capable of detecting proteins in samples with no more than about 1.0 mg/mL, no more than about 0.9 mg/mL, no more than about 0.8 mg/mL, no more than about 0.7 mg/mL, no more than about 0.6 mg/mL, no more than about 0.5 mg/mL, no more than about 0.4 mg/mL, no more than about 0.3 mg/mL, no more than about 0.2 mg/mL, or no more than about 0.1 mg/mL total protein concentration.
  • Sample collection volumes of CSF may be small, ranging from about 1 mL to about 10 mL, limiting the number of assays that may be performed with each sample.
  • the methods and compositions disclosed herein may be well suited for detecting proteins in small sample volumes.
  • the protein corona analysis assays disclosed herein may be capable of detecting proteins in sample volumes of no more than about 1000 ⁇ L, no more than about 900 ⁇ L, no more than about 800 ⁇ L, no more than about 700 ⁇ L, no more than about 600 ⁇ L, no more than about 500 ⁇ L, no more than about 400 ⁇ L, no more than about 300 ⁇ L, no more than about 200 ⁇ L, no more than about 100 ⁇ L, no more than about 50 ⁇ L, no more than about 20 ⁇ L, no more than about 10 ⁇ L, no more than about 5 ⁇ L, no more than about 2 ⁇ L, or no more than about 1 ⁇ L.
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a CSF sample.
  • a protein corona analysis may identify low abundance proteins in a CSF sample in no more than about 2 hours, no more than about 2.5 hours, no more than about 3 hours, no more than about 3.5 hours, no more than about 4 hours, no more than about 4.5 hours, no more than about 5 hours, no more than about 5.5 hours, no more than about 6 hours, no more than about 6.5 hours, no more than about 7 hours, no more than about 7.5 hours, no more than about 8 hours, no more than about 8.5 hours, no more than about 9 hours, no more than about 9.5 hours, or no more than about 10 hours from first contacting the CSF sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a CSF sample in no more than about 8 hours from first contacting the biological sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a CSF sample in no more than about 8 hours from first contacting the CSF sample with a particle. In some embodiments, a protein corona analysis may identify at least about 500 low abundance proteins in a CSF sample in no more than about 4 hours from first contacting the CSF sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a CSF sample in no more than about 4 hours from first contacting the CSF sample with a particle.
  • the methods disclosed herein are superior to other methods of assaying for proteins in CSF and provide sampling of proteins in CSF across a wide dynamic range in a short period of time.
  • the methods disclosed herein utilize one or more of the particles to rapidly enrich for proteins in a CSF sample.
  • one or more of the particle types disclosed herein can be used in a method of assaying for a protein of interest (high abundance protein, medium abundance protein, or low abundance protein) in a CSF sample, the method including incubating said one or more particle types in the CSF sample allowing for formation of a biomolecule corona on the particle and enrichment of proteins in the CSF sample, separating the particle from unbound biomolecules in the CSF sample (e.g., by magnetic separation), and trypsinizing and analyzing the biomolecules in the corona for the protein of interest. Further, using said methods allows for efficient enrichment of the protein of interest in the biomolecule coronas formed around the particle type.
  • a protein of interest may be enriched in a biomolecule corona by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the CSF sample that has not been contacted to a particle.
  • a protein of interest may be enriched in a biomolecule corona by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to the CSF sample that has not been contacted to a particle
  • any one or more of the particles disclosed herein can result in capturing at least about 500, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1000 proteins, at least about 1050 proteins, at least about 1100 proteins, at least about 1150 proteins, or at least about 1200 distinct proteins from a CSF sample.
  • Synovial Fluid Sample The present disclosure provides methods of assaying for proteins in a synovial fluid sample using one or more of the particles disclosed herein (e.g., and one or more of the particles disclosed in TABLE 1).
  • the relatively high concentration of high abundance proteins (e.g., albumin) relative to total protein in SF may confound protein detection using traditional means (e.g., ELISA, immunofluorescence, gel electrophoresis, or chromatography).
  • the methods and compositions disclosed herein may be well suited for detecting low abundance proteins in samples with high concentrations of high abundance proteins.
  • the protein corona analysis assays disclosed herein may be capable of detecting low abundance proteins in samples with at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 70% by weight of a high abundance protein relative to total protein content.
  • the protein corona analysis assays disclosed herein may be performed on a sample comprising a relatively high concentration of high abundance proteins without the need for additional fractionation or depletion steps.
  • Sample collection volumes of SF may be small, ranging from about 2 mL to about 20 mL, limiting the number of assays that may be performed with each sample.
  • the methods and compositions disclosed herein may be well suited for detecting proteins in small sample volumes.
  • the protein corona analysis assays disclosed herein may be capable of detecting proteins in sample volumes of no more than about 1000 ⁇ L, no more than about 900 ⁇ L, no more than about 800 ⁇ L, no more than about 700 ⁇ L, no more than about 600 ⁇ L, no more than about 500 ⁇ L, no more than about 400 ⁇ L, no more than about 300 ⁇ L, no more than about 200 ⁇ L, no more than about 100 ⁇ L, no more than about 50 ⁇ L, or no more than about 20 ⁇ L, no more than about 10 ⁇ L, no more than about 5 ⁇ L, no more than about 2 ⁇ L, or no more than about 1 ⁇ L.
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a SF sample.
  • a protein corona analysis may identify low abundance proteins in a SF sample in no more than about 2 hours, no more than about 2.5 hours, no more than about 3 hours, no more than about 3.5 hours, no more than about 4 hours, no more than about 4.5 hours, no more than about 5 hours, no more than about 5.5 hours, no more than about 6 hours, no more than about 6.5 hours, no more than about 7 hours, no more than about 7.5 hours, no more than about 8 hours, no more than about 8.5 hours, no more than about 9 hours, no more than about 9.5 hours, or no more than about 10 hours from first contacting the SF sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a SF sample in no more than about 8 hours from first contacting the biological sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a SF sample in no more than about 8 hours from first contacting the SF sample with a particle. In some embodiments, a protein corona analysis may identify at least about 500 low abundance proteins in a SF sample in no more than about 4 hours from first contacting the SF sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a SF sample in no more than about 4 hours from first contacting the SF sample with a particle.
  • the methods disclosed herein are superior to other methods of assaying for proteins in synovial fluid and provide sampling of proteins in synovial fluid across a wide dynamic range in a short period of time.
  • the methods disclosed herein utilize one or more of the particles to rapidly enrich for proteins in a synovial fluid sample.
  • one or more of the particle types disclosed herein can be used in a method of assaying for a protein of interest (high abundance protein, medium abundance protein, or low abundance protein) in a synovial fluid sample, the method including incubating said one or more particle types in the synovial fluid sample allowing for formation of a biomolecule corona on the particle and enrichment of proteins in the synovial fluid sample, separating the particle from unbound biomolecules in the synovial fluid sample (e.g., by magnetic separation), and trypsinizing and analyzing the biomolecules in the corona for the protein of interest. Further, using said methods allows for efficient enrichment of the protein of interest in the biomolecule coronas formed around the particle type.
  • a protein of interest may be enriched in a biomolecule corona by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the SF sample that has not been contacted to a particle.
  • a protein of interest may be enriched in a biomolecule corona by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to the SF sample that has not been contacted to a particle
  • any one or more of the particles disclosed herein can result in enrichment of at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2750, at least about 3000, at least about 3250, at least about 3500, at least about 3750, or at least about 4000 from a synovial fluid sample.
  • Urine Sample The present disclosure provides methods of assaying for proteins in a urine sample using one or more of the particles disclosed herein (e.g., and one or more of the particles disclosed in TABLE 1).
  • the relatively low abundance of proteins in urine may confound protein detection using traditional means (e.g., ELISA, immunofluorescence, gel electrophoresis, or chromatography).
  • the methods and compositions disclosed herein may be well suited for detecting proteins in samples with low protein concentration.
  • the protein corona analysis assays disclosed herein may be capable of detecting proteins in samples with no more than about 10 mg/mL, no more than about 5.0 mg/mL, no more than about 4.0 mg/mL, no more than about 3.0 mg/mL, no more than about 2.0 mg/mL, no more than about 1.0 mg/mL, no more than about 0.9 mg/mL, no more than about 0.8 mg/mL, no more than about 0.7 mg/mL, no more than about 0.6 mg/mL, no more than about 0.5 mg/mL, no more than about 0.4 mg/mL, no more than about 0.3 mg/mL, no more than about 0.2 mg/mL, no more than about 0.1 mg/mL, no more than about 0.05 mg/mL, or no more than about 0.02 mg/mL total protein concentration.
  • the relatively high concentration of high abundance proteins (e.g., albumin) relative to total protein in urine may confound protein detection using traditional means.
  • the methods and compositions disclosed herein may be well suited for detecting low abundance proteins in samples with high concentrations of high abundance proteins.
  • the protein corona analysis assays disclosed herein may be capable of detecting low abundance proteins in samples with at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% by weight of a high abundance protein relative to total protein content.
  • the protein corona analysis assays disclosed herein may be performed on a sample comprising a relatively high concentration of high abundance proteins without the need for additional fractionation or depletion steps.
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a urine sample.
  • a protein corona analysis may identify low abundance proteins in a urine sample in no more than about 2 hours, no more than about 2.5 hours, no more than about 3 hours, no more than about 3.5 hours, no more than about 4 hours, no more than about 4.5 hours, no more than about 5 hours, no more than about 5.5 hours, no more than about 6 hours, no more than about 6.5 hours, no more than about 7 hours, no more than about 7.5 hours, no more than about 8 hours, no more than about 8.5 hours, no more than about 9 hours, no more than about 9.5 hours, or no more than about 10 hours from first contacting the urine sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a urine sample in no more than about 8 hours from first contacting the biological sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a urine sample in no more than about 8 hours from first contacting the urine sample with a particle. In some embodiments, a protein corona analysis may identify at least about 500 low abundance proteins in a urine sample in no more than about 4 hours from first contacting the urine sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a urine sample in no more than about 4 hours from first contacting the urine sample with a particle.
  • the methods disclosed herein are superior to other methods of assaying for proteins in urine and provide sampling of proteins in urine across a wide dynamic range in a short period of time.
  • the methods disclosed herein utilize one or more of the particles to rapidly enrich for proteins in a urine sample.
  • one or more of the particle types disclosed herein can be used in a method of assaying for a protein of interest (high abundance protein, medium abundance protein, or low abundance protein) in a urine sample, the method including incubating said one or more particle types in the urine sample allowing for formation of a biomolecule corona on the particle and enrichment of proteins in the urine sample, separating the particle from unbound biomolecules in the urine sample (e.g., by magnetic separation), and trypsinizing and analyzing the biomolecules in the corona for the protein of interest. Further, using said methods allows for efficient enrichment of the protein of interest in the biomolecule coronas formed around the particle type.
  • a protein of interest may be enriched in a biomolecule corona by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the urine sample that has not been contacted to a particle.
  • a protein of interest may be enriched in a biomolecule corona by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to the urine sample that has not been contacted to a particle.
  • any one or more of the particles disclosed herein can result in enrichment of at least about 291, at least about 294, at least about 300, at least about 350, at least about 400, at least about 450, at least about 470, at least about 500, at least about 550, at least about 600, at least about 618, at least about 650, at least about 700, at least about 709, at least about 718, at least about 750, at least about 800, at least about 838, at least about 840, at least about 847, at least about 850, at least about 900, at least about 950, at least about 984, at least about 1000, at least about 1050, at least about 1100, at least about 1150, or at least about 1200 from a urine sample.
  • a fluidized solid e.g., a tissue homogenate
  • a fluidized solid may comprise inhomogeneities, which may confound protein detection using traditional means (e.g., ELISA, immunofluorescence, gel electrophoresis, or chromatography).
  • the methods and compositions disclosed herein may be well suited for detecting proteins in heterogeneous samples.
  • the protein corona analysis assays disclosed herein may be performed on a fluidized solid without the need for additional purification, fractionation, or depletion steps.
  • the methods disclosed herein are superior to other methods of assaying for proteins in fluidized solid and provide sampling of proteins in fluidized solid across a wide dynamic range in a short period of time.
  • the methods disclosed herein utilize one or more of the particles to rapidly enrich for proteins in a fluidized solid sample.
  • one or more of the particle types disclosed herein can be used in a method of assaying for a protein of interest (high abundance protein, medium abundance protein, or low abundance protein) in a fluidized solid sample, the method including incubating said one or more particle types in the fluidized solid sample allowing for formation of a biomolecule corona on the particle and enrichment of proteins in the fluidized solid sample, separating the particle from unbound biomolecules in the fluidized solid sample (e.g., by magnetic separation), and trypsinizing and analyzing the biomolecules in the corona for the protein of interest. Further, using said methods allows for efficient enrichment of the protein of interest in the biomolecule coronas formed around the particle type.
  • a protein of interest may be enriched in a biomolecule corona by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the fluidized solid sample that has not been contacted to a particle.
  • a protein of interest may be enriched in a biomolecule corona by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to the fluidized solid sample that has not been contacted to a
  • any one or more of the particles disclosed herein can result in enrichment of at least about 291, at least about 294, at least about 300, at least about 350, at least about 400, at least about 450, at least about 470, at least about 500, at least about 550, at least about 600, at least about 618, at least about 650, at least about 700, at least about 709, at least about 718, at least about 750, at least about 800, at least about 838, at least about 840, at least about 847, at least about 850, at least about 900, at least about 950, at least about 984, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a fluidized solid sample.
  • a protein corona analysis may identify low abundance proteins in a fluidized solid sample in no more than about 2 hours, no more than about 2.5 hours, no more than about 3 hours, no more than about 3.5 hours, no more than about 4 hours, no more than about 4.5 hours, no more than about 5 hours, no more than about 5.5 hours, no more than about 6 hours, no more than about 6.5 hours, no more than about 7 hours, no more than about 7.5 hours, no more than about 8 hours, no more than about 8.5 hours, no more than about 9 hours, no more than about 9.5 hours, or no more than about 10 hours from first contacting the fluidized solid sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a fluidized solid sample in no more than about 8 hours from first contacting the biological sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a fluidized solid sample in no more than about 8 hours from first contacting the fluidized solid sample with a particle. In some embodiments, a protein corona analysis may identify at least about 500 low abundance proteins in a fluidized solid sample in no more than about 4 hours from first contacting the fluidized solid sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a fluidized solid sample in no more than about 4 hours from first contacting the fluidized solid sample with a particle.
  • the present disclosure provides methods of assaying for proteins in a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample) using one or more of the particles disclosed herein (e.g., and one or more of the particles disclosed in TABLE 1).
  • a cell culture sample may comprise inhomogeneities, which may confound protein detection using traditional means (e.g., ELISA, immunofluorescence, gel electrophoresis, or chromatography).
  • the methods and compositions disclosed herein may be well suited for detecting proteins in heterogeneous samples.
  • the protein corona analysis assays disclosed herein may be performed on a cell culture sample without the need for additional purification, fractionation, or depletion steps.
  • the methods disclosed herein are superior to other methods of assaying for proteins in a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample) and provide sampling of proteins in a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample) across a wide dynamic range in a short period of time.
  • the methods disclosed herein utilize one or more of the particles to rapidly enrich for proteins in a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample).
  • one or more of the particle types disclosed herein can be used in a method of assaying for a protein of interest (high abundance protein, medium abundance protein, or low abundance protein) in a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample), the method including incubating said one or more particle types in the cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample) allowing for formation of a biomolecule corona on the particle and enrichment of proteins in the cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample), separating the particle from unbound biomolecules in the a cell culture sample (a cellular sample, a cell permeated sample, or an extracellular sample) (e.g., by magnetic separation), and trypsinizing and analyzing the biomolecules in the corona for the protein of interest.
  • a protein of interest high abundance protein, medium abundance protein, or low abundance protein
  • a cell culture sample a cellular sample, a
  • a protein of interest may be enriched in a biomolecule corona by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the cell culture sample that has not been contacted to a particle.
  • a protein of interest may be enriched in a biomolecule corona by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold relative to the cell culture sample that has not been contacted to a particle
  • any one or more of the particles disclosed herein can result in capturing at least about 100, at least about 200, at least about 300, at least about 350, at least about 400, at least about 450, at least about 470, at least about 500, at least about 550, at least about 600, at least about 618, at least about 650, at least about 700, at least about 709, at least about 718, at least about 750, at least about 800, at least about 838, at least about 840, at least about 847, at least about 850, at least about 900, at least about 950, at least about 984, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about
  • a protein corona analysis assay may be used to rapidly identify low abundance proteins in a cell culture sample.
  • a protein corona analysis may identify low abundance proteins in a cell culture sample in no more than about 2 hours, no more than about 2.5 hours, no more than about 3 hours, no more than about 3.5 hours, no more than about 4 hours, no more than about 4.5 hours, no more than about 5 hours, no more than about 5.5 hours, no more than about 6 hours, no more than about 6.5 hours, no more than about 7 hours, no more than about 7.5 hours, no more than about 8 hours, no more than about 8.5 hours, no more than about 9 hours, no more than about 9.5 hours, or no more than about 10 hours from first contacting the cell culture sample with a particle.
  • a protein corona analysis may identify at least about 500 low abundance proteins in a cell culture sample in no more than about 8 hours from first contacting the biological sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a cell culture sample in no more than about 8 hours from first contacting the cell culture sample with a particle. In some embodiments, a protein corona analysis may identify at least about 500 low abundance proteins in a cell culture sample in no more than about 4 hours from first contacting the cell culture sample with a particle. In some embodiments, a protein corona analysis may identify at least about 1000 low abundance proteins in a cell culture sample in no more than about 4 hours from first contacting the cell culture sample with a particle.
  • a tissue sample may be collected through a biopsy, dissection, fine needle aspiration, or surgical excision.
  • a tissue sample may be fluidized, for example by homogenization or centrifugation.
  • a fluid sample e.g., blood, serum, plasma, urine, or semen
  • a sample collection tube Commercially available collection tubes may be used for the collection of a fluid sample.
  • collection tubes that may be used to collect a sample of this disclosure include EDTA collection tubes, collection tubes comprising a nucleic acid stabilizing agent (e.g., Streck tubes), serum-separating tubes (SST), sodium citrate collection tubes, citrate, theophylline, adenosine and dipyridamole (CTAD) collection tubes, lithium/sodium heparin collection tubes, sodium fluoride collection tubes, acid citrate dextrose (ACD) collection tubes, or sodium polyanethol sulfonate (SPS) collection tubes.
  • a nucleic acid stabilizing agent e.g., Streck tubes
  • SST serum-separating tubes
  • CAD adenosine and dipyridamole
  • CAD adenosine and dipyridamole
  • lithium/sodium heparin collection tubes lithium/sodium heparin collection tubes
  • sodium fluoride collection tubes e.g., acid citrate dextrose (ACD) collection tubes
  • a biofluid may be collected in a sample collection tube comprising stabilizing reagents (e.g., preservatives, protease inhibitors, phosphatase inhibitors, or crosslinkers).
  • the stabilizing reagents may stabilize the biofluid to be stored unfrozen.
  • the biofluid may be stored at an ambient temperature, at a room temperature, or at any temperature from 0° C. to 37° C.
  • the biofluid collected in the sample collection tube comprising a stabilizing reagent may be stored unfrozen (e.g., at a temperature of from 0° C.
  • the biofluid may be analyzed (e.g., via corona analysis) or frozen for later use.
  • a biofluid sample may be collected in a collection tube comprising a nucleic acid stabilizing agent (e.g., a Streck tube) and stored at room temperature overnight, or the biofluid sample may be collected in the blood collection tube comprising a nucleic acid stabilizing agent and shipped without ice or dry ice.
  • the unfrozen biofluid may be processed at separate facility from where the sample was collected.
  • the biofluids collected in collection tubes and stored unfrozen may be suitable for analysis by protein corona analysis.
  • at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 proteins may be detected in the biofluid using corona analysis.
  • At least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, or at least 5000 peptides may be detected in the biofluid using corona analysis.
  • a frozen biofluid may be shipped on ice, on dry ice, or with liquid nitrogen.
  • the frozen biofluid may be thawed and processed at separate facility from where the sample was collected.
  • a biofluid may be collected in a tube comprising a nucleic acid stabilizing agent (e.g., a Streck tube) at a medical facility and shipped under ambient conditions to a research facility for corona analysis.
  • a nucleic acid stabilizing agent e.g., a Streck tube
  • a biofluid may be collected in a sample collection tube and frozen.
  • a biofluid may be frozen within about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours.
  • Biofluids that have been collected in sample collection tubes and frozen may be suitable for analysis by protein corona analysis.
  • At least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 proteins may be detected in the biofluid using corona analysis.
  • At least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, or at least 5000 peptides may be detected in the biofluid using corona analysis.
  • following storage at a temperature below 0° C. at least 5000, at least 6000, at least 7000, at least 7100, at least 7200, at least 7300, at least 7400, at least 7500, at least 7600, at least 7700, at least 7800, at least 7900, at least 8000, at least 8500, or at least 9000 protein features may be detected in the biofluid using corona analysis.
  • the methods disclosed herein provide for collection of a biofluid in a Streck blood collection tube, incubation with particles to form coronas, and subsequent analysis of the proteins in the coronas at a later time point (e.g., up to 14 days after incubation of said sample in the Streck blood collection tube at room temperature).
  • the particles and methods of use thereof disclosed herein can bind a large number of unique proteins in a biological sample (e.g., a biofluid).
  • biological samples that may be analyzed using the protein corona analysis methods described herein include biofluid samples (e.g., cerebral spinal fluid (CSF), synovial fluid (SF), urine, plasma, serum, tears, semen, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, sweat or saliva), fluidized solids (e.g., a tissue homogenate), or samples derived from cell culture.
  • CSF cerebral spinal fluid
  • SF synovial fluid
  • urine plasma
  • serum serum
  • tears semen
  • whole blood milk
  • a particle disclosed herein can be incubated with any biological sample disclosed herein to form a protein corona comprising at least 100 unique proteins, at least 120 unique proteins, at least 140 unique proteins, at least 160 unique proteins, at least 180 unique proteins, at least 200 unique proteins, at least 220 unique proteins, at least 240 unique proteins, at least 260 unique proteins, at least 280 unique proteins, at least 300 unique proteins, at least 320 unique proteins, at least 340 unique proteins, at least 360 unique proteins, at least 380 unique proteins, at least 400 unique proteins, at least 420 unique proteins, at least 440 unique proteins, at least 460 unique proteins, at least 480 unique proteins, at least 500 unique proteins, at least 520 unique proteins, at least 540 unique proteins, at least 560 unique proteins, at least 580 unique proteins, at least 600 unique proteins, at least 620 unique proteins, at least 640 unique proteins, at least 660 unique proteins, at least 680 unique proteins, at least 700 unique proteins, at least 720 unique proteins, at least 740 unique proteins, at least 760 unique
  • particles can be multiplexed in order to bind and identify large numbers of proteins in a biological sample.
  • Protein corona analysis of the biomolecule corona may compress the dynamic range of the analysis compared to a total protein analysis method.
  • compositions and methods disclosed herein can be used to identify various biological states in a particular biological sample.
  • a biological state can refer to an elevated or low level of a particular protein or a set of proteins.
  • a biological state can refer to identification of a disease, such as cancer.
  • One or more particle types can be incubated with CSF, allowing for formation of a protein corona.
  • Said protein corona can then be analyzed by gel electrophoresis or mass spectrometry in order to identify a pattern of proteins. Analysis of protein corona (e.g., by mass spectrometry or gel electrophoresis) may be referred to as corona analysis.
  • the pattern of proteins can be compared to the same methods carried out on a control sample. Upon comparison of the patterns of proteins, it may be identified that the first CSF sample comprises an elevated level of markers corresponding to a particular type of brain cancer.
  • the particles and methods of use thereof can thus be used to diagnose a particular disease state.
  • dynamic range is the ratio of the highest concentration of a molecule in a sample to the lowest concentration of a molecule in a sample.
  • the dynamic range may be quantified by a number of methods consistent with evaluating the ratio of the highest concentration of a molecule in a sample to the lowest concentration of a molecule in a sample.
  • the dynamic range may be quantified as a ratio of the top decile of proteins in a sample to the bottom decile of proteins in the sample.
  • the dynamic range may be quantified by a ratio of the concentration of the highest concentration detectable in a sample to the lowest concentration detectable in the sample.
  • the dynamic range may be quantified as a span of the interquartile range (a ratio of the 75 th percentile and 25 th percentile) of proteins in a sample.
  • the dynamic range may be quantified by measuring the slope of fitted data in a plot of all concentrations of proteins detected in a sample (e.g., using a method disclosed herein such as serial interrogation with a particle) against published concentrations of said proteins (e.g., using the Carr database of plasma proteins).
  • the dynamic range may be quantified by measuring the slope between any two points in a plot of protein concentration detected in a sample (e.g., using a method disclosed herein such as serial interrogation with a particle) against published concentrations of said protein (e.g., using the Carr database of plasma proteins).
  • the dynamic range captures the range of concentrations, or orders of magnitude, at which a molecule is detectable in the sample.
  • “dynamic range” may refer to the ratio of the highest detectable concentration of a protein in a sample to the lowest detectable concentration of another protein in the sample.
  • the dynamic range captures the range of concentrations, or orders of magnitude, at which proteins are detectable in the sample.
  • the phrase “compressing the dynamic range” or “compression of the dynamic range” refers to a decrease in the ratio of the highest concentration of a molecule in a sample to the lowest concentration of a molecule in a sample.
  • the dynamic range is compressed when the range of concentrations, or orders of magnitude, at which a molecule is detectable in a sample is decreased.
  • a particle type of the present disclosure can be used to serially interrogate a sample. Upon incubation of the particle type in the sample, a biomolecule corona comprising forms on the surface of the particle type.
  • the dynamic range may span a wider range of concentrations, or more orders of magnitude, than if the proteins are directed on the surface of the particle type.
  • using the particle types disclosed herein compresses the dynamic range of proteins in a sample. Without being limited by theory, this effect may be observed due to more capture of higher affinity, lower abundance proteins in the biomolecule corona of the particle type and less capture of lower affinity, higher abundance proteins in the biomolecule corona of the particle type.
  • a dynamic range of a proteomic analysis assay may be the slope of a plot of a protein signal measured by the proteomic analysis assay as a function of total abundance of the protein in the sample.
  • Compressing the dynamic range may comprise decreasing the slope of the plot of a protein signal measured by a proteomic analysis assay as a function of total abundance of the protein in the sample relative to the slope of the plot of a protein signal measured by a second proteomic analysis assay as a function of total abundance of the protein in the sample.
  • the protein corona analysis assays disclosed herein may compress the dynamic range relative to the dynamic range of a total protein analysis method (e.g., mass spectrometry, gel electrophoresis, or liquid chromatography).
  • the dynamic range of a proteomic analysis assay may be the ratio of the signal produced by highest abundance proteins (e.g., the highest 10% of proteins by abundance) to the signal produced by the lowest abundance proteins (e.g., the lowest 10% of proteins by abundance).
  • Compressing the dynamic range of a proteomic analysis may comprise decreasing the ratio of the signal produced by the highest abundance proteins to the signal produced by the lowest abundance proteins for a first proteomic analysis assay relative to that of a second proteomic analysis assay.
  • the protein corona analysis assays disclosed herein may compress the dynamic range relative to the dynamic range of a total protein analysis method (e.g., mass spectrometry, gel electrophoresis, or liquid chromatography).
  • Compressing the dynamic range of a proteomic analysis may be achieved by serial interrogation.
  • a method for compressing the dynamic range of a proteomic analysis may comprise serially interrogating a sample (e.g., a biological sample) with particles and performing a protein corona analysis of the proteomic data captured by the particles.
  • a method for compressing the dynamic range of a proteomic analysis may comprise depleting high abundance proteins from a sample or enriching low abundance proteins in a sample, serially interrogating the sample with a single particle type multiple times or various particle types, and performing a protein corona analysis of the particles.
  • a method for compressing the dynamic range of a proteomic analysis may comprise fractioning a sample and interrogating a fraction that is enriched for low abundance proteins or depleted of high abundance proteins using the methods of serially interrogating the sample with various particle types, as disclosed herein.
  • serial interrogation of a sample may be performed before, after, or in lieu of sample fractionation or sample depletion.
  • serial interrogation methods disclosed herein may increase the detection of low abundance proteins by protein corona analysis.
  • a serial interrogation method may comprise depleting a sample of high abundance proteins that impact the ability to assay for low abundance proteins.
  • the serial interrogation methods disclosed herein may increase the detection of low abundance proteins by protein corona analysis, because the compressed dynamic range of proteins captured in the protein corona allows for locally concentrating a low abundance protein on the particle surface instead of sampling for said low abundance protein directly in the sample where confounding high abundance proteins are present.
  • a serial interrogation method may comprise enriching low abundance proteins in biomolecule corona formed on particles.
  • the methods disclosed herein for serially interrogating a sample with various sample types may be preceded by depletion or fractionation. These methods of depletion can include depletion using a depletion kit. These methods of depletion can also include depletion with a novel particle disclosed herein (e.g., any one of the particles in TABLE 1).
  • a corona analysis after serially interrogating a sample with various particle types may be performed on a biological sample that has been depleted prior to corona analysis.
  • a corona analysis after serially interrogating a sample with various particle types may be performed on individual fractions of fractionated biological sample.
  • a corona analysis after serially interrogating a sample with various particle types may be performed on one or more of the particle types separated from a serially interrogated sample. Fractionation may be used to deplete a sample, and serial interrogation of the sample with various particle types and corona analysis may be performed on the sample that has been fractioned. Fractionation or depletion of a biological sample prior to corona analysis may increase the sensitivity, accuracy, or specificity of the corona analysis.
  • serial interrogation, fractionation, or depletion of a biological sample prior to corona analysis may enable detection of one or more biological molecules that could not be detected in a sample that is directly assayed for total protein content (e.g., by a gel analysis or mass spectrometry).
  • Serial interrogation of a sample with various particle types and corona analysis of an individual fraction of a fractioned sample may enable detection of a desired subset of biological molecules.
  • a sample may be depleted then serially interrogated with various particle types, which are then analyzed by protein corona analysis.
  • a sample may be fractioned, and one or more fractions may be serially interrogated with various particle types, which are then analyzed by protein corona analysis.
  • a sample (e.g., a biological sample) may be serially interrogated with one or more particle types of the present disclosure.
  • a particle type may be separated from the sample, and a protein corona analysis may be performed.
  • Serial interrogation may comprise contacting a sample with a particle type to forming protein corona on the particle, separating the particle type, and repeating the contacting and the separating one or more times on the same sample with the same particle type, a different particle type, or a combination thereof.
  • a protein corona analysis may be performed on one or more of the separated particle types.
  • Serial interrogation of a sample may be well suited for small sample sizes.
  • a protein corona analysis using a panel of particle types may be performed using a sample volume as low as about of no more than about 1000 ⁇ L, no more than about 900 ⁇ L, no more than about 800 ⁇ L, no more than about 700 ⁇ L, no more than about 600 ⁇ L, no more than about 500 ⁇ L, no more than about 400 ⁇ L, no more than about 300 ⁇ L, no more than about 200 ⁇ L, no more than about 100 ⁇ L, no more than about 50 ⁇ L, or no more than about 20 ⁇ L, no more than about 10 ⁇ L, no more than about 5 ⁇ L, no more than about 2 ⁇ L, or no more than about 1 ⁇ L.
  • a panel of particle types e.g., a panel of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more particle types
  • Serial interrogation of a sample may increase the number of proteins that can be identified by protein corona analysis as compared to interrogation of the sample with a single particle type.
  • a biomolecule composition of a sample may not change substantially during serial interrogation.
  • the biomolecule composition of a sample prior to contacting the sample with first particle type may be substantially the same as the biomolecule composition of the sample following separation of the first particle type from the sample.
  • the biomolecule composition of a sample prior to contacting the sample with first particle type may be substantially the same as the biomolecule composition of the sample following separation of the second particle type, the third particle type, the fourth particle type, the fifth particle type, or more particle types, from the sample.
  • the biomolecule composition of the sample may be substantially the same if the total biomolecule mass changes by no more than about 1% by mass, no more than about 2% by mass, no more than about 3% by mass, no more than about 4% by mass, no more than about 5% by mass, no more than about 6% by mass, no more than about 7% by mass, no more than about 8% by mass, no more than about 9% by mass, no more than about 10% by mass, no more than about 11% by mass, no more than about 12% by mass, no more than about 13% by mass, no more than about 14% by mass, or no more than about 15% by mass.
  • the total biomolecule mass of the sample may change by less than about 1% by mass.
  • the biomolecule composition of the sample may be substantially the same if a mass of a subset of biomolecules changes by no more than about 1% by mass, no more than about 2% by mass, no more than about 3% by mass, no more than about 4% by mass, no more than about 5% by mass, no more than about 6% by mass, no more than about 7% by mass, no more than about 8% by mass, no more than about 9% by mass, no more than about 10% by mass, no more than about 11% by mass, no more than about 12% by mass, no more than about 13% by mass, no more than about 14% by mass, or no more than about 15% by mass.
  • the subset of biomolecules may comprise a medium abundance protein. In some embodiments, the subset of biomolecules may comprise a low abundance protein.
  • serial interrogation may be performed by contacting the sample with a first particle type, allowing biological molecules to bind to the first particle type, and separating the first particle type along with the bound biological molecules from the biofluid.
  • the particles may be separated from the biofluid using centrifugation or magnetic separation.
  • the biofluid may be further interrogated by contacting the fluid with a second particle type, allowing biological molecules to bind to the second particle type, and separating the second particle type along with the bound biological molecules from the biofluid.
  • serial interrogation may be performed by sequentially contacting a biological sample with the same or different particle types, allowing biological molecules to bind to the particle type, and separating each particle type and the bound biological molecules before adding the next particle type. Each separated particle type and bound biological molecules may constitute a fraction.
  • serial interrogation may be performed with at least 1, at least 2, 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, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 particle types.
  • serial interrogation with a particle type may be repeated using the sample particle type.
  • corona analysis may be performed on a fraction of the serially interrogated biological sample. In some embodiments, corona analysis may be performed on a remaining supernatant of the biological sample following serial interrogation.
  • Depletion Provided herein are methods of depleting a sample of highly abundant proteins and compositions for doing so. These methods of depleting a sample of high abundance proteins can be carried out prior to the methods described herein of serially interrogating a sample with various particle types, which are then analyzed using the protein corona analysis methods disclosed herein.
  • one or more nanoparticles can be used to deplete a biological sample of highly abundant proteins.
  • Depletion of a sample with particles may include round-by-round depletion of the sample, wherein within each round, a different type of particle (e.g., a particle with a unique material composition) may be used for the depletion.
  • plasma depletion kits, columns, chromatography, or other systems may be used to deplete a biological sample of highly abundant proteins. Depletion followed by serial interrogation of the sample with various particle types and protein corona analysis may increase the number of proteins that can be identified as compared to carrying out the same method in an undepleted sample. However, depletion may not be necessary and serial interrogation of the sample with various particle types and protein corona analysis may be used to identify various proteins in a shorter assay time than if the sample was depleted first.
  • Sample depletion may comprise reducing a level of at least one biological molecule (e.g., a protein) in the biofluid sample.
  • sample depletion may comprise reducing a level of a highly abundant protein in the sample.
  • the highly abundant protein may be albumin, IgG, or a ribosomal protein.
  • sample depletion may be performed by contacting the biofluid with a first particle type, allowing biological molecules to bind to the first particle type, and separating the first particle type along with the bound biological molecules from the biofluid.
  • the particles may be separated from the biofluid using centrifugation or magnetic separation.
  • the biofluid may be further depleted by contacting the fluid with a second particle type, allowing biological molecules to bind to the second particle type, and separating the second particle type along with the bound biological molecules from the biofluid.
  • Depletion may be performed on a biofluid by contacting the biofluid with different particle types and separating each particle type before contacting the biofluid with the next particle type, in a round-by-round manner.
  • depletion may be performed with at least 1, at least 2, 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, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 particle types.
  • sample depletion may be performed using chromatography or electrophoresis.
  • sample depletion may comprise depleting the sample using a commercially available kit.
  • a kit that may be used to deplete a sample may be a spin column-based depletion kit, an albumin depletion kit, an immunodepletion kit, or an abundant protein depletion kit.
  • a kit of the present disclosure may comprise a particle type to serially interrogate a sample.
  • the kit may be pre-packaged in discrete aliquots.
  • the kit can comprise a plurality of different particle types that can be used to serially interrogate a sample.
  • the plurality of particle types can be pre-packaged where each particle type of the plurality is packaged separately.
  • the plurality of particle types can be packaged together to contain combination of particle types in a single package.
  • Depleting a sample may comprise precipitating a component from a sample.
  • depleting a sample may comprise precipitating an undesired component (e.g. a high abundance protein).
  • An undesired component may be precipitated, and the supernatant may be further processed (e.g., by protein corona analysis, serial interrogation, fractionation, or depletion).
  • a precipitated undesired component may be removed from the supernatant by filtration, centrifugation, ultracentrifugation, or gravitational separation.
  • Methods of precipitating an undesired component may comprise cryo-precipitation or chemical precipitation (e.g., Cohn process or ethanol precipitation).
  • depleting a sample may comprise precipitating a desired component (e.g., a low abundance protein).
  • a desired component may be precipitated, the supernatant may be discarded, and the precipitate may be resuspended for further processing (e.g., serial interrogation with various particle types, which are then assayed by protein corona analysis, fractionation, or depletion).
  • a precipitated desired component may be separated from the supernatant by filtration, centrifugation, ultracentrifugation, or gravitational separation.
  • Methods of precipitating a desired component may comprise cryo-precipitation or chemical precipitation (e.g., Cohn process or ethanol precipitation).
  • an untreated sample may comprise an undesired precipitate. An undesired precipitate may be removed from the supernatant by filtration, centrifugation, ultracentrifugation, or gravitational separation.
  • Depleting a sample may comprise separating and undesired component from a desired component (e.g., separating a high abundance protein from a low abundance protein).
  • components may be separated based on a biochemical property (e.g., binding affinity, charge, or size).
  • depletion may be performed using chromatography or gel electrophoresis.
  • a sample may be separated into two or more fractions based on a biochemical property. A first fraction may comprise a desired component, and a second fraction may comprise an undesired component. Fractions comprising desired components may be further processed (e.g., by serial interrogation with various particle types and corona analysis, further fractionation, or depletion).
  • Non-limiting examples of depletion methods may include ion-exchange chromatography, affinity chromatography, size exclusion chromatography, gel electrophoresis, or reverse phase chromatography. While these methods are listed by way of example, one of skill in the art could envision numerous other methods that may be used for sample depletion.
  • depletion can result in removal of at least 50%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100%, from 5% to 20%, from 5% to 100%, from 10% to 95%, from 15% to 90%, from 20% to 85%, from 25% to 80%, from 30% to 75%, from 35% to 70%, from 40% to 65%, from 45% to 60%, from 50% to 55%, from 60% to 70%, from 60% to 80%, or from 50% to 90% of high abundance proteins from a biological sample.
  • fractionation Provided herein are methods of separating components of a sample (e.g., a biological sample) into fractions.
  • a first fraction may differ from a second fraction in biomolecule composition.
  • a first fraction may be enriched in high abundance proteins
  • a second fraction may be enriched in low abundance proteins, relative to the unfractioned sample.
  • a fraction may be further interrogated using the methods and compositions disclosed herein (e.g., serially interrogating the sample followed by protein corona analysis of each particle type used in the serial interrogation).
  • fractionation may facilitate protein corona analysis by separating high abundance proteins from low abundance proteins, thereby increasing the detection of low abundance proteins.
  • fractionation may facilitate protein corona analysis by separating a sample into fractions that are well-suited for a particular particle type. For example, a first fraction may be interrogated with a first particle type, and a second fraction may be interrogated with a second particle type. Fractionation followed by protein corona analysis may increase the number of proteins that can be identified as compared to protein corona analysis of an unfractioned sample.
  • Sample fractionation may comprise separating components of a biological sample.
  • components may be separated based on a biochemical property (e.g., binding affinity, charge, or size).
  • fractionation may be performed using chromatography or gel electrophoresis.
  • One or more fractions may be further processed (e.g., by serially interrogating the sample followed by protein corona analysis of each particle type used in the serial interrogation, further fractionation, or depletion).
  • Non-limiting examples of fractionation methods may include ion-exchange chromatography, affinity chromatography, size exclusion chromatography, gel electrophoresis, or reverse phase chromatography. While these methods are listed by way of example, one of skill in the art could envision numerous other methods that may be used for sample fractionation.
  • the methods disclosed herein include isolating one or more particle types from one or more than one sample (e.g., a biological sample or a serially interrogated sample).
  • the particle types can be rapidly isolated or separated from the sample using a magnetic.
  • multiple samples that are spatially isolated can be processed in parallel.
  • the methods disclosed herein provide for isolating or separating a particle type from unbound protein in a sample.
  • a particle type may be separated by a variety of means, including but not limited to magnetic separation, centrifugation, filtration, or gravitational separation.
  • particle panels may be incubated with a plurality of spatially isolated samples, wherein each spatially isolated sample is in a well in a well plate (e.g., a 96-well plate).
  • a well plate e.g., a 96-well plate
  • the particle types in each of the wells of the well plate can be separated from unbound protein present in the spatially isolated samples by placing the entire plate on a magnet. This simultaneously pulls down the superparamagnetic particles in the particle panel. The supernatant in each sample can be removed to remove the unbound protein.
  • steps incubate, pull down
  • the methods and compositions of the present disclosure provide identification and measurement of particular proteins in the biological samples by processing of the proteomic data via digestion of coronas formed on the surface of particles.
  • proteins that can be identified and measured include highly abundant proteins, proteins of medium abundance, and low-abundance proteins.
  • a low abundance protein may be present in a sample at concentrations at or below about 10 ng/mL.
  • a high abundance protein may be present in a sample at concentrations at or above about 10 ⁇ g/mL.
  • a protein of moderate abundance may be present in a sample at concentrations between about 10 ng/mL and about 10 ⁇ g/mL.
  • proteins that are highly abundant proteins include albumin, IgG, and the top 14 proteins in abundance that contribute 95% of the mass in plasma.
  • any proteins that may be purified using a conventional depletion column may be directly detected in a sample using the particle panels disclosed herein.
  • proteins may be any protein listed in published databases such as Keshishian et al. (Mol Cell Proteomics. 2015 September; 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 August; 11(4):431-48. doi: 10.1586/14789450.2014.901157. Epub 2014 Mar. 24.).
  • proteins that can be measured and identified using the methods and compositions 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.
  • proteins that can be measured and identified using the particle panels disclosed herein are any proteins or protein groups listed in the open targets database for a particular disease indication of interest (e.g., prostate cancer, lung cancer, or Alzheimer's disease).
  • proteomic data of the biological sample can be identified, measured, and quantified using a number of different analytical techniques.
  • proteomic data can be analyzed using SDS-PAGE or any gel-based separation technique.
  • Peptides and proteins can also be identified, measured, and quantified using an immunoassay, such as ELISA.
  • proteomic data can be identified, measured, and quantified using 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.
  • FIG. 29 shows a computer system that is programmed or otherwise configured to implement methods provided herein.
  • the computer system 901 can regulate various aspects of the assays disclosed herein, which are capable of being automated (e.g., movement of any of the reagents disclosed herein on a substrate).
  • the computer system 901 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 901 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 905 , which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 901 also includes memory or memory location 910 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 915 (e.g., hard disk), communication interface 920 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 925 , such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 910 , storage unit 915 , interface 920 and peripheral devices 925 are in communication with the CPU 905 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 915 can be a data storage unit (or data repository) for storing data.
  • the computer system 901 can be operatively coupled to a computer network (“network”) 930 with the aid of the communication interface 920 .
  • the network 930 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 930 in some cases is a telecommunication and/or data network.
  • the network 930 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 930 in some cases with the aid of the computer system 901 , can implement a peer-to-peer network, which may enable devices coupled to the computer system 901 to behave as a client or a server.
  • the CPU 905 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 910 .
  • the instructions can be directed to the CPU 905 , which can subsequently program or otherwise configure the CPU 905 to implement methods of the present disclosure. Examples of operations performed by the CPU 905 can include fetch, decode, execute, and writeback.
  • the CPU 905 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 901 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • the storage unit 915 can store files, such as drivers, libraries and saved programs.
  • the storage unit 915 can store user data, e.g., user preferences and user programs.
  • the computer system 901 in some cases can include one or more additional data storage units that are external to the computer system 901 , such as located on a remote server that is in communication with the computer system 901 through an intranet or the Internet.
  • the computer system 901 can communicate with one or more remote computer systems through the network 930 .
  • the computer system 901 can communicate with a remote computer system of a user.
  • remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system 901 via the network 930 .
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 901 , such as, for example, on the memory 910 or electronic storage unit 915 .
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 905 .
  • the code can be retrieved from the storage unit 915 and stored on the memory 910 for ready access by the processor 905 .
  • the electronic storage unit 915 can be precluded, and machine-executable instructions are stored on memory 910 .
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as 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 the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including 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.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may 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.
  • the computer system 901 can include or be in communication with an electronic display 935 that comprises a user interface (UI) 940 for providing, for example a readout of the proteins identified using the methods disclosed herein.
  • UI user interface
  • Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 905 .
  • Determination, analysis or statistical classification is done by methods known in the art, including, but not limited to, for example, a wide variety of supervised and unsupervised data analysis and clustering approaches such as hierarchical cluster analysis (HCA), principal component analysis (PCA), Partial least squares Discriminant Analysis (PLSDA), machine learning (also known as random forest), logistic regression, decision trees, support vector machine (SVM), k-nearest neighbors, naive bayes, linear regression, polynomial regression, SVM for regression, K-means clustering, and hidden Markov models, among others.
  • HCA hierarchical cluster analysis
  • PCA principal component analysis
  • PLSDA Partial least squares Discriminant Analysis
  • machine learning also known as random forest
  • logistic regression decision trees
  • SVM support vector machine
  • k-nearest neighbors naive bayes
  • linear regression polynomial regression
  • SVM for regression
  • K-means clustering K-means clustering
  • hidden Markov models among others.
  • the computer system can perform various aspects of analyzing the protein sets or protein corona of the present disclosure, such as, for example, comparing/analyzing the biomolecule corona of several samples to determine with statistical significance what patterns are common between the individual biomolecule coronas to determine a protein set that is associated with the biological state.
  • the computer system can be used to develop classifiers to detect and discriminate different protein sets or protein corona (e.g., characteristic of the composition of a protein corona).
  • Data collected from the presently disclosed sensor array can be used to train a machine learning algorithm, specifically an algorithm that receives array measurements from a patient and outputs specific biomolecule corona compositions from each patient. Before training the algorithm, raw data from the array can be first denoised to reduce variability in individual variables.
  • Machine learning can be generalized as the ability of a learning machine to perform accurately on new, unseen examples/tasks after having experienced a learning data set.
  • Machine learning may include the following concepts and methods.
  • Supervised learning concepts may include AODE; Artificial neural network, such as Backpropagation, Autoencoders, Hopfield networks, Boltzmann machines, Restricted Boltzmann Machines, and Spiking neural networks; Bayesian statistics, such as Bayesian network and Bayesian knowledge base; Case-based reasoning; Gaussian process regression; Gene expression programming; Group method of data handling (GMDH); Inductive logic programming; Instance-based learning; Lazy learning;
  • Unsupervised learning concepts may include; Expectation-maximization algorithm; Vector Quantization; Generative topographic map; Information bottleneck method; Artificial neural network, such as Self-organizing map; Association rule learning, such as, Apriori algorithm, Eclat algorithm, and FPgrowth algorithm; Hierarchical clustering, such as Singlelinkage clustering and Conceptual clustering; Cluster analysis, such as, K-means algorithm, Fuzzy clustering, DBSCAN, and OPTICS algorithm; and Outlier Detection, such as Local Outlier Factor.
  • Semi-supervised learning concepts may include; Generative models; Low-density 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 adapted to implement a method described herein.
  • the system includes a central computer server that is programmed to implement the methods described herein.
  • the server includes a central processing unit (CPU, also “processor”) which can be a single core processor, a multi core processor, or plurality of processors for parallel processing.
  • the server also includes memory (e.g., random access memory, read-only memory, flash memory); electronic storage unit (e.g.
  • the memory, storage unit, interface, and peripheral devices are in communication with the processor through a communications bus (solid lines), such as a motherboard.
  • the storage unit can be a data storage unit for storing data.
  • the server is operatively coupled to a computer network (“network”) with the aid of the communications interface.
  • the network can be the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a telecommunication or data network.
  • the network in some cases, with the aid of the server, can implement a peer-to-peer network, which may enable devices coupled to the server to behave as a client or a server.
  • the storage unit can store files, such as subject reports, and/or communications with the data about individuals, or any aspect of data associated with the present disclosure.
  • the computer server can communicate with one or more remote computer systems through the network.
  • the one or more remote computer systems may be, for example, personal computers, laptops, tablets, telephones, Smart phones, or personal digital assistants.
  • the computer system includes a single server. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the internet.
  • the server can be adapted to store measurement data or a database as provided herein, patient information from the subject, such as, for example, medical history, family history, demographic data and/or other clinical or personal information of potential relevance to a particular application. Such information can be stored on the storage unit or the server and such data can be transmitted through a network.
  • Methods as described herein can be implemented by way of machine (or computer processor) executable code (or software) stored on an electronic storage location of the server, such as, for example, on the memory, or electronic storage unit.
  • the code can be executed by the processor.
  • the code can be retrieved from the storage unit and stored on the memory for ready access by the processor.
  • the electronic storage unit can be precluded, and machine-executable instructions are stored on memory.
  • the code can be executed on a second computer system.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, 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 the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming.
  • All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • the physical elements that carry such waves, such as wired or wireless likes, optical links, or the like, also may be considered as media bearing the software.
  • terms such as computer or machine “readable medium” can refer to any medium that participates in providing instructions to a processor for execution.
  • the computer systems described herein may comprise computer-executable code for performing any of the algorithms or algorithms-based methods described herein.
  • the algorithms described herein will make use of a memory unit that is comprised of at least one database.
  • Data relating to the present disclosure can be transmitted over a network or connections for reception and/or review by a receiver.
  • the receiver can be but is not limited to the subject to whom the report pertains; or to a caregiver thereof, e.g., a health care provider, manager, other health care professional, or other caretaker; a person or entity that performed and/or ordered the analysis.
  • the receiver can also be a local or remote system for storing such reports (e.g. servers or other systems of a “cloud computing” architecture).
  • a computer-readable medium includes a medium suitable for transmission of a result of an analysis of a biological sample using the methods described herein.
  • Machine executable code can be stored on an electronic storage unit, such as 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 the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide nontransitory storage at any time for the software programming.
  • All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • the physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software.
  • terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including 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.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may 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.
  • the method of determining a set of proteins associated with the disease or disorder and/or disease state include the analysis of the corona of the at least two samples.
  • This determination, analysis or statistical classification is done by methods known in the art, including, but not limited to, for example, a wide variety of supervised and unsupervised data analysis, machine learning, deep learning, and clustering approaches including hierarchical cluster analysis (HCA), principal component analysis (PCA), Partial least squares Discriminant Analysis (PLS-DA), random forest, logistic regression, decision trees, support vector machine (SVM), k-nearest neighbors, naive bayes, linear regression, polynomial regression, SVM for regression, K-means clustering, and hidden Markov models, among others.
  • HCA hierarchical cluster analysis
  • PCA principal component analysis
  • PLS-DA Partial least squares Discriminant Analysis
  • SVM support vector machine
  • k-nearest neighbors naive bayes
  • linear regression polynomial regression
  • SVM for regression
  • machine learning algorithms are used to construct models that accurately assign class labels to examples based on the input features that describe the example.
  • machine learning can be used to associate the protein corona with various disease states (e.g. no disease, precursor to a disease, having early or late stage of the disease, etc.).
  • one or more machine learning algorithms are employed in connection with a method of the invention to analyze data detected and obtained by the protein corona and sets of proteins derived therefrom.
  • machine learning can be coupled with the sensor array described herein to determine not only if a subject has a pre-stage of cancer, cancer or does not have or develop cancer, but also to distinguish the type of cancer.
  • a method of serially interrogating a sample comprising: a) contacting the sample to a first particle type and incubating the first particle type with the sample to form a first biomolecule corona, wherein the first biomolecule corona forms upon incubation of the first particle type with the sample and wherein the first biomolecule corona comprises a protein; b) separating the first particle type comprising the first biomolecule corona from the sample; c) contacting the sample to a second particle type and incubating the second particle type with the sample to form a second biomolecule corona, wherein the second biomolecule corona forms upon incubation of the second particle type with the sample, wherein the second biomolecule corona comprises a protein, and further wherein (i) the first particle type and the second particle type are a same particle type or (ii) the first particle type and the second particle type are different; d) separating the second particle type comprising the second biomolecule corona from the sample; e) assaying the first biomolecule corona
  • the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the first biomolecule corona. 5. The method of embodiment 2, wherein the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio of a top decile of proteins to a bottom decile of proteins in the plurality of proteins in the first biomolecule corona. 6. The method of embodiment 2, wherein the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio comprising a span of the interquartile range of proteins in the plurality of proteins in the first biomolecule corona. 7.
  • the dynamic range of the plurality of proteins in the first biomolecule corona is a first ratio comprising a slope of fitted data in a plot of all concentrations of proteins in the plurality of proteins in the first biomolecule corona versus known concentrations of the same proteins in the sample.
  • the known concentrations of the same proteins in the sample are obtained from a database.
  • the dynamic range of a plurality of proteins in the sample is a second ratio of: a) a signal produced by a higher abundance protein of the plurality of proteins in the sample, as measured by a total protein analysis method; and b) a signal produced by a lower abundance protein of the plurality of proteins in the sample, as measured by a total protein analysis method.
  • the dynamic range of the plurality of proteins in the sample, as measured by a total protein analysis method is a second ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the sample.
  • the decreased first ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, or at least 10-fold less than the second ratio.
  • the method further comprises: determining the composition and concentration of a plurality of proteins in the second biomolecule corona. 18.
  • the method of any one of embodiments 1-17, wherein the determining the composition and concentration of the plurality of proteins in the first biomolecule corona or determining the composition and concentration of the plurality of proteins in the second biomolecule corona comprises performing mass spectrometry. 19. The method of any one of embodiments 1-18, the method further comprising repeating step a), b), and e) with one or more additional particle types to form one or more additional biomolecule coronas. 20. The method of embodiment 19, wherein the one or more additional particle types are the same as the first particle type or the second particle type. 21. The method of embodiment 19, wherein the one or more additional particle types are different from the first particle type, the second particle type, or a combination thereof 22.
  • the method of any one of embodiments 19-21, wherein the one or more additional biomolecule coronas comprise proteins from the sample.
  • the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises at least 100 distinct proteins.
  • the method of any one of embodiments 19-23, wherein the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises at least 200 distinct proteins, at least 300 distinct proteins, at least 400 distinct proteins, at least 500 distinct proteins, at least 1000 distinct proteins, at least 2000 distinct proteins, or at least 5000 distinct proteins. 25.
  • the method of any one of embodiments 19-24, wherein the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises a different composition of proteins, a different concentration of a subset of proteins, or a combination thereof 26.
  • the method of any one of embodiments 19-25 upon incubating the one or more additional particle types with the sample to form one or more additional biomolecule coronas, a dynamic range of a plurality of proteins in the one or more additional biomolecule coronas is compressed relative to a dynamic range of a plurality of proteins in the sample, as measured by a total protein analysis method. 27.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of: a) a signal produced by a higher abundance protein of the plurality of proteins in the one or more additional biomolecule coronas; and b) a signal produced by a lower abundance protein of the plurality of proteins in the one or more additional biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of a concentration of the highest abundance protein to a concentration of the lowest abundance protein in the plurality of proteins in the one or more biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio of a top decile of proteins to a bottom decile of proteins in the plurality of proteins in the one or more biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio comprising a span of the interquartile range of proteins in the plurality of proteins in the one or more biomolecule coronas.
  • the dynamic range of the plurality of proteins in the one or more additional biomolecule coronas is an additional ratio comprising a slope of fitted data in a plot of all concentrations of proteins in the plurality of proteins in the one or more biomolecule coronas versus known concentrations of the same proteins in the sample.
  • the known concentrations of the same proteins in the sample are obtained from a database.
  • the compressing the dynamic range comprises a decreased additional ratio relative to the second ratio. 34.
  • the decreased first ratio is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, or at least 10-fold less than the second ratio.
  • the total protein analysis method comprises direct quantification of the plurality of proteins in the sample by mass spectrometry, gel electrophoresis, or liquid chromatography.
  • any one of embodiments 1-35 wherein the sample comprises a sample volume of no more than about 1000 ⁇ L, no more than about 900 ⁇ L, no more than about 800 ⁇ L, no more than about 700 ⁇ L, no more than about 600 ⁇ L, no more than about 500 ⁇ L, no more than about 400 ⁇ L, no more than about 300 ⁇ L, no more than about 200 ⁇ L, no more than about 100 ⁇ L, no more than about 50 ⁇ L, no more than about 20 ⁇ L, no more than about 10 ⁇ L, no more than about 5 ⁇ L, no more than about 2 ⁇ L, or no more than about 1 ⁇ L. 37. The method of any one of embodiments 1-36, wherein the sample comprises a biological sample. 38.
  • the biological sample comprises a biofluid.
  • the biofluid is selected from the group consisting of plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, and cell culture samples. 40.
  • the method of any one of embodiments 38-39, wherein the biofluid is plasma or serum. 41. The method of any one of embodiments 38-39, wherein the biofluid is cerebrospinal fluid. 42. The method of any one of embodiments 19-41, wherein the first particle type, the second particle type, the one or more additional particle types, or any combination thereof are selected from TABLE 1. 43. The method of any one of embodiments 1-42, comprising removing high abundance proteins from the sample by depleting or fractioning the sample prior to step a). 44. The method of any one of embodiments 19-43, wherein the one or more additional biomolecule coronas comprises a protein. 45.
  • the method of any one of embodiments 19-44, wherein the first biomolecule corona, the second biomolecule corona, the one or more additional biomolecule coronas, or any combination thereof comprises a lipid, a nucleic acid, a polysaccharide, or any combination thereof 46.
  • 47. The method of any one of embodiments 1-46, wherein the first particle type and the second particle type differ by at least one physicochemical property and wherein the first biomolecule corona comprises a distinct but overlapping set of proteins relative to the second biomolecule corona. 48.
  • composition of the plurality of proteins and the concentration of a protein of the plurality of proteins is determined based on the distinct but overlapping set of proteins in the first biomolecule corona and the second biomolecule corona.
  • the method of any one of embodiments 19-48, wherein the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, or any combination thereof 50.
  • the method of any one of embodiments 1-49, wherein the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is a superparamagnetic particle. 51.
  • the method of any one of embodiments 1-50, wherein the first particle type, the second particle type, the one or more additional particle types, or any combination thereof is selected from TABLE 1. 52. The method of any one of embodiments 1-51, wherein the first particle type and the second particle type are the same particle type and wherein at least 70%, at least 80%, at least 90%, or at least 95% of proteins assayed in the first biomolecule corona and the second biomolecule corona are the same. 53.
  • a high throughput method of analyzing a plurality of biofluids comprising: a) contacting a first biofluid and a second biofluid of the plurality of biofluids with a particle type; b) in a first volume, separating the particle type from a first biofluid of the plurality of biofluids and determining the composition and concentration of a plurality of proteins in a first biomolecule corona corresponding to the particle type; and c) in a second volume, separating the particle type from a second biofluid of the plurality of biofluids and determining the composition and concentration of a plurality of proteins in a second biomolecule corona corresponding to the particle type, wherein the first biofluid and the second biofluid are different biofluids.
  • a biofluid of the plurality of biofluids is selected from the group consisting of plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, and cell culture samples. 56.
  • biofluid is plasma or serum.
  • biofluid is cerebrospinal fluid.
  • a biomolecule corona forms upon contacting the particle type with the sample and wherein the first biomolecule corona comprises a protein.
  • the biomolecule corona comprises a lipid, a nucleic acid, a polysaccharide, or any combination thereof 60.
  • any one of embodiments 54-60 wherein the particle type is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, or any combination thereof 62.
  • the separating comprises magnetic separation, filtration, gravitational separation, or centrifugation.
  • 65 The method of any one of embodiments 1-64, wherein the contacting occurs in vitro.
  • 66 The method of any one of embodiments 38-53 or 55-65, wherein the method further comprises identifying a biological state of the biofluid processing the composition and concentration of the plurality of proteins in the biomolecule corona using a trained algorithm to identify the biological state.
  • the biofluid is isolated from a subject.
  • the subject is a human. 69.
  • the method of embodiment 67 wherein the subject is a non-human animal. 70. The method of any one of embodiments 67-69, wherein the subject has a condition. 71. The method of embodiment 70, wherein the condition is a disease state. 72. The method of embodiment 71, wherein the disease state is cancer. 73. The method of embodiment 72, wherein the cancer is prostate cancer, colorectal cancer, lung cancer, or breast cancer. 74. The method of embodiment 71, wherein the disease state is Alzheimer's disease. 75.
  • the separating comprises magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density or gradient-based centrifugation, gravitational separation, or any combination thereof 76.
  • a method of analyzing a biofluid comprising: a) contacting a biofluid in a biofluid collection tube with a particle type, wherein the blood collection tube comprises a stabilization reagent, wherein the stabilization reagent stabilizes cell-free nucleic acid molecules in the biofluid; and b) incubating the biofluid and the particle type in the biofluid collection tube to permit binding of proteins of the biofluid to the particle type, thereby forming a biomolecule corona comprising proteins bound to the particle type, wherein the biomolecule corona comprises a population of proteins that is also detected when the particle type is incubated in the biofluid when stored in EDTA and permitted to form a control biomolecule corona; and wherein the biofluid and the particle type are incubated in the biofluid collection tube at from about 20° C.
  • the incubating comprises incubating at room temperature.
  • the incubating comprises incubating for up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, or up to 3 days. 84.
  • the method of any one of embodiments 76-83, wherein the incubating comprises incubating for up to 14 days.
  • the biomolecule corona comprises a unique protein that is absent from the control biomolecule corona.
  • the biomolecule corona comprises at least 100 distinct proteins, at least 200 distinct proteins, at least 300 distinct proteins, at least 400 distinct proteins, at least 500 distinct proteins, at least 1000 distinct proteins, at least 2000 distinct proteins, or at least 5000 distinct proteins.
  • the particle type binds 100 or more distinct proteins 88 .
  • biofluid comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, fluid from swabbings, bronchial aspirants, fluidized solids, fine needle aspiration samples, tissue homogenates, or cell culture samples.
  • the biofluid comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter
  • biofluid is plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, crevicular fluid, semen, prostatic fluid, sputum, fecal matter, bronchial lavage, a fluid from swabbing, a bronchial aspirant, a fluidized solid, a fine needle aspiration sample, a tissue homogenate, or a cell culture sample. 90.
  • the method of embodiment 90, wherein separating comprises magnetic separation, filtration, gravitational separation, or centrifugation.
  • 92. The method of any one of embodiments 76-91, further comprising determining the composition and concentration of proteins in the biomolecule corona.
  • 93. The method of any one of embodiments 76-92, further comprising determining the composition and concentration of proteins in the control biomolecule corona.
  • 94 The method of any one of embodiments 76-93, wherein the determining the composition and concentration of proteins comprises mass spectrometry, gel electrophoresis, or liquid chromatography. 95.
  • the stabilization reagent comprises a metabolic inhibitor, a protease inhibitor, a phosphatase inhibitor, a nuclease inhibitor, a preservative agent, or a combination thereof 96.
  • the metabolic inhibitor comprises glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodium fluoride, or K2C2O4. 97.
  • the protease inhibitor comprises antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, EDTA, pepstatin, VdLPFFVdL, 1,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, or egg white ovostatin egg white cystatin.
  • the protease inhibitor comprises antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor
  • phosphatase inhibitor comprises calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, or sodium orthovanadate. 99.
  • nuclease inhibitor comprises diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide, vanadyl-ribonucleoside complexes, macaloid, ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl) phosphene hydrochloride, or a divalent cation such as Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, or Cu+2.
  • the preservative agent comprises diazolidinyl urea, imidazolidinyl urea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethyl glycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, 5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7dioxabicyclo[3.3.0]octane, or quaternary adamantine.
  • the biofluid collection tube is a blood collection tube.
  • the particle type is a nanoparticle or a microparticle.
  • the particle type is selected from the group consisting of a nanoparticle, a microparticle, a micelle, a liposome, an iron oxide particle, a graphene particle, a silica particle, a protein-based particle, a polystyrene particle, a silver particle, a gold particle, a metal particle, a quantum dot, a superparamagnetic particle, and any combination thereof 104.
  • 105. The method of any one of embodiments 76-104, further comprising contacting the biofluid with one or more additional particle types, incubating the biofluid with the one or more additional particle types to permit binding of proteins of the biofluid to the one or more additional particle type, thereby forming one or more additional biomolecule coronas comprising proteins bound to the one or more additional particle types.
  • 106. The method of embodiment 105, wherein the one or more additional particle types binds a population of proteins that is distinct but overlapping with the population of proteins in the biomolecule proteins of the particle type. 107.
  • a method of analyzing of a plurality of biofluids comprising: (a) separately contacting each biofluid of the plurality of biofluids with a nanoparticle (b) isolating the first nanoparticle and analyzing a first protein corona corresponding to a first biofluid of the plurality of biofluids; and (c) isolating the second nanoparticle and analyzing a second protein corona corresponding to a second biofluid of the plurality of biofluids, wherein step (b) and step (c) occur simultaneously.
  • the nanoparticle is made of a material selected from a lipid, a polymer, a metal, or any combination thereof 8.
  • the polymer comprises PS, PLA, PGA, PLGA, or PVP.
  • the lipid comprises EPC, DOPC, DOPG, or DPPG.
  • the metal comprises iron oxide, gold, or silver.
  • the method of embodiment 1, wherein the nanoparticle comprises a positive surface charge.
  • the method of embodiment 1, wherein the nanoparticle comprises a negative surface charge.
  • the method of embodiment 1, wherein the nanoparticle comprises a neutral surface charge.
  • the nanoparticle comprises a size of 1-400 nm. 15.
  • biofluid comprises plasma, serum, CSF, urine, tear, or saliva. 16. The method of embodiment 1, wherein the method further comprises comparing the first protein corona to a first control protein corona identified by contacting the first biofluid with the first nanoparticle. 17. The method of embodiment 1, wherein the method further comprising comparing the second protein corona to a second control protein corona identified by contacting the second biofluid with the second nanoparticle. 18. The method of embodiment 1, wherein the method further comprises correlating the first protein corona to a first biological state. 19. The method of embodiment 1, wherein the method further comprises correlating the second protein corona to a second biological state. 1.
  • a method of identifying a biological state of a sample of a subject comprising: incubating the sample with a depletion nanoparticle, thereby generating a depleted sample; incubating the depleted sample with a first nanoparticle to generate a protein corona; generating protein data corresponding to a protein in the protein corona associated with the first nanoparticle; and processing the protein data using a trained algorithm to identify the biological state of the subject.
  • a method of identifying a biological state of a protein sample from a subject comprising: fractionating the protein sample, thereby generating a fractionated sample comprising a subset of proteins in the protein sample; incubating the depleted sample with a first nanoparticle to generate a protein corona; generating protein data corresponding to a protein of the protein corona associated with the first nanoparticle; and processing the protein data using a trained algorithm to identify the biological state of the subject, wherein fractionating the sample comprises separating high abundance proteins with a method selected from the group consisting of Cohn's method, column chromatograph, ion-exchange chromatography, affinity chromatography, size exclusion chromatography, centrifugation, filtration, ultrafiltration and cryo-precipitation.
  • incubating the sample with the depletion nanoparticle comprises the incubating the sample with the depletion nanoparticle for at least about 30 minutes and extracting the depletion nanoparticle from the sample. 4. The method of embodiment 3, wherein incubating the sample with the depletion nanoparticle for at least about 30 min and the extracting the depletion nanoparticle from the sample removes at least about 80% of high abundance proteins from the sample. 5. The method of embodiment 1, further comprising analyzing a protein corona associated with the depletion nanoparticle. 6.
  • the incubating the sample with the depletion nanoparticle comprises incubating the sample with the depletion nanoparticle for at least about 60 minutes and extracting the depletion nanoparticle from the sample at least two times. 7. The method of embodiment 6, wherein the incubating the sample with the depletion nanoparticle for at least about 60 minutes and removing the depletion nanoparticle from the sample at least two times removes 99% of high abundance proteins. 8. The method of embodiment 1 or 2, wherein a concentration of the first nanoparticle in the depleted sample is from 1-50 mg/mL. 9. The method of embodiment 1 or 2, wherein a concentration of the first nanoparticle in the depleted sample is from 2.5-10 mg/mL. 10.
  • the depletion nanoparticle comprises at least one different physicochemical property from the first nanoparticle.
  • the different physicochemical property comprises size, surface charge, or a chemical moiety.
  • the generating protein data corresponding to the protein in the protein corona associated with the first nanoparticle comprises determining a concentration of each unique protein in the protein corona and correlating the concentration of each unique protein in the protein corona to the biological state.
  • processing the protein data using a trained algorithm to identify the biological state of the subject comprises associating the biological state with a biomolecule fingerprint.
  • the sample is plasma or serum.
  • processing the protein data comprises analyzing the protein corona, analyzing the sample prior to depletion, or analyzing the depleted sample. 16.
  • processing the protein data comprises analyzing the protein corona, analyzing the protein sample prior to fractionation, or analyzing the fractionated sample. 17.
  • a method for assaying a biological sample comprising (a) obtaining a biological sample comprising a plurality of biomolecules, which plurality of biomolecules comprises protein; (b) enriching said biological sample based on one or more features selected from the group consisting of: size, charge, hydrophobicity, structure and affinity, to yield a processed biological sample; (c) generating at least a first subset of biomolecules and a second subset of biomolecules from said processed biological sample of (b) with the aid of one or more nanoparticles, wherein said first subset of biomolecules associates with said one or more nanoparticles and said second subset of biomolecules does not associate with said one or more nanoparticles; and (d) separately conducting one or more assays on at least said first subset of biomolecules or said second subset of biomolecules.
  • the method of embodiment 17, further comprising (e) isolating said first subset of biomolecules from said second subset of biomolecules.
  • said one or more assays comprises an assay selected from the group consisting of: mass spectrometry and enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • said one or more assays comprise mass spectrometry.
  • the enriching said biological sample comprises fractionation to yield said processed biological sample. 22.
  • the fractionation comprises separating high abundance proteins with a method selected from the group consisting of Cohn's method, ion-exchange chromatography, affinity chromatography, size exclusion chromatography, centrifugation, filtration, ultrafiltration and cryo-precipitation.
  • the enriching said biological sample to yield said processed biological sample comprises depleting one or more proteins.
  • the enriching said biological sample to yield said processed biological sample comprises depleting one or more proteins.
  • one or more depletion nanoparticles are used to yield said processed biological sample.
  • said one or more depletion nanoparticles of (b) are different from said one or more nanoparticles of (c). 26.
  • said one or more assays are conducted on said first subset, which assay is ELISA.
  • said biological sample is a biofluid selected from the group consisting of: plasma, serum, urine, cerebrospinal fluid, tears, saliva, whole blood, nipple aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, sweat, pericrevicular fluid, semen, prostatic fluid, sputum, synovial fluid, fecal matter, trabecular fluid, bronchial lavage, fluid from swabbings, and bronchial aspirants.
  • CSF cerebrospinal fluid
  • CSF cerebrospinal fluid
  • a trypsin digestion kit (iST 96X, PreOmics, Germany) was used according to the protocols provided. Briefly, after the last round of wash buffer was pipetted out from the plate, about 50 ⁇ L of lyse buffer was added to each well and the sample was heated at about 95° C. for about 10 minutes with agitation. After cooling down the plates to room temperature, trypsin digest buffer was added and the plate was incubated at about 37° C. for about 3 hours with shaking. The digestion process was stopped with a stop buffer. The supernatant was separated from the particles by a magnetic collector and further cleaned up by a peptide cleanup cartridge included in the kit. The peptide was eluted with about 75 ⁇ L of elution buffer twice and combined. Peptide concentration was measured by a quantitative colorimetric peptide assay kit from Pierce.
  • the peptide eluates were lyophilized and reconstituted in about 0.1% TFA.
  • An about 2 ⁇ g aliquot (based on submitted protein quant values) was analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Fusion Lumos.
  • Peptides were loaded on a trapping column and eluted over a 75 ⁇ m analytical column at 350 nL/min; both columns were packed with Luna C18 resin (Phenomenex).
  • a 2 hour gradient was employed in all cases except for sample 33295 that was also acquired with a 1 hour gradient.
  • the mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 60,000 FWHM resolution and 15,000 FWHM resolution, respectively. APD was turned on. The instrument was run with a 3 second cycle for MS and MS/MS.
  • the mass spectrometry data were processed through the MaxQuant software v1.6.0.16. Swissprot was used to search for protein identifications with 1% protein and peptide false discovery rate (FDR). Peak areas were calculated for detected peptides.
  • the MaxQuant results were parsed into the Scaffold software for visualization.
  • FIG. 1 shows the total number of proteins identified in different CSF samples.
  • a set of 10 individual CSF samples were interacted with P39 to create 10 protein coronas for mass spectrometry analysis.
  • the total number of identified proteins was counted for each sample: for CSF1—about 710 proteins; for CSF2—about 850 proteins; for CSF3—about 620 proteins; for CSF4—about 760 proteins; for CSF5—about 700 proteins; for CSF6—about 620 proteins; for CSF7—about 690 proteins; for CSF8—about 760 proteins; for CSF9—about 710 proteins; and for CSF10—about 640 proteins.
  • the data shows significantly higher number of proteins than obtained with the same particle in plasma.
  • the CSF proteins cluster together because the majority of them are not measurable in plasma.
  • FIG. 2A and FIG. 2B CSF and plasma/serum protein corona demonstrated good separation of the two types of samples.
  • Four samples of serum or plasma protein corona were clustered on the left four nodes of the heatmap. All CSF protein corona was clustered on the right side of the heatmap.
  • FIG. 3 shows the spectral counts associated with CSF protein corona and plasma protein corona.
  • FIG. 4 shows the total number of proteins identified in CSF protein corona derived from different particles. A pooled CSF sample was used for all particles. More than 1000 unique proteins were identified with all particles.
  • This example describes the identification of proteins in urine protein corona.
  • FIG. 5 a substantial number of proteins were identified in each urine sample.
  • a set of 10 individual urine samples (U1 to U10) and 1 control plasma sample (PC) were interacted with P39 to create 11 protein coronas for mass spec analysis.
  • the total number of identified proteins identified in each urine sample was higher than the number of protein identified in plasma for every urine sample tested.
  • This example describes sample preparation for identification of peptide corona.
  • proteomic data from a peptide corona plasma samples were first denatured and reduced before digestion with trypsin. The resulting peptides were incubated with about 100 ⁇ L of paramagnetic particles in microtiter plates. The plates were sealed and incubated at about 37° C. for about 1 hour with shaking at 300 rpm. The peptide corona was washed 3 times with a TE buffer with magnetic separation. Bound peptides were eluted with 1% TFA in 50% of acetonitrile. The peptide eluates were lyophilized and reconstituted in 0.1% TFA before submitting for mass spectrometry analysis.
  • This example describes multiplexing measurement of protein corona.
  • Protein corona can be multiplexed in two different ways: particle mixing and post-digestion mixing.
  • the particles were prepared at about equal concentrations and pre-mixed before being plated to the microtiter plate at about 100 ⁇ L.
  • Biological samples were prepared as usual and mixed with the particles completely by pipetting the mixture up and down for at least about 10 times. The plates were sealed and incubated at about 37° C. for about 1 hour with shaking at 300 rpm. The rest of the assay was performed the same way as CSF protein corona, as described in EXAMPLE 1.
  • the protein corona assay was performed the same way as CSF protein corona, as described above. After digested peptides were eluted out, peptides derived from different particles were combined and mixed before lyophilization. The peptide elutes were lyophilized and reconstituted in 0.1% TFA before submitting for mass spectrometry analysis.
  • FIG. 6A and FIG. 6B show the proteins identified from multiplexing measurement of protein coronas derived from particles P39, HX42 and HX56.
  • a pooled plasma sample that was diluted 5 ⁇ with TE buffer was used in this experiment for particles and particle combinations.
  • FIG. 6A shows the total number of proteins identified with different conditions. With single particle protein corona (P39, HX42 and HX56), 234, 303 and 353 unique proteins can be identified for the three particles, respectively.
  • FIG. 6B shows the overlap of proteins between particle run separately (“P39+HX42+HX56”), particle mixing (“P39+HX42+HX56 NP Mixing”), and post-digestion mixing (“P39+HX42+HX56 Post Digestion Mixing”).
  • This example describes the identification of a variety of proteins in particle protein corona.
  • Proteins identified in the corona of 23 different particle types were compared to the cellular concentration of the identified protein.
  • FIG. 7A shows the broad range of the proteome in biofluids. This shows that broad interrogation is difficult and slow. Proteins are ranked based on concentration of identified proteins from plasma or serum extracted from the Plasma Proteome Project as described in Geyer, et al. Mol Syst Biol 13, 942-15 (2017), which is incorporated herein by reference in its entirety.
  • FIG. 7B shows the particle biosensors of the present disclosure (listed on the X-axis label and in a legend to the right of FIG. 7B ) are able to interrogate the proteome with depth, breadth, and efficiency. Each point represents a protein identified in a corona of the corresponding particle and the plasma concentration of that protein as found in literature.
  • Proteins can be identified at a wide range of concentrations in the protein corona of a number of different particle types.
  • the particle biosensors rapidly interrogate the proteome across its wide dynamic range (e.g., by assaying proteins present at concentrations at about 0.001 ug/ml up to almost 10 5 ⁇ g/ml).
  • This example describes a procedure for particle-based serial interrogation.
  • An example of a workflow for such serial interrogation is provided in TABLE 2. Particle types can be changed depending on experimental design.
  • FIG. 9 shows a gel of a plasma sample that has been serially interrogated with particles.
  • the sample was interrogated with P39 particles, the particles were extracted, and the resulting corona were run on the gel (P39 Corona 1).
  • the supernatant was interrogated with P39 particles a second time and a third time, the particles were extracted each time, and the resulting corona were run on the gel (P39 Corona 2 and P39 Corona 3).
  • the supernatant was then interrogated with HX-42 particles, the particles were extracted, and the resulting corona were run on the gel (P39 and HX-42 Corona 4).
  • the sample was interrogated with HX-42 particles, the particles were extracted, and the resulting corona were run on the gel (HX-42 Corona 1).
  • the supernatant was interrogated with HX-42 particles a second time and a third time, the particles were extracted each time, and the resulting corona were run on the gel (HX-42 Corona 2 and HX-42 Corona 3).
  • the supernatant was then interrogated with P39 particles, the particles were extracted, and the resulting corona were run on the gel (HX-42 and P39 Corona 4).
  • Separated particles were treated with 8M urea to release proteins from the particles to be run on the gel. Plasma denatured with 8M Urea (HP1 Urea) was run as a control.
  • the multiple steps allow for the analysis of more diverse proteins once the abundant proteins are first removed. Different coronas were created during serial interrogation. Change of protein abundance on protein corona was observed even with high abundance proteins in plasma samples.
  • This example describes a depletion assay followed by serial incubation and assaying with particles.
  • the PureProteomeTM Human Albumin/Immunoglobulin depletion kit from EMD Millipore Sigma was used.
  • the original buffer of the beads was removed, the beads were washed twice with TE, and 4 ⁇ diluted plasma was added and vortexed. Then, the beads and plasma were incubated at about room temperature with rotation for about 1 hour. The supernatant was removed.
  • plasma samples were depleted using magnetic beads against albumin and immunoglobulin (Ig).
  • Depleted plasma was depleted using a series of two HPLC columns to (1) deplete most abundant proteins and (2) to deplete moderate abundance proteins.
  • Depleted and undepleted samples were interrogated with particles to form protein corona.
  • FIG. 10 shows that protein corona derived from undepleted and depleted plasma had different proteins bound to particles based on gel image.
  • Undepleted or depleted plasma samples were interrogated with P39 particles, and the protein corona were assayed.
  • Red arrows indicate proteins (exemplified by bands) which differ in protein coronas derived from undepleted vs. depleted plasma samples.
  • Particles were treated with 8M urea to release proteins from the particles to be run on the gel.
  • This example describes a procedure for depletion with spin columns.
  • the depletion spin column is equilibrated to about room temperature, 100 ⁇ L of sample is directly added to the resin slurry in the column, the mixture is incubated in the column with gentle end-over-end mixing for about 10 minutes at about room temperature, and then the mixture is centrifuged at 1,000 ⁇ g for about 2 minutes. Then, the flow through is collected.
  • This example describes protein depletion and coronal analysis of synovial fluid samples.
  • Synovial fluid (SF) samples were incubated with particles to form particle protein corona. 100 ⁇ L of sample was incubated with 100 ⁇ L of particles for 1 hour. Following incubation, particles were pelleted using a magnet. Supernatants were removed using a pipette. The pellet was washed with PBS and re-pelleted three times. Proteins bound to the particles were digested with a trypsin digestion kit (iST 96X, PreOmics, Germany) according to the protocols provided in the kit and summarized in EXAMPLE 1. Peptide concentrations were measured by a quantitative colorimetric peptide assay kit from Thermo Fisher Scientific. Mass spectrometry was performed on the eluted peptides following the method described in EXAMPLE 1.
  • FIG. 11 shows corona analysis experimental results from a single synovial fluid (SF) sample assayed with nine different particles. Specific particle treatments are indicated on the x-axis.
  • a corona analysis (“Proteograph”) assay was also run with synovial fluid that had not been contacted to particles (“neat SF”) as a control. The number of unique proteins identified by all nine particles was also plotted (“All NP”).
  • Protein corona were prepared and analyzed for tissue samples and fine needle aspiration (FNA) samples as described in EXAMPLE 9 with respect to synovial fluid samples.
  • FNA fine needle aspiration
  • FIG. 12 shows corona analysis experimental results for two types of samples assayed with particles.
  • Fine needle aspiration (FNA) samples were prepared using 1 mL syringes with 25 G needles. FNA samples were lysed with Mammalian Protein Extraction Reagent (M-PER) and buffer exchanged into TE buffer before corona analysis. Due to the small mass provided by FNA, a single particle (P-039) was used to assess the FNA-corona.
  • Tissue samples were also lysed with M-PER and buffer exchanged to TE buffer before corona analysis. 1004 of tissue samples were incubated with 1004 of particles to form tissue-particle corona. Peptides were run on an Orbitrap Lumos instrument with 1 hour liquid chromatography (LC) gradient. Corona analysis was also performed on a neat tissue sample that had not been contacted to a particle (“Tissue neat”) as a control.
  • M-PER Mammalian Protein Extraction Reagent
  • Protein corona were prepared and analyzed for matched plasma samples and serum samples as described in EXAMPLE 9 with respect to synovial fluid samples. Depleted plasma was depleted using a series of two HPLC columns to (1) deplete most abundant proteins and (2) to deplete moderate abundance proteins.
  • FIG. 13 shows corona analysis experimental results from a matched pair of plasma and serum samples assayed with particles.
  • Plasma and serum in 5 ⁇ dilutions (“Plasma 5 ⁇ ” and “Serum 5 ⁇ ,” respectively) and undiluted plasma and serum (“Undiluted Plasma” and “Undiluted Serum,” respectively) were tested. Bars for each particle treatment correspond to, from left to right, Plasma 5 ⁇ , Plasma neat, Serum 5 ⁇ , Serum Neat, and Depleted plasma. Additionally, an aliquot of the plasma sample was deeply depleted with IgY14 and Supermix columns and tested with neat and diluted samples. Each sample was assayed against each of seven particles in separate wells. A set of samples without particles (“No NP”) were also tested as controls.
  • No NP samples without particles
  • This example describes protein corona analysis of samples collected in blood collection tubes comprising a nucleic acid stabilizing agent.
  • FIG. 33 summarizes the methods of processing biological sample collected in EDTA sample collection tubes (left) or Streck sample collection tubes (right).
  • FIG. 14 shows results of experiments comparing blood samples collected in EDTA blood collection tubes (“EDTA”) to samples collected in Streck blood collection tubes (“STRECK”). Analysis of recovered peptide mass was performed on blood samples collected from three patients (#1, #2, and #3). Each sample was assayed with particles (“P39 CORONA”) and without particles (“NEAT PLASMA”). Bar height indicates the mean protein mass recovered in each sample. No different in peptide yield was observed between EDTA and STRECK plasma corona. Both EDTA and Streck plasma samples that had not been contacted to particles yielded similar peptide mass. Streck plasma samples assayed with particles also yielded similar peptide mass to EDTA plasma samples assayed with particles given the typical coefficient of variation of peptide quantification observed (about 10% to 15%).
  • EDTA EDTA blood collection tubes
  • STRECK Streck blood collection tubes
  • FIG. 15 shows mean number of proteins identified by corona analysis experiments performed on the samples. Bar height indicates the mean number of proteins identified in each sample. No difference in number of identified proteins was observed between EDTA and STRECK plasma corona. Similar numbers of proteins were identified in both EDTA and Streck plasma samples that had not been contacted to particles. More proteins were identified in Streck plasma samples assayed with particles as compared to EDTA plasma samples assayed with particles.
  • FIG. 16 shows mean number of peptides detected by corona analysis experiments performed on the samples. Bar height indicates the mean number of peptides detected in each sample. No difference in number of identified peptides was observed between EDTA and STRECK plasma corona. More peptides were detected in Streck plasma samples assayed with particles as compared to EDTA plasma samples assayed with particles.
  • FIG. 17 shows mean number of features identified by corona analysis experiments performed on the samples. Bar height indicates the mean number of features identified in each sample. No difference in number of features was observed between EDTA and STRECK plasma corona. A similar number of features was detected in Streck plasma samples assayed with particles as compared to EDTA plasma samples assayed with particles.
  • FIG. 18 shows a summary of the experimental results presented in FIG. 14 - FIG. 17 .
  • Circles represent the proteins identified in each sample under the indicated condition. Overlapping regions of the circles indicate proteins that were identified in both the corresponding EDTA sample and Streck sample. Non-overlapping regions of the circles indicate proteins that were identified in only the EDTA sample or only the Streck sample assayed under corresponding conditions. Circles positioned toward the left in each pair represent EDTA samples, and circles positioned toward the right in each pair represent Streck samples. Each patient sample (#1, #2, and #3) resulted in similar numbers of common proteins between the Streck samples assayed in the absence of particles and the EDTA samples assayed in the absence of particles.
  • FIG. 19 shows numbers of individual proteins of selected protein types identified by corona analysis in each patient sample. Numbers of proteins of the select protein types identified in each of the three patient samples in only Streck samples assayed with particles (“Streck”) or only EDTA samples assayed with particles (“EDTA”) are plotted. The number of proteins of selected protein type identified in each of the three patient samples in both Streck samples assayed with particles and EDTA samples assayed with particles (“Overlap”) are also plotted. Numbers of identified glycoproteins, nuclear-related proteins, DNA-related proteins, and RNA-related proteins were the highest among the “Overlap” data which demonstrated common proteins shared by Streck samples and EDTA samples.
  • FIG. 20 shows a distribution of protein group intensity for the samples plotted in FIG. 14 - FIG. 19 .
  • EDTA and STRECK plasma samples showed similar peptide yields, protein counts, peptide counts, feature counts and protein categories when they were assayed with or without particles in, indicating the suitability of samples collected in sample collection tubes comprising a nucleic acid stabilizing agent in protein corona analysis assays.
  • FIG. 21 shows the percent coefficient of variation (% CV) of protein group intensities for the samples plotted in FIG. 14 - FIG. 20 .
  • EDTA and STRECK plasma samples showed similar reproducibility when they were assayed with or without particles in, indicating the suitability of samples collected in sample collection tubes comprising a nucleic acid stabilizing agent in protein corona analysis assays.
  • FIG. 22A and FIG. 22B show the number of common and unique proteins identified in the samples plotted in FIG. 14 - FIG. 21 assayed in the presence of particles. Numbers in overlapping regions of each circle indicate proteins that were identified in each sample represented by an overlapping circle.
  • FIG. 22A shows the number of common and unique proteins identified in each patient sample collected in EDTA blood collection tubes
  • FIG. 22B shows the number of common and unique proteins identified in each patient sample collected in Streck blood collection tubes. Samples collected in Streck blood collection tubes and EDTA blood collection tubes demonstrated similar consistency among replicate samples.
  • FIG. 23 shows the coefficient of variation (CV) of protein group intensities for the samples plotted in FIG. 14 - FIG. 22 .
  • FIG. 24 shows plots of the intensities of common protein groups identified in samples collected in both Streck blood collection tubes (STRECK Product #218962) and EDTA blood collection tubes and plotted in FIG. 14 - FIG. 23 .
  • Protein group intensities of Streck samples are plotted on the x-axes.
  • Protein group intensities of EDTA samples are plotted on the y-axes.
  • R 2 values (“Rsq”) of the linear fits are provided in the lower right corner of each plot.
  • FIG. 25A and FIG. 25B show numbers of protein groups identified in the samples collected in both Streck blood collection tubes and EDTA blood collection tubes in FIG. 14 - FIG. 24 .
  • FIG. 25A shows the number of protein groups containing at least one plasma protein identified in each sample.
  • FIG. 25B shows the number of protein groups identified in each sample that do not contain at least one plasma protein identified in each sample. Classification of proteins as plasma proteins was based on the set of 5,304 plasma proteins identified by Keshishian et al. ( Mol. Cell Proteomics 14, 2375-2393 (2015)), which is incorporated herein by reference in its entirety.
  • This example describes protein corona analysis of lung cancer samples collected in tubes comprising a nucleic acid stabilizing agent.
  • Samples were collected from subjects participating in a lung cancer study. Samples from each subject were collected in EDTA plasma collection tubes and Streck plasma collection tubes. Collected samples were processed for protein corona analysis. Samples collected in EDTA sample collection tubes were analyzed on site. Samples collected in Streck sample collection tubes were shipped overnight and analyzed at a remote facility. Samples from 20 subjects were analyzed with a single particle type. Each sample was assayed in triplicate. MS analysis was performed on each sample and replicates using a TF Lumos with 1 hour data-dependent acquisition (DDA) runs.
  • DDA data-dependent acquisition
  • FIG. 26 shows the overlap of proteins identified experimentally in biological samples collected into an EDTA sample collection tube (“EDTA”) or a Streck sample collection tube (“Streck”). While more proteins were identified in samples collected in EDTA tubes, the proteins identified in samples collected in Streck tubes almost completely overlapped with the proteins identified in the samples collected in EDTA tubes.
  • EDTA EDTA sample collection tube
  • Streck Streck sample collection tube
  • FIG. 27 shows mean protein group intensities of proteins identified in samples collected in EDTA sample collection tubes and samples collected in Streck sample collection tubes. Protein group intensities were plotted for proteins present in the Carr plasma proteome database and identified in both the analyzed in the samples collected in EDTA sample collection tubes (y-axis) and the samples collected in Streck sample collection tubes (x-axis).
  • FIG. 28A and FIG. 28B show Spearman ( FIG. 28A ) and Pearson ( FIG. 28B ) correlation coefficients between the 115 protein groups identified in FIG. 27 . Both the Spearman and Pearson correlation coefficients indicate a strong positive correlation in protein group intensity between samples collected in EDTA tubes and samples collected in Streck tubes. Together these results indicated that collection of biological samples in Streck tubes are suitable for large-scale proteomic profiling using particle sample preparation methods.
  • This example describes corona analysis via serial interrogation of a cerebrospinal fluid sample.
  • a cerebrospinal fluid sample is collected from a subject.
  • a first particle type selected from the particles provided in TABLE 1 is added to 100 ⁇ L of the cerebrospinal fluid sample and incubated to form protein corona.
  • the first particle type is separated from the cerebrospinal fluid sample by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a second particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the second particle type is the same as the first particle type.
  • the second particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a third particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the third particle type is the same as the first particle type.
  • the third particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • the supernatant is re-interrogated with up to ten particle types selected from the particles provided in TABLE 1, and the protein corona formed on each particle type are analyzed by mass spectrometry. Proteins are identified from the mass spectrometry analysis of the protein corona formed on each of the up to ten particle types.
  • This example describes corona analysis via serial interrogation of a synovial fluid sample.
  • a synovial fluid sample is collected from a subject.
  • a first particle type selected from the particles provided in TABLE 1 is added to 100 ⁇ L of the synovial fluid sample and incubated to form protein corona.
  • the first particle type is separated from the synovial fluid sample by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a second particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the second particle type is the same as the first particle type.
  • the second particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a third particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the third particle type is the same as the first particle type.
  • the third particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • the supernatant is re-interrogated with up to ten particle types selected from the particles provided in TABLE 1, and the protein corona formed on each particle type are analyzed by mass spectrometry. Proteins are identified from the mass spectrometry analysis of the protein corona formed on each of the up to ten particle types.
  • This example describes corona analysis via serial interrogation of a homogenized tissue sample.
  • a homogenized tissue sample is collected from a subject.
  • a first particle type selected from the particles provided in TABLE 1 is added to 100 ⁇ L of the homogenized tissue sample and incubated to form protein corona.
  • the first particle type is separated from the homogenized tissue sample by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a second particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the second particle type is the same as the first particle type.
  • the second particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a third particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the third particle type is the same as the first particle type.
  • the third particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • the supernatant is re-interrogated with up to ten particle types selected from the particles provided in TABLE 1, and the protein corona formed on each particle type are analyzed by mass spectrometry. Proteins are identified from the mass spectrometry analysis of the protein corona formed on each of the up to ten particle types.
  • This example describes corona analysis via serial interrogation of a plasma sample.
  • a plasma sample is collected from a subject.
  • a first particle type selected from the particles provided in TABLE 1 is added to 100 ⁇ L of the plasma sample and incubated to form protein corona.
  • the first particle type is separated from the plasma sample by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a second particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the second particle type is the same as the first particle type.
  • the second particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a third particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the third particle type is the same as the first particle type.
  • the third particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • the supernatant is re-interrogated with up to ten particle types selected from the particles provided in TABLE 1, and the protein corona formed on each particle type are analyzed by mass spectrometry. Proteins are identified from the mass spectrometry analysis of the protein corona formed on each of the up to ten particle types.
  • This example describes corona analysis via serial interrogation of a urine sample.
  • a urine sample is collected from a subject.
  • a first particle type selected from the particles provided in TABLE 1 is added to 100 ⁇ L of the urine sample and incubated to form protein corona.
  • the first particle type is separated from the urine sample by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a second particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the second particle type is the same as the first particle type.
  • the second particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • a third particle type selected from the particles provided in TABLE 1 is added to the supernatant and incubated to form protein corona.
  • the third particle type is the same as the first particle type.
  • the third particle type is separated from the supernatant by centrifugation or magnetic pulldown.
  • the supernatant is collected.
  • the pellet containing the particles is washed and resuspended, and the corona are analyzed by mass spectrometry.
  • the supernatant is re-interrogated with up to ten particle types selected from the particles provided in TABLE 1, and the protein corona formed on each particle type are analyzed by mass spectrometry. Proteins are identified from the mass spectrometry analysis of the protein corona formed on each of the up to ten particle types.
  • This example describes protein detection by ELISA in plasma samples collected in EDTA or blood collection tubes comprising a nucleic acid stabilizing agent.
  • Plasma samples were collected from human subjects into either EDTA blood collection tubes or Streck blood collection tubes. Paired EDTA and Streck samples were collected from each of 20 patients for comparison. Concentrations of three high importance proteins, C-reactive protein (CRP), Calgranulin A and B (“S100”), and MUC16/CA125 (“CA125”), were measured in each sample by enzyme-linked immunosorbent assay (ELISA).
  • CRP C-reactive protein
  • S100 Calgranulin A and B
  • CA125 MUC16/CA125
  • Calgranulin A and B are calcium-binding proteins that are secreted in a heterodimeric form by activated immune cells, such as monocytes, granulocytes, and neutrophils and are expressed in larger amounts in inflammatory diseases such as rheumatoid arthritis, and in some types of cancer.
  • CA125 is a component of the ocular surface, the respiratory tract and the female reproductive tract and has been shown to play a role in advancing tumorigenesis and tumor proliferation by several different mechanisms.
  • FIG. 30A - FIG. 30C show correlation of three protein concentrations as measured by enzyme-linked immunosorbent assay (ELISA) between plasma samples collected in EDTA blood collection tubes (x-axis) and plasma samples collected in Streck sample collection tubes (y-axis).
  • FIG. 30A shows the correlation of C-reactive protein (CRP).
  • FIG. 30B shows the correlation of a Calgranulin A and B heterodimer (“S100”).
  • FIG. 30C shows the correlation of MUC16/CA125 (“CA125”). Protein concentrations in each sample were measured in duplicate and averaged between the two repeats. Concentrations of CRP, S100, and CA125 were well correlated between samples collected in EDTA blood collection tubes and samples collected in Streck blood collection tubes.
  • FIG. 31 shows the percent coefficient of variation (% CV) of CRP (left two box and whiskers), S100 (middle two box and whiskers), and CA125 (right two box and whiskers) of protein concentrations detected by ELISA in plasma samples collected in either EDTA blood collection tubes or Streck blood collection tubes.
  • FIG. 32 shows the matched pair difference in protein concentration detected by ELISA in samples collected in either EDTA blood collection tubes or Streck blood collection tubes for CRP (left plot), S100 (middle plot), and CA125 (right plot). The difference in protein concentration in samples collected in EDTA blood collection tubes and samples collected in Streck blood collection tubes was low.
  • This example describes dynamic range compression of plasma using protein corona analysis.
  • measured and identified protein feature intensities were compared to the published values for the concentration of the same protein.
  • the resulting peptide features for each protein was selected with the maximum MS-determined intensity of all possible features for a protein (using the OpenMS MS data processing tools to extract monoisotopic peak values), and then the intensities were modeled against the published abundance levels for those same proteins.
  • FIG. 34 shows correlation of the maximum intensities of particle corona proteins and plasma proteins to the published concentration of the same proteins.
  • the blue plotted lines are linear regression models to the data and the shaded regions represent the standard error of the model fit.
  • FIG. 35 shows the dynamic range compression of a protein corona analysis assay with mass spectrometry as compared to mass spectrometry without particle corona formation. Protein intensities of common proteins identified in particle corona in the plasma samples assayed in FIG.
  • Nanoparticle MS In Intensity are plotted against the protein intensity identified by mass spectrometry of plasma without particles (“Plasma MS In Intensity”).
  • the lightest dotted line shows a slope of 1, indicating the dynamic range of mass spectrometry without particles.
  • the slopes of the linear fits to the protein intensity was 0.12, 0.36, and 0.093 for S-003, S-007, and S-011 particles, respectively.
  • the grayed area indicates the standard error region of the regression fit.
  • the particle coronas contain more proteins at lower abundances (measured or reported) than does plasma.
  • the dynamic range of those measured values was compressed (the slope of the regression model is reduced) for particle measurements as compared to plasma measurements. This was consistent with previous observations that particle can effectively compress the measured dynamic range for abundances in the resulting corona as compared to the original dynamic range in plasma, which could be attributable to the combination of absolute concentration of a protein, its binding affinity to particles, and its interactions with neighboring proteins.
  • This example describes serial interrogation of a sample with particles as compared to parallel interrogation of a sample with particles.
  • a first sample having a low sample volume of 10 ⁇ L is serially interrogated with two or more particle types.
  • a first particle type is contacted to the first sample, the first sample and the first particle type are incubated to allow formation of a first biomolecule corona on the first particle type, and the first particle type is separated from the first sample.
  • the remainder of the first sample is contacted to a second particle type, the remainder of the first sample and the second particle type are incubated to allow formation of a second biomolecule corona on the first particle type, and the second particle type is separated from the remainder of the first sample.
  • the contacting, incubating, and separating are optionally repeated with additional particle types.
  • the biomolecule corona on the first particle type, the second particle type, and any additional particle types are analyzed by assaying for proteins in the corona, for example, by mass spectrometry.
  • a second sample, from the same source as the first sample, is split into 10 ⁇ L multiple aliquots for parallel interrogation with two or more particle types.
  • the sample is split into a number of aliquots greater than or equal to the number of particle types to be used in the method of assaying for proteins.
  • a first particle type is contacted to a first aliquot, the first aliquot and the first particle type are incubated to allow formation of a first biomolecule corona on the first particle type, and the first particle type is separated from the first aliquot.
  • a second particle type is contacted to a second aliquot, the second aliquot and the second particle type are incubated to allow formation of a second biomolecule corona on the second particle type, and the second particle type is separated from the second aliquot.
  • the contacting, incubating, and separating are performed with additional particle types in additional aliquots.
  • the biomolecule corona on the first particle type, the second particle type, and any additional particle types are analyzed by assaying for proteins in the corona, for example, by mass spectrometry.
  • serial interrogation with two or more particle types of the first sample uses a smaller sample volume (10 ⁇ L total) for protein corona analysis than parallel interrogation of the second sample, which uses 10 ⁇ L of sample per particle type to be assayed. Even while using less total sample, serial interrogation of the first sample enables detection of a range of proteins that is comparable to or broader than the range detected by parallel interrogation.
  • a biomolecule corona formed on a particle e.g., HX-42 may contain a different population of proteins when contacted to a sample first (“HX-42 corona 1”) than when contacted to a sample following 3 rounds of serial interrogation with another particle type (“P39 & HX-42 Corona 4”).
  • Serial interrogation of the first sample enables detection of a range of low abundance proteins that comparable to or broader than the range detected by parallel interrogation.
  • serial interrogation of the first sample may result in depletion of low abundance, high affinity proteins in early rounds of interrogation, allowing for adsorption of low abundance, low affinity proteins into the biomolecule corona of particle types used in later rounds of interrogation.

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