Systems and methods for biomolecular assays

Abstract

In one aspect, the present disclosure provides a method comprising: (a) selectively enriching a first plurality of types of biomolecules in a first fluid composition; (b) selectively enriching a second plurality of types of biomolecules in a second fluid composition; and (c) performing an assay downstream of the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules, wherein the first fluid composition comprises a first pH, the second fluid composition comprises a second pH, and the first pH and the second pH are different or the same. Systems, kits, compositions, and computer-readable media for performing a biomolecule assay are also disclosed.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,591, filed Jun. 15, 2022; U.S. Provisional Patent Application No. 63 / 379,275, filed Oct. 12, 2022; and U.S. Provisional Patent Application No. 63 / 491,012, filed Mar. 17, 2023, the entireties of each of which are incorporated herein by reference.

Background Art

[0002] Background Biological samples, such as biofluids, contain a wide variety of proteins, and the biological state can be indicated by the presence, processing, and relative abundance of those proteins. High-abundance proteins and other proteins can mask signals of other proteins in an assay. Sample preparation, such as dilution, can further mask relative signals in an assay.

Summary of the Invention

Means for Solving the Problems

[0003] In some aspects, the present disclosure provides a method comprising: (a) selectively enriching a first plurality of types of biomolecules in a first fluid composition; (b) selectively enriching a second plurality of types of biomolecules in a second fluid composition; and (c) performing an assay downstream of the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules, wherein the first fluid composition comprises a first pH, the second fluid composition comprises a second pH, and the first pH and the second pH are different or the same. In some embodiments, the second infinite dilution limit enthalpy or free energy of solvation of a second biomolecule within a second subset of biomolecules is different when the second biomolecule is in the second fluid composition compared to when the second biomolecule is in the first fluid composition.

[0004] In some embodiments, the first fluid composition includes a first set of strong physical properties that mediate the selective enrichment of a first plurality of types of biomolecules, the second fluid composition includes a second set of strong physical properties that mediate the selective enrichment of a second plurality of types of biomolecules, and the first set of strong physical properties is different from the second set of strong physical properties.

[0005] In some embodiments, the first fluid composition includes a first ratio between a first sample volume and a first surface area of a first surface, the second fluid composition includes a second ratio between a second sample volume and a second surface area of a second surface, and the first ratio is different from the second ratio.

[0006] In some embodiments, the first fluid composition and the second fluid composition include different temperatures.

[0007] In some embodiments, the first fluid composition includes a first ionic strength, the second fluid composition includes a second ionic strength, and the first ionic strength is different from the second ionic strength.

[0008] In some embodiments, the first fluid composition and the second fluid composition include different salts.

[0009] In some embodiments, the first fluid composition and the second fluid composition include different solvents.

[0010] In some embodiments, the first fluid composition, the second fluid composition, or both include a buffer solution containing tris(hydroxymethyl)aminomethane.

[0011] In some embodiments, the first fluid composition, the second fluid composition, or both include a buffer solution containing citrate.

[0012] In some embodiments, the first fluid composition, the second fluid composition, or both include a pH between 2 and 4.

[0013] In some embodiments, the first fluid composition, the second fluid composition, or both contain a pH between 5 and 7.

[0014] In some embodiments, the first fluid composition, the second fluid composition, or both contain a pH between 9 and 10.

[0015] In some embodiments, the first fluid composition, the second fluid composition, or both contain a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0016] In some embodiments, the first fluid composition, the second fluid composition, or both contain a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0017] In some embodiments, the first fluid composition has a pH between 9 and 10 and the second fluid composition has a pH between 6 and 8.

[0018] In some embodiments, the first fluid composition and the second fluid composition have a pH between 9 and 10.

[0019] In some embodiments, the method further includes diluting a sample to create the first composition, the second composition, or both.

[0020] In some embodiments, diluting includes adding a buffer.

[0021] In some embodiments, the buffer contains a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0022] In some embodiments, the buffer contains a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0023] In some embodiments, the sample and the buffer contain different pHs.

[0024] In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules.

[0025] In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per milliliter of sample.

[0026] In some embodiments, the sample contains biomolecules derived from up to about 1000, 100, 10, or 1 cell.

[0027] In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters.

[0028] In some embodiments, the sample contains a complex biological sample.

[0029] In some embodiments, the complex biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal mucus, ear discharge, gastric juice, pancreatic juice, trabecular bone fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a swab, bronchial aspirate, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0030] In some embodiments, the biological sample includes plasma.

[0031] In some embodiments, the step of selectively enriching the first plurality of types of biomolecules therein comprises contacting a first fluid composition with a first surface and adsorbing the first plurality of types of biomolecules onto the first surface.

[0032] In some embodiments, the step of selectively enriching the second plurality of types of biomolecules therein comprises contacting a second fluid composition with a second surface and adsorbing the second plurality of types of biomolecules onto the second surface.

[0033] In some embodiments, the first surface, the second surface, or both contain particles.

[0034] In some embodiments, the particles are porous particles.

[0035] In some embodiments, the particles are microparticles.

[0036] In some embodiments, the particles are nanoparticles.

[0037] In some embodiments, the particles contain a magnetic material.

[0038] In some embodiments, the particles contain a paramagnetic material.

[0039] In some embodiments, the paramagnetic material is a superparamagnetic material.

[0040] In some embodiments, the paramagnetic material contains iron oxide.

[0041] In some embodiments, the step of selectively enriching the first plurality of types of biomolecules therein comprises contacting a first fluid composition with a first plurality of types of surfaces and adsorbing the first plurality of types of biomolecules onto the first plurality of types of surfaces.

[0042] In some embodiments, the step of selectively enriching the second plurality of types of biomolecules therein comprises contacting a second fluid composition with a second plurality of types of surfaces and adsorbing the second plurality of types of biomolecules onto the second plurality of types of surfaces.

[0043] In some embodiments, the first plurality of types of surfaces contain the same-sign charge.

[0044] In some embodiments, the types of the second plurality of surfaces include charges of the same sign.

[0045] In some embodiments, the types of the first plurality of surfaces have the same charge, and the types of the second plurality of surfaces have the same charge.

[0046] In some embodiments, the charges of the types of the first plurality of surfaces are opposite to the charges of the types of the second plurality of surfaces.

[0047] In some embodiments, the types of the first plurality of surfaces have a zeta potential of the same sign.

[0048] In some embodiments, the types of the second plurality of surfaces have a zeta potential of the same sign.

[0049] In some embodiments, the types of the first plurality of surfaces and the types of the second plurality of surfaces have a zeta potential of the same sign.

[0050] In some embodiments, the types of the first plurality of surfaces each have a zeta potential less than -5 mV, less than -10 mV, less than -15 mV, or less than -20 mV.

[0051] In some embodiments, the types of the second plurality of surfaces each have a zeta potential less than -5 mV, less than -10 mV, less than -15 mV, or less than -20 mV.

[0052] In some embodiments, the types of the first plurality of surfaces each have a zeta potential greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0053] In some embodiments, the types of the second plurality of surfaces each have a zeta potential greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0054] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both include acidic functional groups.

[0055] In some embodiments, the acidic functional groups include Bronsted-Lowry acid or Lewis acid functional groups.

[0056] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both include carboxylic acid groups, acrylic acid groups, methacrylic acid groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphoric acid groups, phosphoric acid diester groups, boronic acid groups, boronic acid ester groups, boric acid groups, boric acid ester groups, silica groups, silanol groups, thiol groups, polymers, or any combination thereof.

[0057] In some embodiments, the first plurality of surface types includes a first surface including a carboxylic acid functionalized surface and a second surface including a silanol functionalized surface.

[0058] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both include primary amine groups, secondary amine groups, tertiary amine groups, quaternary amine groups, cyclic secondary amine groups, primary amide groups, secondary amide groups, tertiary amide groups, imine groups, pyridyl groups, pyrimidine groups, pyrrolidinium groups, imidazole groups, guanidine groups, guanidinium groups, carbamoyl groups, ammonium groups, pyridinium groups, or any combination thereof.

[0059] In some embodiments, each of the first plurality of surface types includes an amine group.

[0060] In some embodiments, the method further includes washing a first plurality of types of biomolecules with a first washing composition and washing a second plurality of types of biomolecules with a second washing composition.

[0061] In some embodiments, the first fluid composition and the first cleaning composition include at least one common strong physical property.

[0062] In some embodiments, the first fluid composition and the first cleaning composition include at least one common solvent.

[0063] In some embodiments, the first fluid composition and the first cleaning composition include at least one different strong physical property.

[0064] In some embodiments, the second fluid composition and the second cleaning composition include at least one common strong physical property.

[0065] In some embodiments, the second fluid composition and the second cleaning composition include at least one common solvent.

[0066] In some embodiments, the second fluid composition and the second cleaning composition include at least one different strong physical property.

[0067] In some embodiments, the first cleaning composition and the second cleaning composition include at least one common strong physical property.

[0068] In some embodiments, the first cleaning composition and the second cleaning composition are the same.

[0069] In some embodiments, the first cleaning composition and the second cleaning composition include at least one different strong physical property.

[0070] In some embodiments, the first cleaning composition releases from the first surface a first plurality of types of biomolecules adsorbed on the first surface.

[0071] In some embodiments, the method further includes a step of purifying a first plurality of types of biomolecules to produce a first purified composition.

[0072] In some embodiments, the purification step includes drying a plurality of types of biomolecules to remove the first washing composition.

[0073] In some embodiments, the method further includes the step of reconstructing the first purified composition using the first reconstruction composition to produce the first reconstructed composition.

[0074] In some embodiments, the second washing composition releases a second plurality of types of biomolecules deposited on the second surface from the second surface.

[0075] In some embodiments, the method further includes the step of purifying the second plurality of types of biomolecules to produce the second purified composition.

[0076] In some embodiments, the purification step includes drying the second plurality of types of biomolecules to remove the second washing composition.

[0077] In some embodiments, the method further includes the step of reconstructing the second purified composition using the second reconstruction composition to produce the second reconstructed composition.

[0078] In some embodiments, the downstream assay includes mass spectrometry.

[0079] In some embodiments, the mass spectrometry includes LC-MS / MS.

[0080] In some embodiments, the downstream assay includes protein sequencing.

[0081] In some embodiments, the downstream assay comprises contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both, with a pair of antibodies capable of binding to at least one type of biomolecule among the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, wherein the pair of antibodies comprises a complementary single-stranded nucleic acid sequence bound thereto such that when the pair of antibodies binds to at least one type of biomolecule, a complementary nucleic acid hybridizes to form a double-stranded nucleic acid, and the double-stranded nucleic acid forms a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction and generate a detectable signal.

[0082] In some embodiments, the downstream assay comprises contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both, with one or more aptamers capable of binding to at least one type of biomolecule among the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, wherein the one or more aptamers are bound to a surface via a cleavable linker.

[0083] In some embodiments, the surface is a particle surface.

[0084] In some embodiments, the cleavable linker is a linker cleavable by light.

[0085] In some embodiments, the method further comprises contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both, with a polymeric competitor configured to reduce dissociation of a complex composed of one or more aptamers and the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, in a fluid composition.

[0086] In some embodiments, the polymeric competitor is further configured to bind to a type of biomolecule that is different from the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both.

[0087] In some embodiments, the polymeric competitor is a polyanionic polymer.

[0088] In some embodiments, the downstream assay includes nucleic acid sequencing.

[0089] In some embodiments, (a) and (b) are performed on one machine.

[0090] In some embodiments, (a) and (b) are performed on different machines.

[0091] In some embodiments, (a) and (b) are performed in parallel.

[0092] In some embodiments, (a) and (b) are performed sequentially.

[0093] In some embodiments, the first fluid composition and the second fluid composition are part of the same sample.

[0094] In some embodiments, the first fluid composition and the second fluid composition are derived from different samples.

[0095] In some embodiments, the step of selectively enriching is performed in parallel on the first fluid composition and the second fluid composition.

[0096] In some embodiments, the step of selectively enriching is performed sequentially on the first fluid composition and the second fluid composition.

[0097] In some embodiments, the step of selectively enriching is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are enriched per hour.

[0098] In some embodiments, the step of selectively enriching is carried out at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are enriched per hour.

[0099] In some embodiments, contacting is carried out in parallel with respect to the first fluid composition and the second fluid composition.

[0100] In some embodiments, contacting is carried out continuously with respect to the first fluid composition and the second fluid composition.

[0101] In some embodiments, contacting is carried out at a rate at which at least 100, 1000, 10000, 100000, or 1000000 biomolecules deposit per hour.

[0102] In some embodiments, contacting is carried out at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules deposit per hour.

[0103] In some embodiments, the step of washing is carried out in parallel with respect to the first fluid composition and the second fluid composition.

[0104] In some embodiments, the step of washing is carried out continuously with respect to the first fluid composition and the second fluid composition.

[0105] In some embodiments, the step of washing is carried out at a rate at which at least 100, 1000, 10000, 100000, or 1000000 biomolecules are washed per hour.

[0106] In some embodiments, the washing step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule is washed per hour.

[0107] In some embodiments, the purification step is carried out in parallel with respect to the first fluid composition and the second fluid composition.

[0108] In some embodiments, the purification step is carried out sequentially with respect to the first fluid composition and the second fluid composition.

[0109] In some embodiments, the purification step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 are purified per hour.

[0110] In some embodiments, the purification step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule is purified per hour.

[0111] In some embodiments, the reconstitution step is carried out in parallel with respect to the first fluid composition and the second fluid composition.

[0112] In some embodiments, the reconstitution step is carried out sequentially with respect to the first fluid composition and the second fluid composition.

[0113] In some embodiments, the reconstitution step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 are reconstituted per hour.

[0114] In some embodiments, the reconstitution step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule is reconstituted per hour.

[0115] In some embodiments, the downstream assay is performed in parallel on the first fluid composition and the second fluid composition.

[0116] In some embodiments, the downstream assay is performed sequentially on the first fluid composition and the second fluid composition.

[0117] In some embodiments, the downstream assay is performed at a rate such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples are assayed per hour.

[0118] In some embodiments, the downstream assay is performed at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule are assayed per hour.

[0119] In some embodiments, the downstream assay is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour.

[0120] In some embodiments, the downstream assay is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour.

[0121] In some embodiments, the method is performed in parallel on the first fluid composition and the second fluid composition.

[0122] In some embodiments, the method is performed sequentially on the first fluid composition and the second fluid composition.

[0123] In some embodiments, the method is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0124] In some embodiments, the method is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour.

[0125] In some embodiments, the method is performed at a rate at which at least 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour.

[0126] In some embodiments, the method is performed at a rate at which at least 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour.

[0127] In some embodiments, when the first fluid composition and the second fluid composition are HeLa cell extracts, at least about 100, 1000, 10000, 100000, or 1000000 biomolecules are identified in the downstream assay.

[0128] In some embodiments, when the downstream assay is performed before the steps of selectively enriching (a) and (b) for the first fluid composition and the second fluid composition, the first plurality of biomolecule types and the second plurality of biomolecule types together include at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0129] In some embodiments, the first plurality of biomolecule types are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the abundance of the first biomolecule in the first fluid composition is greater than that of the second biomolecule.

[0130] In some embodiments, the second plurality of biomolecule types are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the abundance of the first biomolecule in the second fluid composition is greater than that of the second biomolecule.

[0131] In some embodiments, the first likelihood that a biomolecule present in a low abundance among the first plurality of biomolecule types or the second plurality of biomolecule types is identified in the downstream assay is higher than the second likelihood that a biomolecule present in a low abundance in the first fluid composition or the second fluid composition is identified in the downstream assay, and the biomolecule present in a low abundance comprises a first set of biomolecules or a second set of biomolecules that are less than about 1 weight percent, less than about 10-1 weight percent, less than about 10-2 weight percent, less than about 10-3 weight percent, less than about 10-4 weight percent, less than about 10-5 weight percent, less than about 10-6 weight percent, less than about 10-7 weight percent, less than about 10-8 weight percent, less than about 10-9 weight percent, or less than about 10-10 weight percent.

[0132] In some embodiments, the first likelihood is at least 2, 5, 10, 102, 102, 103, 104, 105, 106, 107, 108, 109, or 1010 times higher than the second likelihood.

[0133] In some embodiments, one or more biomolecules in the first plurality of biomolecule types, the second plurality of biomolecule types, or both, are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent downstream.

[0134] In some embodiments, one or more biomolecules in the first plurality of biomolecule types, the second plurality of biomolecule types, or both, are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent downstream.

[0135] In some embodiments, one or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 peptides.

[0136] In some embodiments, the one or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins.

[0137] In some embodiments, the first plurality of biomolecule types, the second plurality of biomolecule types, or both comprise one or more polyamino acids.

[0138] In some embodiments, the method further comprises identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first fluid composition.

[0139] In some embodiments, the method further comprises identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second fluid composition.

[0140] In some embodiments, the method further comprises identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first wash composition.

[0141] In some embodiments, the method further comprises identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second wash composition.

[0142] In some embodiments, the method further comprises identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first reconstitution composition.

[0143] In some embodiments, the method further includes identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstitution composition.

[0144] In some embodiments, the first surface is disposed in a first lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers.

[0145] In some embodiments, the second surface is disposed in a second lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers.

[0146] In some embodiments, the one or more modifiers include a pH modifier, an ionic strength modifier, a viscosity modifier, or any combination thereof.

[0147] In some embodiments, the first pH and the second pH are different. In some embodiments, the first pH and the second pH are the same.

[0148] In some aspects, the present disclosure provides a method comprising: (a) selectively enriching a first plurality of types of biomolecules in a first fluid composition; (b) selectively enriching a second plurality of types of biomolecules in a second fluid composition; and (c) performing an assay downstream of the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules, wherein the first fluid composition and the second fluid composition are different such that the infinite dilution limit enthalpy or free energy of solvation of a first biomolecule among the first plurality of types of biomolecules is different when the first biomolecule is in the first fluid composition compared to when the biomolecule is in the second fluid composition.

[0149] In some embodiments, the present disclosure provides a kit comprising a first reagent having a first pH, a second reagent having a second pH, a first surface configured to selectively enrich a first plurality of types of biomolecules in a first fluid composition comprising the first surface and the first reagent, and a second surface configured to selectively enrich a second plurality of types of biomolecules in a second fluid composition comprising the second surface and the second reagent, wherein the first fluid composition comprises the first reagent, the second fluid composition comprises the second reagent, and the first pH and the second pH are different or the same.

[0150] In some embodiments, the present disclosure provides a composition comprising a suspension, the suspension comprising: a first particle comprising (i) a first paramagnetic moiety and (ii) a first surface chemistry; a second particle comprising (i) a second paramagnetic moiety and (ii) a second surface chemistry; and a plurality of biomolecules adsorbed to the first particle and the second particle, wherein the second surface chemistry and the first surface chemistry are different.

[0151] In some embodiments, the first particle and the second particle have the same charge.

[0152] In some embodiments, the first particle and the second particle have the same sign of zeta potential.

[0153] In some embodiments, the same sign is a negative sign.

[0154] In some embodiments, the same sign is a positive sign.

[0155] In some embodiments, the first particle and the second particle each have a zeta potential of less than -5 mV, less than -10 mV, less than -15 mV, or less than -20 mV.

[0156] In some embodiments, the first particle and the second particle each have a zeta potential of greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0157] In some embodiments, the first particle, the second particle, or both contain acidic functional groups.

[0158] In some embodiments, the acidic functional group includes a Bronsted-Lowry acid or a Lewis acid functional group.

[0159] In some embodiments, the first particle, the second particle, or both contain a carboxylic acid group, an acrylic acid group, a methacrylic acid group, an acetal group, a hemiacetal group, a hemiketal group, a sulfonic acid group, a sulfinic acid group, a thiocarboxylic acid group, a phosphonic acid group, a phosphoric acid group, a phosphoric acid diester group, a boronic acid group, a boronic acid ester group, a boric acid group, a boric acid ester group, a silica group, a silanol group, a thiol group, a polymer, or any combination thereof.

[0160] In some embodiments, the first particle, the second particle, or both contain carboxylic acid functionalization, silanol functionalization, or both.

[0161] In some embodiments, the first particle, the second particle, or both contain a primary amine group, a secondary amine group, a tertiary amine group, a quaternary amine group, a cyclic secondary amine group, a primary amide group, a secondary amide group, a tertiary amide group, an imine group, a pyridyl group, a pyrimidine group, a pyrrolidinium group, an imidazole group, a guanidine group, a guanidinium group, a carbamoyl group, an ammonium group, a pyridinium group, or any combination thereof.

[0162] In some embodiments, the first particle, the second particle, or both contain an amine group.

[0163] In some embodiments, the first surface chemistry structure, the second surface chemistry structure, or both contain acidic functional groups.

[0164] In some embodiments, the acidic functional group includes a Bronsted-Lowry acid or a Lewis acid functional group.

[0165] In some embodiments, the first surface chemistry structure, the second surface chemistry structure, or both include a carboxylic acid group, an acrylic acid group, a methacrylic acid group, an acetal group, a hemiacetal group, a hemiketal group, a sulfonic acid group, a sulfinic acid group, a thiocarboxylic acid group, a phosphonic acid group, a phosphoric acid group, a phosphoric acid diester group, a boronic acid group, a boronic acid ester group, a boric acid group, a boric acid ester group, a silica group, a silanol group, a polymer, or any combination thereof.

[0166] In some embodiments, the suspension is stable for at least about 1, 5, 10, 15, 30, or 60 minutes.

[0167] In some embodiments, the time constant for destabilization of the suspension is at least about 1, 5, 10, 15, 30, or 60 minutes.

[0168] In some embodiments, the average aggregation number of the first and second particles in the suspension is at most about 1000, 100, or 10.

[0169] In some embodiments, the suspension includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles per about 1 part of the second particles.

[0170] In some embodiments, the suspension includes at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles per about 1 part of the second particles.

[0171] In some embodiments, the suspension includes about 15 parts of the first particles per about 6 parts of the second particles.

[0172] In some embodiments, part is part by weight, part by volume, or part by surface area.

[0173] In some embodiments, part is part by weight.

[0174] In some embodiments, the suspension includes at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 distinct types of particles.

[0175] In some embodiments, the suspension comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 distinct particles.

[0176] In some embodiments, the first particle, the second particle, or both are nanoparticles.

[0177] In some embodiments, the first particle, the second particle, or both are microparticles.

[0178] In some embodiments, the first particle, the second particle, or both are porous.

[0179] In some embodiments, the first size of the first particle is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle.

[0180] In some embodiments, the first size of the first particle is at most about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle.

[0181] In some embodiments, the size of the first particle is within ±40% of the size of the second particle, the size of the first particle is within ±30% of the size of the second particle, the size of the first particle is within ±25% of the size of the second particle, the size of the first particle is within ±20% of the size of the second particle, the size of the first particle is within ±15% of the size of the second particle, or the size of the first particle is within ±10% of the size of the second particle.

[0182] In some embodiments, the first size is a first diameter and the second size is a second diameter.

[0183] In some embodiments, the first size is a first average size and the second size is a second average size.

[0184] In some embodiments, the first average size and the second average size are a size average value or a size median.

[0185] In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is from about 3:2 to about 2:3.

[0186] In some embodiments, the plurality of biomolecules comprises up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules.

[0187] In some embodiments, the plurality of biomolecules comprises up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per mL.

[0188] In some embodiments, the plurality of biomolecules comprises biomolecules derived from up to about 1000, 100, 10, or 1 cells.

[0189] In some aspects, the disclosure provides a method comprising assaying a plurality of biomolecules in a composition disclosed herein, the method including identifying one or more of the plurality of biomolecules.

[0190] In some embodiments, the assaying step includes performing mass spectrometry.

[0191] In some embodiments, the mass spectrometry includes LC-MS / MS.

[0192] In some embodiments, the assaying step includes performing protein sequencing.

[0193] In some embodiments, the step of assaying comprises contacting a plurality of biomolecules with a pair of antibodies capable of binding to at least one biomolecule among the plurality of biomolecules, wherein the pair of antibodies comprises a complementary single-stranded nucleic acid sequence bound thereto such that a nucleic acid complementary to the at least one biomolecule to which the pair of antibodies binds hybridizes to form a double-stranded nucleic acid, and the double-stranded nucleic acid forms a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction and is configured to generate a detectable signal.

[0194] In some embodiments, the step of assaying comprises contacting a plurality of biomolecules with one or more aptamers capable of binding to at least one biomolecule among the plurality of biomolecules, wherein the one or more aptamers are bound to a surface via a cleavable linker.

[0195] In some embodiments, the surface is a particle surface.

[0196] In some embodiments, the cleavable linker is a linker cleavable by light.

[0197] In some embodiments, the method further comprises contacting the plurality of biomolecules with a polymeric competitor configured to reduce dissociation of a complex composed of one or more aptamers and a biomarker in a fluid composition.

[0198] In some embodiments, the polymeric competitor is further configured to bind to a biomolecule different from the biomarker.

[0199] In some embodiments, the polymeric competitor is a polyanionic polymer.

[0200] In some embodiments, the step of assaying comprises performing nucleic acid sequencing.

[0201] In some embodiments, the sample comprises a plurality of biomolecules.

[0202] In some embodiments, the sample is a complex biological sample.

[0203] In some embodiments, the complex biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal mucus, earwax, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a cotton swab, bronchial aspirate fluid, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0204] In some embodiments, the biological sample includes plasma.

[0205] In some embodiments, the plurality of biomolecules includes one or more polyamino acids.

[0206] In some embodiments, the step of assaying is performed at a rate at which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100 samples are assayed per hour.

[0207] In some embodiments, the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour.

[0208] In some embodiments, the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour.

[0209] In some embodiments, the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour.

[0210] In some embodiments, when the suspension contains a HeLa cell extract, at least about 100, 1000, 10000, or 100000 biomolecules are identified in the step of assaying.

[0211] In some embodiments, when the step of assaying is performed on a plurality of biomolecules in a biological sample in the absence of the first and second particles, the plurality of biomolecules includes at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0212] In some embodiments, the first probability of identifying a biomolecule with a low abundance among a plurality of biomolecules in the step of assaying is higher than the second probability of identifying a biomolecule with a low abundance in a biological sample in the absence of the first and second particles in the step of assaying, and the biomolecule with a low abundance constitutes less than about 1 weight percent, less than about 10-1 weight percent, less than about 10-2 weight percent, less than about 10-3 weight percent, less than about 10-4 weight percent, less than about 10-5 weight percent, less than about 10-6 weight percent, less than about 10-7 weight percent, less than about 10-8 weight percent, less than about 10-9 weight percent, or less than about 10-10 weight percent of the first set of biomolecules or the second set of biomolecules.

[0213] In some embodiments, the first probability is at least 2, 5, 10, 102, 102, 103, 104, 105, 106, 107, 108, 109, or 1010 times higher than the second probability.

[0214] In some embodiments, one or more of the plurality of biomolecules are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent in the step of assaying.

[0215] In some embodiments, one or more of the plurality of biomolecules in the assay step are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent.

[0216] In some aspects, the present disclosure is a system for analyzing a plurality of biological samples, comprising: (a) a plurality of compartments including a first compartment and a second compartment; (b) a plurality of reagent storage units including a first reagent having a first pH and a second reagent having a second pH, wherein the first pH and the second pH are different or the same; (c) a plurality of substrates including a first substrate having a first surface chemistry and a second substrate having a second surface chemistry; (d) one or more transfer devices operably connected to the plurality of compartments, the plurality of reagent storage units, and the plurality of substrates; and (e) at least one processor, and a computer including instructions executable by the at least one processor to perform steps including: (i) using one or more transfer devices to generate a first fluid composition within a first compartment including the first substrate, the first reagent, and a first plurality of biomolecules, wherein the first plurality of biomolecules adsorb to the first substrate; (ii) using one or more transfer devices to generate a second fluid composition within a second compartment including the second substrate, the second reagent, and a second plurality of biomolecules, wherein the second plurality of biomolecules adsorb to the second substrate; and (iii) using one or more transfer devices to prepare the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry.

[0217] In some embodiments, the present disclosure relates to a system for analyzing a plurality of biological samples, the system comprising: (a) a compartment; (b) a reagent storage unit containing reagents; (c) a plurality of substrates including a first substrate having a first surface chemistry and a second substrate having a second surface chemistry, wherein the first surface chemistry is different from the second surface chemistry; (d) one or more transfer devices operably connected to the compartment, the reagent storage unit, and the plurality of substrates; and (e) at least one processor, and a computer including instructions executable by the at least one processor to perform steps including: (i) using one or more transfer devices to generate a fluid composition within the compartment that includes the plurality of substrates, the reagents, and a plurality of biomolecules, wherein the plurality of biomolecules adsorb to the substrates; and (ii) using one or more transfer devices to prepare the plurality of biomolecules for mass spectrometry.

[0218] In some embodiments, the present disclosure provides a method comprising: (a) forming a first suspension comprising a first plurality of particles and a first portion of a biological sample, wherein the first plurality of particles includes a first particle and a second particle, the first particle has a first functionalized surface chemistry that is different from a second functionalized surface chemistry of the second particle, and the first particle and the second particle both have a negative charge; (b) forming a second suspension comprising a second plurality of particles and a second portion of the biological sample, wherein the second plurality of particles includes a third particle and a fourth particle, the third particle has a third functionalized surface chemistry that is different from a fourth functionalized surface chemistry of the fourth particle, and the third particle and the fourth particle both have a positive charge; (c) enriching biomolecules adsorbed to the first plurality of particles and biomolecules adsorbed to the second plurality of particles; and (d) performing a downstream assay on the enriched biomolecules.

[0219] In some embodiments, the first suspension and the second suspension each have a pH between 9 and 10.

[0220] In some embodiments, the first suspension and the second suspension each contain a Tris buffer.

[0221] In some embodiments, the first suspension has a pH between 9 and 10.

[0222] In some embodiments, the first suspension contains a Tris buffer.

[0223] In some embodiments, the second suspension has a pH between 6 and 8.

[0224] In some embodiments, the first particles are magnetic particles and include an outer layer surface-functionalized with silanol, and the second particles are magnetic particles and include an outer layer surface-functionalized with carboxylic acid.

[0225] In some embodiments, the third particles are magnetic particles and include an outer layer surface-functionalized with amine, and the fourth particles are magnetic particles and include an outer layer surface-functionalized with amine.

[0226] In some embodiments, the outer layer of the third particles contains a polymer.

[0227] In some embodiments, the first plurality of particles has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles.

[0228] In some embodiments, the second plurality of particles has a ratio of about 10 to about 15 parts by weight of the third particles to about 1 part by weight of the fourth particles.

[0229] In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is about 3:2 to about 2:3.

[0230] In some embodiments, the ratio of the average diameter of the third particles to the average diameter of the fourth particles is about 3:2 to about 2:3.

[0231] In some embodiments, each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle.

[0232] In some aspects, the present disclosure provides a kit comprising: (a) a first composition comprising a first particle and a second particle, wherein the first particle has a first functionalized surface chemical structure different from the second functionalized surface chemical structure of the second particle, both the first particle and the second particle have a negative charge, and the first composition is a solid; and (b) a second composition comprising a third particle and a fourth particle, wherein the third particle has a third functionalized surface chemical structure different from the fourth functionalized surface chemical structure of the fourth particle, both the third particle and the fourth particle have a positive charge, and the second composition is a solid.

[0233] In some embodiments, the kit further comprises a buffer solution having a pH between 9 and 10.

[0234] In some embodiments, the kit further comprises a Tris buffer solution having a pH between 9 and 10.

[0235] In some embodiments, both the first composition and the second composition in the kit are lyophilized.

[0236] In some embodiments, the first particle in the kit is a magnetic particle and comprises an outer layer surface-functionalized with silanol, and the second particle in the kit is a magnetic particle and comprises an outer layer surface-functionalized with carboxylic acid.

[0237] In some embodiments, the third particle in the kit is a magnetic particle and comprises an outer layer surface-functionalized with amine, and the fourth particle in the kit is a magnetic particle and comprises an outer layer surface-functionalized with amine.

[0238] In some embodiments, the outer layer of the third particle in the kit comprises a polymer.

[0239] In some embodiments, the first composition in the kit has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles.

[0240] In some embodiments, the second composition in the kit has a ratio of about 10 to about 15 parts by weight of the third particles to about 1 part by weight of the fourth particles.

[0241] In some embodiments, the ratio of the average diameter of the first particles in the kit to the average diameter of the second particles in the kit is from about 3:2 to about 2:3.

[0242] In some embodiments, the ratio of the average diameter of the third particles in the kit to the average diameter of the fourth particles in the kit is from about 3:2 to about 2:3.

[0243] In some embodiments, each of the first, second, third, and fourth particles in the kit is a superparamagnetic iron oxide nanoparticle.

[0244] In some aspects, the present disclosure provides a suspension comprising (a) a first particle comprising an outer layer surface-functionalized with a negative charge, (b) a biological sample, and (c) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein biomolecules adsorb to the outer layer of the first particle.

[0245] In some aspects, the present disclosure provides a suspension comprising (a) a first particle comprising a first outer layer surface-functionalized with a negative charge, (b) a second particle comprising a second outer layer surface-functionalized with a negative charge, wherein the second outer layer is different from the first outer layer, (c) a biological sample, and (d) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein biomolecules adsorb to the first outer layer of the first particle and the second outer layer of the particle.

[0246] In some embodiments, the first outer layer comprises silanol and the second outer layer comprises carboxylic acid.

[0247] In one aspect, the present disclosure provides a suspension comprising: (a) a first particle having a first outer layer surface-functionalized with a positive charge; (b) a second particle having a second outer layer surface-functionalized with a positive charge, wherein the second outer layer is different from the first outer layer; (c) a biological sample; and (d) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein biomolecules adsorb to the first outer layer of the first particle and the second outer layer of the particle.

[0248] In some embodiments, the first outer layer and the second outer layer comprise amine groups.

[0249] In some embodiments, the biomolecules comprise at least 100 different peptides or proteins.

[0250] In some embodiments, the first particle and optionally the second particle are magnetic.

[0251] In some embodiments, the buffer is a Tris buffer.

[0252] In one aspect, the present disclosure provides a method comprising: (a) contacting and incubating a composition with one or more surfaces, wherein the composition comprises a biological sample and a buffer configured to maintain the pH of the composition between 8 and 11, and biomolecules from the biological sample adsorb to the one or more surfaces; (b) enriching the biomolecules adsorbed to the surface; and (c) assaying the enriched biomolecules to identify one or more of the biomolecules enriched from the biological sample.

[0253] In some embodiments, the buffer is configured to maintain the pH of the composition between 9 and 10.

[0254] In some embodiments, the buffer is configured to maintain the pH of the composition at about 9.5.

[0255] In some embodiments, the buffer is a Tris buffer.

[0256] In some embodiments, the biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal mucus, earwax, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a swab, bronchial aspirate fluid, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0257] In some embodiments, the biological sample is plasma or serum.

[0258] In some embodiments, the biological sample is a cell culture medium.

[0259] In some embodiments, the composition includes 60% by volume or less of the biological sample.

[0260] In some embodiments, the composition includes 25% by volume or less of the biological sample.

[0261] In some embodiments, the composition includes 15% by volume or less of the biological sample.

[0262] In some embodiments, the composition includes at least 1% by volume of the biological sample.

[0263] In some embodiments, the composition includes at least 4% by volume of the biological sample.

[0264] In some embodiments, the composition includes at least 10% by volume of the biological sample.

[0265] In some embodiments, the composition includes at least 20% by volume of the biological sample.

[0266] In some embodiments, the step of assaying includes SDS-PAGE, gel-based separation techniques, immunoassays, ELISA, high performance liquid chromatography, mass spectrometry, Edman degradation, or immunoaffinity techniques.

[0267] In some embodiments, the step of assaying includes mass spectrometry.

[0268] In some embodiments, the step of assaying includes LC-MS / MS.

[0269] In some embodiments, the step of assaying includes quantifying a biomolecule.

[0270] In some embodiments, the step of assaying includes identifying one or more proteins in a biological sample.

[0271] In some embodiments, at least 100 different proteins are identified in the biological sample.

[0272] In some embodiments, at least 250 different proteins are identified in the biological sample.

[0273] In some embodiments, at least 500 different proteins are identified in the biological sample.

[0274] In some embodiments, one or more surfaces are composed of particles dispersed in the composition during incubation.

[0275] In some embodiments, the average diameter of the nanoparticles is less than 500 nanometers.

[0276] In some embodiments, the particles are magnetic.

[0277] In some embodiments, the particles include an outer layer of poly(dimethylaminopropylmethacrylamide) (PDMAPMA).

[0278] In some embodiments, the particles include an outer layer of silica.

[0279] In some embodiments, the particles comprise a carboxylic acid-functionalized outer layer.

[0280] In some embodiments, the particles comprise an amine-functionalized outer layer.

[0281] In some embodiments, the particles comprise a polymer outer layer.

[0282] In some embodiments, the particles comprise a polyacrylamide outer layer.

[0283] In some embodiments, the particles comprise an acrylic acid polymer.

[0284] In some embodiments, the method further comprises: (a) contacting and incubating a second composition with one or more second surfaces, wherein the second composition comprises a biological sample and biomolecules derived from the biological sample adsorb to the one or more second surfaces; (b) enriching the biomolecules adsorbed to the one or more second surfaces; and (c) assaying the enriched biomolecules in the biological sample to identify one or more of the enriched biomolecules from the one or more second surfaces.

[0285] In some embodiments, the second composition has a pH between 5 and 9.

[0286] In some embodiments, the second composition has a pH between 6 and 8.

[0287] In some embodiments, the second composition has a pH between 8 and 11.

[0288] In some embodiments, the second composition has a pH between 9 and 10.

[0289] In some embodiments, the second composition does not contain a buffer.

[0290] In some embodiments, the second composition comprises a buffer different from the buffer configured to maintain the pH of the composition between 8 and 11.

[0291] In some embodiments, the second composition comprises the same buffer as the composition.

[0292] In some embodiments, the step of incubating the composition and the step of incubating the second composition are performed simultaneously.

[0293] In some embodiments, the enriched biomolecule is identified by the assay with a coefficient of variation of up to 30%.

[0294] In some embodiments, the enriched biomolecule is identified by the assay with a coefficient of variation of up to 25%.

[0295] In some embodiments, the method further comprises a step of digesting the protein adsorbed on the first surface before the step of assaying the biomolecule.

[0296] In some embodiments, the method further comprises a step of treating the protein in the biomolecule corona with a reducing agent before the step of assaying the biomolecule.

[0297] In some embodiments, the method further comprises a step of treating the protein in the biomolecule corona with an alkylating agent before the step of assaying the biomolecule.

[0298] In some aspects, the present disclosure provides a suspension comprising (a) a first particle having a first functionalized surface chemistry, (b) a second particle having a second functionalized surface chemistry, (c) a biological sample, and (d) a buffer configured to maintain a pH of about 8 to about 11, wherein the first functionalized surface chemistry is different from the second functionalized surface chemistry of the second particle, and wherein both the first particle and the second particle have a negative charge.

[0299] In some embodiments, the buffer is configured to maintain a pH between 9 and 10.

[0300] In some embodiments, the buffer is a Tris buffer having a pH between 9 and 10.

[0301] In some embodiments, the first particles are magnetic particles and include an outer layer surface-functionalized with silanol, and the second particles are magnetic particles and include an outer layer surface-functionalized with carboxylic acid.

[0302] In some embodiments, the first composition has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles.

[0303] In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is from about 3:2 to about 2:3.

[0304] In some embodiments, the first particles and the second particles are superparamagnetic iron oxide nanoparticles.

[0305] In some aspects, the present disclosure provides a suspension comprising (a) first particles having a first functionalized surface chemistry, (b) second particles having a second functionalized surface chemistry, and (c) a biological sample, wherein the first functionalized surface chemistry is different from the second functionalized surface chemistry of the second particles, and wherein both the first particles and the second particles have a positive charge.

[0306] In some embodiments, the first particles are magnetic particles and include an outer layer surface-functionalized with amine, and the second particles are magnetic particles and include an outer layer surface-functionalized with amine.

[0307] In some embodiments, the outer layer of the first particles includes a polymer.

[0308] In some embodiments, the suspension has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles.

[0309] In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is from about 3:2 to about 2:3.

[0310] In some embodiments, the first particles and the second particles are superparamagnetic iron oxide nanoparticles.

[0311] In some embodiments, the biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal discharge, ear discharge, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a swab, bronchial aspirate fluid, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0312] In some embodiments, the biological sample includes plasma or serum.

[0313] In some embodiments, the biological sample is a cell-free biological sample.

[0314] Incorporation by reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, the present specification shall supersede and / or prevail over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0315] The novel features of the present disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that describes illustrative embodiments (wherein the principles of the present disclosure are utilized) and the accompanying drawings.

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Modes for Carrying Out the Invention

[0352] Detailed Description The human genome contains approximately 20,000 genes, but some researchers estimate that the human proteome contains over one million proteins expressed from these genes. A large number of different proteoforms can be expressed from the repertoire of various transcriptional, translational, and post-translational mechanisms (e.g., alternative splice forms, allelic variations, and protein modifications) that produce proteins different from those containing the standard sequences expressed from genes. Thus, only a small fraction of the vast number of proteins presumed to exist in the human proteome have been meaningfully identified and / or quantified in the human body.

[0353] Some of the challenges in protein identification and quantification are related to the rarity of certain proteins. For example, human plasma contains protein species over a dynamic range exceeding 12 orders of magnitude, where the top few proteins (e.g., albumin, transferrin, complement proteins, apolipoproteins, and alpha-2-macroglobulin) constitute 95% of the protein mass in plasma, and the majority of protein species constitute the remaining 5%. Some protein species are present in the range of a few nanograms per milliliter (e.g., transforming growth factor beta-1-induced transcript 1 protein is present at about 10 ng / ml, fructose-bisphosphate aldolase A is present at about 20 ng / ml, thioredoxin is present at about 18 ng / ml, and L-selectin is present at about 92 ng / ml), and some proteins are expected to be present at levels even below that range. Liquid chromatography combined with mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS / MS) has grown as a ubiquitous detection platform due to their speed, sensitivity, and breadth of application. Protein species can be identified using LC-MS and LC-MS / MS, but due to the probabilistic nature of this method, only a small fraction of the ion species generated at once from a given sample can be selected for mass spectrum acquisition. As a result, the presence of species that are extremely abundant compared to rare species can create a overwhelming number of signals that make it difficult to capture rare species.

[0354] Some aspects of the PROTEOGRAPH (trademark) technology aim to solve some of these problems by "compressing" the dynamic range of protein species in a sample. Some aspects of the PROTEOGRAPH (trademark) technology function based on non-specifically binding proteins to the surface of nanoparticles to form a protein corona. It does not require the existence of a specific entity configured to bind to a single specific protein (as in an immunoassay, for example). Non-specific binding can capture a wide variety of proteins while bringing about a compression of the dynamic range of the proteins binding to the nanoparticle surface. In other words, the relative abundance of proteins in the sample on the nanoparticle surface can be modified such that, compared to the original sample, the abundance of rare proteins becomes relatively higher and the abundance of extremely abundant proteins becomes relatively lower. The proteins can then be separated from the sample and analyzed, for example, using mass spectrometry. The compression of the dynamic range enables rare proteins to constitute a higher proportion of the ion species, thereby making it possible to increase the probability of detecting those rare proteins in an MS experiment. Although the above example has been described with respect to proteins, other classes of biomolecules (such as lipids, sugars, etc.) can similarly be targeted. Another aspect of the PROTEOGRAPH (trademark) technology includes the controlled automation of the PROTEOGRAPH (trademark) workflow to increase speed / throughput and accuracy / reliability.

[0355] By introducing PROTEOGRAPH (trademark) technology, the number of proteins that can be detected from a sample increases, but there is another problem of finding biomarkers and / or therapeutic targets among those proteins. As the number of proteins that can be considered for diagnostic or therapeutic potential increases, the sample size can also increase to effectively screen for relevant proteins. Due to individual differences in biological properties among humans, thousands of proteins can have varying levels between two individuals in a plasma sample. Therefore, it is possible to experiment with samples from hundreds or thousands of individuals to identify significant and systematic signals with clinical importance. Thus, some aspects of the present disclosure provide systems, compositions, and methods that provide greater depth (e.g., an increase in the number of biomolecules detected and an increase in the dynamic range) and throughput to identify more elusive details present in the biological signature of complex biological samples. By increasing the depth, more obscure biological signatures (e.g., those associated with very rare proteins) can be made more visible. By increasing the throughput, data spurious variations (e.g., those associated with known and unknown sources of error, i.e., standard deviation / error) can be reduced by improving the certainty.

[0356] In some embodiments, the present disclosure provides a composition of a plurality of particles having physicochemically distinct features in a single volume. The physicochemically distinct particles can each enrich / deplete different sections of the biomolecular profile in a biological sample. Thus, by using a plurality of physicochemically distinct particles, it is possible to provide an advantage for profiling over a larger dynamic range of a biological sample. By multiplexing the physicochemically distinct particles together into a single volume, e.g., one well of a 96-well plate, a PROTEOGRAPH™ experiment can be performed with a higher throughput than, for example, when each particle is placed in a separate well. As a simple illustration, multiplexing with two particles per well can improve the overall throughput by a factor of two. In some embodiments, the present disclosure provides systems and methods of using such compositions.

[0357] Another aspect of the present disclosure provides a composition comprising a particle and a solvent environment tailored to the particle. The tailored solvent environment can include certain solvents, salts, pH, etc. that can improve the number of biomolecules identified in the PROTEOGRAPH™ method. Another aspect of the present disclosure provides a composition comprising multiplexed particles and a solvent environment tailored to the multiplexed particles. The tailored solvent environment can increase the number of biomolecules identified, but can also improve the stability and / or dispersibility of the multiplexed particles. In some embodiments, the present disclosure provides systems and methods of using such compositions.

[0358] Another aspect of the present disclosure provides methods and systems for performing high-speed, scalable, deep, and unbiased plasma proteomics. In some cases, the methods and systems can be used to identify known and / or novel biomarkers for diseases. In some cases, for example, as disclosed in PCT / US2023 / 060271, which is hereby incorporated by reference in its entirety, the methods and systems can be used to facilitate the identification of protein variants associated with diseases. Important progress has been made in characterizing the proteomic landscape of lung cancer, such as non-small cell lung cancer (NSCLC) and squamous cell lung cancer, and important protein biomarkers have been identified. However, the number of proteoforms identified as being associated with lung cancer is relatively small. Readout techniques such as high-resolution quantitative mass spectrometry (MS) can be used to infer and quantify peptides and proteins with high confidence (e.g., a false discovery rate (FDR) of less than 1%). However, large-scale LC-MS / MS-based proteomics studies can be difficult because they require very long workflows to achieve deep (e.g., broad detection of proteins across a dynamic range from high-abundance to low-abundance proteins) and unbiased (e.g., hypothesis-free detection) sampling of clinically important biological specimens such as plasma, which have a large dynamic range of protein abundances. LC-MS and LC-MS / MS method systems can provide the ability to infer proteoforms, but peptide identification in LC-MS / MS-based proteomics data can rely on protein databases such as UniProt, and proteoforms that may be present in an individual's proteome may be excluded. In some cases, examples of alternative exon usage can be observed using the compositions, methods, and systems disclosed herein. In some cases, proteoforms resulting from alternative splicing can be identified using the compositions, methods, and systems disclosed herein. In some cases, proteoforms resulting from genetic variations can be identified using the compositions, methods, and systems disclosed herein.In some cases, using the compositions, methods, and systems disclosed herein, proteoforms can be identified based at least in part on a custom protein database generated from genotypic data that matches a subject, such as whole exome sequencing (WES) data. In some cases, using the compositions, methods, and systems disclosed herein, new proteoforms can be discovered. In some cases, using the compositions, methods, and systems disclosed herein, proteoforms that otherwise could not be identified by targeting techniques based on protein affinity can be identified. In some cases, using the compositions, methods, and systems disclosed herein, identifying proteoforms can support enhancing the understanding of human health and disease.

[0359] In some aspects, the present disclosure provides a method for selectively enriching and assaying a plurality of biomolecules in a fluid composition. In some embodiments, the method includes selectively enriching a first plurality of types of biomolecules in a first fluid composition. In some embodiments, the method includes selectively enriching a second plurality of types of biomolecules in a second fluid composition. In some embodiments, the method includes performing an assay downstream of the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules.

[0360] The methods, compositions, and systems disclosed herein can provide, for example, improved proteomics analysis of biological samples. In some embodiments, similar or more protein groups can be detected in a biological sample using fewer mass spectrometry injections and / or a shorter chromatography gradient in LC-MS / MS. For example, similar protein groups can be identified using two 30-minute gradient injections compared to five 30-minute gradient injections. In some embodiments, a similar protein group identification can still be obtained while using a lower sample volume.

[0361] Method for selectively enriching and assaying biomolecules In some embodiments, the step of selectively enriching a first plurality of types of biomolecules in a fluid composition comprises contacting a first fluid composition with a first surface and adsorbing the first plurality of types of biomolecules to the first surface. In some embodiments, the step of selectively enriching a second plurality of types of biomolecules in a fluid composition comprises contacting a second fluid composition with a second surface and adsorbing the second plurality of types of biomolecules to the second surface. In some embodiments, the step of selectively enriching a first plurality of types of biomolecules in a fluid composition comprises contacting a first fluid composition with a first plurality of types of surfaces and adsorbing the first plurality of types of biomolecules to the first plurality of types of surfaces. In some embodiments, the step of selectively enriching a second plurality of types of biomolecules in a fluid composition comprises contacting a second fluid composition with a second plurality of types of surfaces and adsorbing the second plurality of types of biomolecules to the second plurality of types of surfaces.

[0362] In some embodiments, the first plurality of surface types and the second plurality of surface types contain charges of the same sign. In some embodiments, the first plurality of surface types and the second plurality of surface types contain charges of opposite signs. In some embodiments, the first plurality of surface types and the second plurality of surface types have a zeta potential of the same sign. In some embodiments, the first plurality of surface types and the second plurality of surface types have a zeta potential of opposite signs. In some embodiments, the first plurality of surface types, the second plurality of surface types, or both contain acidic functional groups. In some embodiments, the acidic functional groups include Bronsted-Lowry acid or Lewis acid functional groups. In some embodiments, the first plurality of surface types, the second plurality of surface types, or both include carboxylic acid groups, acrylic acid groups, methacrylic acid groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphoric acid groups, phosphoric acid diester groups, boronic acid groups, boronic acid ester groups, boric acid groups, boric acid ester groups, silica groups, silanol groups, polymers, or any combination thereof. In some embodiments, the first plurality of surface types, the second plurality of surface types, or both contain basic functional groups. In some embodiments, the basic functional groups include Bronsted-Lowry acid or Lewis acid functional groups.

[0363] In some embodiments, the first fluid composition is incubated with the first surface at at least 30 °C for at least 20 minutes to adsorb the first plurality of biomolecule types to the first surface. In some embodiments, the second fluid composition is incubated with the second surface at at least 30 °C for at least 20 minutes to adsorb the second plurality of biomolecule types to the second surface. In some embodiments, the first fluid composition is incubated with the first surface at at least 30 °C for at least 20 minutes to adsorb the first plurality of biomolecule types to the first surface, and the second fluid composition is incubated with the second surface at at least 30 °C for at least 20 minutes to adsorb the second plurality of biomolecule types to the second surface.

[0364] In some embodiments, the first plurality of types of biomolecules are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the first biomolecule in the first fluid composition is present in a greater abundance than the second biomolecule. In some embodiments, the second plurality of types of biomolecules are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the abundance of the first biomolecule in the second fluid composition is greater than that of the second biomolecule. For example, as described herein, the first fluid composition and the second fluid composition may each contain the same particle composition, but may also contain different pH, solvents, temperatures, pressures, and the like. The first fluid composition and the second fluid composition can enrich and deplete different biomolecules even though the particle compositions are the same. In some cases, the first particle composition and the second particle composition can be different.

[0365] In some embodiments, the first surface is disposed in a first lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers. In some embodiments, the second surface is disposed in a second lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers. In some embodiments, the one or more modifiers include a pH modifier, an ionic strength modifier, a viscosity modifier, or any combination thereof. In some embodiments, the surface has an average zeta potential between 85% and 115% of the zeta potential of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement when the lyophilized composition is reconstituted in solution.

[0366] In some embodiments, the surface has an average zeta potential between 90% and 110% of the zeta potential of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement when the lyophilized composition is reconstituted in solution. In some embodiments, the surface has an average zeta potential between 95% and 105% of the zeta potential of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement when the lyophilized composition is reconstituted in solution. In some embodiments, the surface has an average zeta potential standard deviation between 85% and 115% of the zeta potential standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement when the lyophilized composition is reconstituted in solution. In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have an average zeta potential standard deviation between 90% and 110% of the zeta potential standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have a zeta potential standard deviation between 95% and 105% of the zeta potential standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have an average diameter between 85% and 115% of the average diameter of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have an average diameter between 90% and 110% of the average diameter of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have an average diameter between 95% and 105% of the average diameter of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have an average diameter between 98% and 102% of the average diameter of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS).In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have a diameter standard deviation between 85% and 115% of the diameter standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have a diameter standard deviation between 90% and 110% of the diameter standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have a diameter standard deviation between 95% and 105% of the diameter standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the particles have a diameter standard deviation between 98% and 102% of the diameter standard deviation of the same particles dissolved in the same solution in the absence of lyophilization, as determined by dynamic light scattering (DLS). The lyophilized composition can have stable physicochemical properties over various periods. In some cases, the period can include a period of at least about 12 days, at least about 14 days, at least about 30 days, at least 40 days, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year. The lyophilized composition can have stable physicochemical properties at various temperatures. In some cases, the temperature can be about room temperature. In some cases, the temperature can be about 37°C. In some cases, the temperature can be about 60°C. In some cases, the temperature can be about -26°C to about 0°C. In some cases, the temperature can be about -10°C to about -5°C. In some cases, the temperature can be about 0°C to 20°C. In some cases, the temperature can be about 0°C to about 10°C. In some cases, the temperature can be about 25°C to about 60°C. In some cases, the temperature can be about 35°C to about 40°C. In some cases, the dry composition or the lyophilized composition is stable at about 37°C for at least 40 days.In some cases, the dry composition or the lyophilized composition is stable at ambient temperature for at least 11 months.

[0367] In some embodiments, the surface is composed of particles dispersed in the composition during incubation. In some embodiments, the particles are nanoparticles. In some embodiments, the average diameter of the nanoparticles is less than 500 nanometers. In some embodiments, the particles are porous particles. In some embodiments, the particles are microparticles. In some embodiments, the particles contain a paramagnetic material. In some embodiments, the paramagnetic material is a superparamagnetic material. In some embodiments, the paramagnetic material includes iron oxide, aluminum, platinum, or any combination thereof. In some embodiments, the particles include an outer layer of poly(dimethylaminopropylmethacrylamide) (PDMAPMA). In some embodiments, the particles include an outer layer of silica. In some embodiments, the particles include a carboxylic acid-functionalized outer layer. In some embodiments, the particles include an amine-functionalized outer layer. In some embodiments, the particles include a polymer outer layer. In some embodiments, the particles include a polyacrylamide outer layer. In some embodiments, the particles include an acrylic acid polymer.

[0368] In some embodiments, the method further includes digesting the protein adsorbed on the first surface before the step of assaying the biomolecule. In some embodiments, the method further includes treating the protein in the biomolecule corona with a reducing agent before the step of assaying the biomolecule. In some embodiments, the method further includes treating the protein in the biomolecule corona with an alkylating agent before the step of assaying the biomolecule.

[0369] In some embodiments, the method further includes washing a first plurality of types of biomolecules with a first washing composition and washing a second plurality of types of biomolecules with a second washing composition. In some embodiments, the first fluid composition and the first washing composition include at least one common strong physical property. In some embodiments, the first fluid composition and the first washing composition include at least one different strong physical property. In some embodiments, the first fluid composition and the first washing composition include at least one common solvent. In some embodiments, the first fluid composition and the first washing composition include at least one different solvent. In some embodiments, the first fluid composition and the first washing composition include approximately the same ionic strength. In some embodiments, the first fluid composition and the first washing composition include different ionic strengths. In some embodiments, the first fluid composition and the first washing composition include at least one common salt. In some embodiments, the first fluid composition and the first washing composition include at least one different salt. In some embodiments, the first fluid composition and the first washing composition include approximately the same pH. In some embodiments, the first fluid composition and the first washing composition include different pHs.

[0370] In some embodiments, the second fluid composition and the second washing composition include at least one common strong physical property. In some embodiments, the second fluid composition and the second washing composition include at least one different strong physical property. In some embodiments, the second fluid composition and the second washing composition include at least one common solvent. In some embodiments, the second fluid composition and the second washing composition include at least one different solvent. In some embodiments, the second fluid composition and the second washing composition include approximately the same ionic strength. In some embodiments, the second fluid composition and the second washing composition include different ionic strengths. In some embodiments, the second fluid composition and the second washing composition include at least one common salt. In some embodiments, the second fluid composition and the second washing composition include at least one different salt.

[0371] In some embodiments, the first cleaning composition and the second cleaning composition include at least one common strong physical property. In some embodiments, the first cleaning composition and the second cleaning composition include at least one different strong physical property. In some embodiments, the first cleaning composition and the second cleaning composition have approximately the same ionic strength. In some embodiments, the first cleaning composition and the second cleaning composition have different ionic strengths. In some embodiments, the first cleaning composition and the second cleaning composition include at least one common salt. In some embodiments, the first cleaning composition and the second cleaning composition include at least one different salt. In some embodiments, the first cleaning composition and the second cleaning composition have approximately the same pH. In some embodiments, the first cleaning composition and the second cleaning composition have different pHs. In some embodiments, the first cleaning composition and the second cleaning composition are the same. In some embodiments, the first cleaning composition and the second cleaning composition are different. Various indicatives, solvents, salts, and pHs are described elsewhere in this specification.

[0372] In some embodiments, the first cleaning composition releases a first plurality of types of biomolecules adsorbed on the first surface from the first surface. In some embodiments, the method further includes a step of purifying the first plurality of types of biomolecules to produce a first purified composition. In some embodiments, the purifying step includes drying the plurality of types of biomolecules to remove the first cleaning composition. In some embodiments, the method further includes a step of reconstituting the first purified composition using a first reconstitution composition to produce a first reconstituted composition. In some embodiments, the second cleaning composition releases a second plurality of types of biomolecules deposited on the second surface from the second surface. In some embodiments, the method further includes a step of purifying the second plurality of types of biomolecules to produce a second purified composition. In some embodiments, the purifying step includes drying the second plurality of types of biomolecules to remove the second cleaning composition. In some embodiments, the method further includes a step of reconstituting the second purified composition using a second reconstitution composition to produce a second reconstituted composition.

[0373] The first or second plurality of types of biomolecules may include peptides. The peptides may include derivatives cleaved by proteolysis of proteins adsorbed on the first or second surface. The peptides may be those cleaved by proteolysis by a protease, such as trypsin or lysin. Drying may include applying a negative pressure (e.g., a negative gauge pressure relative to atmospheric pressure) while heating or cooling the sample during drying, if necessary. Drying can be allowed to proceed to the extent that solvent evaporation is no longer observable, e.g., by a change in mass. Drying can be carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes. Drying can be carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 24, 36, or 48 hours. Drying can be carried out for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes. Drying can be carried out for a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 24, 36, or 48 hours. Drying can be carried out at about room temperature. Drying can be carried out at a temperature of at least -200, -150, -100, -50, -25, 0, 25, 50, 75, or 100 °C. Drying can be carried out at a temperature of a maximum of -200, -150, -100, -50, -25, 0, 25, 50, 75, or 100 °C. Drying can be carried out at a negative gauge pressure of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilopascals (kPa). Drying can be carried out at a negative gauge pressure of a maximum of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kPa. Reconstitution can be carried out by adding a buffer. Various buffer compositions are described elsewhere in this specification. The first reconstitution composition and the second reconstitution composition may be the same or different. The first biological sample and the second biological sample, when reconstituted, may contain peptides at an approximate predetermined concentration.The predetermined concentration can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / μL (biomolecule mass / buffer liquid volume). The predetermined concentration can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / μL (biomolecule mass / buffer liquid volume).

[0374] Various analytical methods for identifying peptide or protein species can be used in the step of assaying (e.g., downstream assay). In some embodiments, the step of assaying includes SDS-PAGE, gel-based separation techniques, immunoassay, ELISA, high performance liquid chromatography, mass spectrometry, Edman degradation, or immunoaffinity techniques. In some embodiments, the step of assaying includes mass spectrometry. In some embodiments, the step of assaying includes LC-MS / MS. In some embodiments, the step of assaying includes quantifying biomolecules. In some embodiments, the step of assaying includes identifying one or more proteins in a biological sample. In some embodiments, the downstream assay includes mass spectrometry. In some embodiments, the downstream assay includes protein sequencing. In some embodiments, the downstream assay includes LC-MS / MS. In some embodiments, the downstream assay includes immunoassay. In some embodiments, the downstream assay includes contacting a first plurality of biomolecule species, a second plurality of biomolecule species, or both with a pair of antibodies capable of binding to at least one biomolecule species among the first plurality of biomolecule species, the second plurality of biomolecule species, or both, wherein the pair of antibodies includes a complementary single-stranded nucleic acid sequence bound thereto such that when the pair of antibodies binds to at least one biomolecule species, a complementary nucleic acid hybridizes to form a double-stranded nucleic acid, and the double-stranded nucleic acid forms a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction and generate a detectable signal. In some embodiments, the downstream assay includes contacting a first plurality of biomolecule species, a second plurality of biomolecule species, or both with one or more aptamers capable of binding to at least one biomolecule species among the first plurality of biomolecule species, the second plurality of biomolecule species, or both, wherein the one or more aptamers are bound to a surface via a cleavable linker. In some embodiments, the cleavable linker is a linker cleavable by light.In some embodiments, the surface is a particle surface. In some embodiments, the method further includes contacting one or more aptamers in a fluid composition with a polymeric competitor configured to reduce dissociation of a complex composed of the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, with the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both. In some embodiments, the polymeric competitor is further configured to bind to a type of biomolecule different from the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both. In some embodiments, the polymeric competitor is a polyanionic polymer. In some embodiments, the polymeric competitor is a polycationic polymer. In some embodiments, the downstream assay includes nucleic acid sequencing.

[0375] In some embodiments, the assaying step is performed at a rate at which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100 samples are assayed per hour. In some embodiments, the assaying step is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour. In some embodiments, the assaying step is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour. In some embodiments, the assaying step is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour. The assay throughput can be measured using a standard, such as a HeLa cell extract. For example, in some embodiments, if the suspension contains a HeLa cell extract, at least about 100, 1000, 10000, or 100000 biomolecules are identified in the assaying step. In some embodiments, when the assaying step is performed on a plurality of biomolecules in a biological sample in the absence of the first and second particles, the plurality of biomolecules includes at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0376] In some embodiments, the first likelihood that a biomolecule present in a low abundance among a plurality of biomolecules is identified in the assaying step is higher than the second likelihood that a biomolecule present in a low abundance in a biological sample is identified in the assaying step in the absence of the first and second particles, where the biomolecule present in a low abundance is less than about 1 weight percent, about 10 -1 weight percent, about 10 -2 weight percent, about 10 -3 weight percent, about 10 -4 weight percent, about 10 -5 weight percent, about 10 -6Less than a mass percentage, about 10 -7 Less than a mass percentage, about 10 -8 Less than a mass percentage, about 10 -9 Less than a mass percentage, or about 10 -10 Comprises less than a mass percentage. In some embodiments, the first possibility is at least 2, 5, 10, 10 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 times larger. In some embodiments, one or more of the plurality of biomolecules are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent in the step of assaying. In some embodiments, one or more of the plurality of biomolecules are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent in the step of assaying.

[0377] In some embodiments, the method includes forming a first suspension comprising a first plurality of particles and a first portion of a biological sample. In some embodiments, the first plurality of particles includes a first particle and a second particle. In some embodiments, the first particle has a first functionalized surface chemistry structure that is different from a second functionalized surface chemistry structure of the second particle. In some embodiments, the first particle has a first functionalized surface chemistry structure that is the same as a second functionalized surface chemistry structure of the second particle. In some embodiments, both the first particle and the second particle have a negative charge. In some embodiments, both the first particle and the second particle have a positive charge. In some embodiments, both the first particle and the second particle have a substantially neutral charge. In some embodiments, both the first particle and the second particle have a substantially zwitterionic charge. In some embodiments, the method includes forming a second suspension comprising a second plurality of particles and a second portion of a biological sample. In some embodiments, the second plurality of particles includes a third particle and a fourth particle. In some embodiments, the third particle has a third functionalized surface chemistry structure that is different from a fourth functionalized surface chemistry structure of the fourth particle. In some embodiments, the third particle has a third functionalized surface chemistry structure that is the same as a fourth functionalized surface chemistry structure of the fourth particle. In some embodiments, both the third particle and the fourth particle have a positive charge. In some embodiments, both the third particle and the fourth particle have a negative charge. In some embodiments, the third particle and the fourth particle have a substantially neutral charge. In some embodiments, the third particle and the fourth particle have a zwitterionic charge. In some embodiments, the method includes enriching biomolecules adsorbed to the first plurality of particles. In some embodiments, the method includes enriching biomolecules adsorbed to the second plurality of particles. In some embodiments, the method includes performing a downstream assay on the enriched biomolecules.

[0378] In some embodiments, the first suspension and the second suspension each have a pH between 9 and 10. In some embodiments, the first suspension and the second suspension each contain a Tris buffer. In some embodiments, the first suspension has a pH between 9 and 10. In some embodiments, the first suspension contains a Tris buffer. In some embodiments, the second suspension has a pH between 6 and 8. The first suspension and the second suspension may have the same or different compositions and / or properties. Various compositional parameters (e.g., buffer, salt, pH, etc.) are described elsewhere in this specification. The suspensions described herein may contain various compositions described elsewhere in this specification.

[0379] In some embodiments, the first particles are magnetic particles. In some embodiments, the first particles include an outer layer surface-functionalized with silanol. In some embodiments, the second particles are magnetic particles. In some embodiments, the second particles include an outer layer surface-functionalized with carboxylic acid. In some embodiments, the third particles are magnetic particles. In some embodiments, the third particles include an outer layer surface-functionalized with amine. In some embodiments, the fourth particles are magnetic particles. In some embodiments, the fourth particles include an outer layer surface-functionalized with amine. In some embodiments, the outer layer of the third particles contains a polymer. In some embodiments, the first plurality of particles has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles. In some embodiments, the second plurality of particles has a ratio of about 10 to about 15 parts by weight of the third particles to about 1 part by weight of the fourth particles. In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is about 3:2 to about 2:3. In some embodiments, the ratio of the average diameter of the third particles to the average diameter of the fourth particles is about 3:2 to about 2:3. In some embodiments, the first particles, the second particles, the third particles, and the fourth particles are each superparamagnetic iron oxide nanoparticles. Various particle compositions are described elsewhere in this specification. The first particles, the second particles, the third particles, and the fourth particles may contain various particles described herein in various ratios. System for selectively enriching and assaying biomolecules

[0380] Various parallel or sequential schemes can be used in the PROTEOGRAPH™ system to increase throughput. In some embodiments, the first fluid composition and the second fluid composition are processed on one machine. In some embodiments, the first fluid composition and the second fluid composition are processed on different machines. In some embodiments, the first fluid composition and the second fluid composition are processed in parallel. In some embodiments, the first fluid composition and the second fluid composition are run sequentially. In some embodiments, the first fluid composition and the second fluid composition are part of the same sample. In some embodiments, the first fluid composition and the second fluid composition are from different samples. In some embodiments, the step of selectively enriching is performed in parallel on the first fluid composition and the second fluid composition. In some embodiments, the step of selectively enriching is performed sequentially on the first fluid composition and the second fluid composition. In some embodiments, the step of selectively enriching is performed at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are enriched per hour. In some embodiments, contacting is performed in parallel on the first fluid composition and the second fluid composition. In some embodiments, contacting is performed sequentially on the first fluid composition and the second fluid composition. In some embodiments, contacting is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules deposit per hour. In some embodiments, contacting is performed at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules deposit per hour. In some embodiments, the step of washing is performed in parallel on the first fluid composition and the second fluid composition. In some embodiments, the step of washing is performed sequentially on the first fluid composition and the second fluid composition. In some embodiments, the step of washing is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are washed per hour.In some embodiments, the washing step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are washed per hour. In some embodiments, the purification step is carried out in parallel with respect to the first fluid composition and the second fluid composition. In some embodiments, the purification step is carried out sequentially with respect to the first fluid composition and the second fluid composition. In some embodiments, the purification step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 are purified per hour. In some embodiments, the purification step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are purified per hour. In some embodiments, the reconstitution step is carried out in parallel with respect to the first fluid composition and the second fluid composition. In some embodiments, the reconstitution step is carried out sequentially with respect to the first fluid composition and the second fluid composition. In some embodiments, the reconstitution step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 are reconstituted per hour. In some embodiments, the reconstitution step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are reconstituted per hour. In some embodiments, the downstream assay is carried out in parallel with respect to the first fluid composition and the second fluid composition. In some embodiments, the downstream assay is carried out sequentially with respect to the first fluid composition and the second fluid composition. In some embodiments, the downstream assay is carried out at a rate such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples are assayed per hour. In some embodiments, the downstream assay is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour.In some embodiments, the downstream assay is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour. In some embodiments, the downstream assay is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour. In some embodiments, the method is performed in parallel on a first fluid composition and a second fluid composition. In some embodiments, the method is performed sequentially on a first fluid composition and a second fluid composition. In some embodiments, the method is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour. In some embodiments, the method is performed at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour. In some embodiments, the method is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour. In some embodiments, the method is performed at a rate such that at least 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour. The rate of biomolecule identification can be measured using an appropriate standard. In some embodiments, when the first fluid composition and the second fluid composition are HeLa cell extracts, at least about 100, 1000, 10000, 100000, or 1000000 biomolecules are identified in the downstream assay.

[0381] In some embodiments, one or more biomolecules in the first plurality of biomolecule types, the second plurality of biomolecule types, or both, in a downstream assay are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent. In some embodiments, one or more biomolecules in the first plurality of biomolecule types, the second plurality of biomolecule types, or both, in a downstream assay are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent. In some embodiments, the enriched biomolecules are identified by the assay with a coefficient of variation of at most 30%. In some embodiments, the enriched biomolecules are identified by the assay with a coefficient of variation of at most 25%. In some embodiments, the enriched biomolecules are identified by the assay with a coefficient of variation of at least 0%.

[0382] In some embodiments, when the downstream assay is performed before the step of selectively enriching the first fluid composition and the second fluid composition for (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together include at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges. In some embodiments, when the downstream assay is performed before the step of selectively enriching the first fluid composition and the second fluid composition for (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together include at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0383] In some embodiments, the first likelihood that a biomolecule present in low abundance in the first plurality of biomolecule types or the second plurality of biomolecule types is identified in the downstream assay is higher than the second likelihood that a biomolecule present in low abundance in the first fluid composition or the second fluid composition is identified in the downstream assay, and the biomolecule present in low abundance is less than about 1 weight percent, about 10 -1 weight percent, about 10 -2 weight percent, about 10 -3less than a mass percentage, about 10 -4 less than a mass percentage, about 10 -5 less than a mass percentage, about 10 -6 less than a mass percentage, about 10 -7 less than a mass percentage, about 10 -8 less than a mass percentage, about 10 -9 less than a mass percentage, or about 10 -10 includes a first set of biomolecules or a second set of biomolecules that are less than a mass percentage. In some embodiments, the first likelihood is at least 2, 5, 10, 10 2 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、or 10 10 times higher.

[0384] In some embodiments, at least 250 different biomolecules are identified in a biological sample. In some embodiments, at least 500 different biomolecules are identified in a biological sample. The biomolecules can be proteins. In some embodiments, one or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 peptides. In some embodiments, one or more biomolecules comprise up to about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 peptides. In some embodiments, one or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins. In some embodiments, one or more biomolecules comprise up to about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins. Compositions and kits for selectively enriching and assaying biomolecules

[0385] In one aspect, the present disclosure provides a suspension for assaying biomolecules derived from a biological sample. The suspension can include first particles comprising (i) a first paramagnetic moiety and (ii) a first surface chemistry. The suspension can include second particles comprising (i) a second paramagnetic moiety and (ii) a second surface chemistry. The second surface chemistry can be different from the first surface chemistry. A plurality of biomolecules can adsorb to the first particles and the second particles. The suspension can be a multiplex of physically distinct particles provided, for example, as a single continuous solution phase in one well.

[0386] The physicochemical properties of the first and second particles can be such that these particles become colloidal stable or semi-stable in the suspension. The physicochemical properties of the first and second particles can be such that these particles disperse in the suspension. The physicochemical properties of the first and second particles can be such that these particles can disperse in the suspension when agitated. The agitation can be, for example, magnetic agitation of the particles if the particles are magnetic particles. Without wishing to be bound by a particular theory, the stability of the colloidal suspension can be partially explained by the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory. From this theory, it is suggested that the stability of the colloidal suspension depends at least on the Bjerrum length, the Debye–Hückel screening length, the charge of the particles, and the size of the particles. The DLVO theory does not explain all of the possible forces that can affect colloidal stability. It is notable, for example, that depletion forces are not explained by this theory. The Bjerrum length is the distance at which electrostatic interactions and thermal energy are equal. The Debye–Hückel screening length is a measure of the extent to which an electrostatic field propagates through a medium and, in an electrolyte solution, depends at least on the dielectric constant and the ionic strength of the solution. Thus, in addition to the charge and size of the particles, the solvent environment of the suspension can define and / or affect the stability of the suspension. The first and second particles can contain charges of the same sign. The charge can be, for example, the zeta potential of the particles. The charges of the same sign can be negative, positive, or nearly neutral. The suspension can be stable for at least about 1, 5, 10, 15, 30, or 60 minutes. The suspension can be stable for up to about 1, 5, 10, 15, 30, or 60 minutes. The time constant for destabilization of the suspension is at least about 1, 5, 10, 15, 30, or 60 minutes. The time constant for destabilization of the suspension is at most about 1, 5, 10, 15, 30, or 60 minutes. The average aggregation number of the first and second particles in the suspension can be at most about 1, 10, or 100. The average aggregation number of the first and second particles in the suspension can be at most about 1000, 100, or 10. The suspension can contain at least about 3, 4, 5, 6, 7, 8, 9, or 10 distinct particles.In some embodiments, the suspension is incubated at a temperature of at least 30 °C.

[0387] The first surface chemistry structure, the second surface chemistry structure, or both may include acidic functional groups. Acidic functional groups include Bronsted-Lowry acid or Lewis acid functional groups. The first surface chemistry structure, the second surface chemistry structure, or both may include carboxylic acid groups, acrylic acid groups, methacrylic acid groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphoric acid groups, phosphoric acid diester groups, boronic acid groups, boronic acid ester groups, boric acid groups, boric acid ester groups, silica groups, silanol groups, polymers, or any combination thereof.

[0388] The suspension may contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particle for about 1 part of the second particle. The suspension may contain up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particle for about 1 part of the second particle. The suspension may contain about 15 parts of the first particle for about 6 parts of the second particle. The parts may be by weight, by volume, or by surface area. The first size of the first particle may be at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle. The first size of the first particle may be up to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle. The first size may be the first diameter, and the second size may be the second diameter. The first size may be the first average size, and the second size may be the second average size. The first average size and the second average size may be the size average value or the size median. The ratio of the average diameter of the first particle to the average diameter of the second particle may be from about 3:2 to about 2:3.

[0389] The first particle, the second particle, or both may be nanoparticles. The first particle, the second particle, or both may be microparticles. The first particle, the second particle, or both may be porous. The plurality of biomolecules may contain up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. The plurality of biomolecules may contain at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. The plurality of biomolecules may contain up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per 1 mL. The plurality of biomolecules may contain at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per 1 mL.

[0390] In some cases, the compositions, methods, and systems disclosed herein may provide spatially or temporally distinct biomolecular compositions of small volume samples (e.g., individual cells). Spatially distinct biomolecular compositions can be obtained by sampling biomolecules from different parts of a cell (e.g., different compartments within a cell) or different parts of a tissue (e.g., healthy cells versus cancerous cells in a tumor, or cells from the epidermis, dermis, and subcutaneous tissue of the skin). Temporally distinct biomolecular compositions can be obtained by sampling biomolecules at different times (e.g., cells before and after treatment with a potential therapeutic agent). In some cases, the systems and methods disclosed herein may provide distinct biomolecular compositions across a population of subjects (e.g., tumor cells from subjects treated with a potential chemotherapeutic agent versus tumor cells from untreated subjects). Different transcriptome or proteomics information between samples can be provided by targeting various biomolecules (e.g., proteins or nucleic acids). The plurality of biomolecules may contain biomolecules derived from up to about 1000, 100, 10, or 1 cell. The plurality of biomolecules may contain biomolecules derived from at least about 1000, 100, 10, or 1 cell.

[0391] The first fluid composition and the second fluid composition may include the same or different intensive properties. Intensive properties may refer to properties that are not affected by the amount of the substance being measured. Intensive properties can be, for example, temperature, pressure, density, concentration of components, pH, ionic strength, heat capacity, viscosity, surface tension. In some embodiments, the first fluid composition includes a first set of strong physical properties that mediate the selective enrichment of a first plurality of types of biomolecules. In some embodiments, the second fluid composition includes a second set of strong physical properties that mediate the selective enrichment of a second plurality of types of biomolecules. In some embodiments, the first set of strong physical properties is different from the second set of strong physical properties. In some embodiments, the first set of strong physical properties is the same as the second set of strong physical properties.

[0392] In some embodiments, the first fluid composition includes a first ratio between a first sample volume and a first surface area of a first surface. In some embodiments, the second fluid composition includes a second ratio between a second sample volume and a second surface area of a second surface. In some embodiments, the first ratio is different from the second ratio. In some embodiments, the first ratio is the same as the second ratio. The ratio can be, for example, at least 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 cm 2 per microliter of sample volume of the surface area of the surface. The ratio can be, for example, at most 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 cm 2 per microliter of sample volume of the surface area of the surface.

[0393] In some embodiments, the first fluid composition and the second fluid composition include different temperatures. In some embodiments, the first fluid composition and the second fluid composition include the same temperature. The temperature can be at least 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 degrees Celsius. The temperature can be at most 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 degrees Celsius.

[0394] In some embodiments, the first fluid composition comprises a first ionic strength and the second fluid composition comprises a second ionic strength. In some embodiments, the first ionic strength and the second ionic strength are different. In some embodiments, the first ionic strength and the second ionic strength are the same. In some embodiments, the first fluid composition comprises a first ion concentration and the second fluid composition comprises a second ion concentration. In some embodiments, the first ion concentration and the second ion concentration are different. In some embodiments, the first ion concentration and the second ion concentration are the same. The ion concentration may be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, or 7 M. The ion concentration may be at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, or 7 M.

[0395] In some embodiments, the first fluid composition and the second fluid composition contain different solvents. In some embodiments, the first fluid composition and the second fluid composition contain the same solvent. The solvent can include, for example, acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane (glyme, DME), dimethyl-formamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide, hexamethylphosphorous triamide, hexane, methanol, methyl t-butyl ether, methylene chloride, N-methyl 2-pyrrolidinone, nitromethane, pentane, petroleum ether, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, triethylamine, water, o-xylene, m-xylene, p-xylene, or any combination thereof. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing tris(hydroxymethyl)aminomethane. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing citrate. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing glycine. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing phosphate. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing carbonate. In some embodiments, the first fluid composition, the second fluid composition, or both contain a buffer solution containing N-cyclohexyl-3-aminopropanesulfonic acid. In some embodiments, the first fluid composition contains an aqueous buffer solution. In some embodiments, the second fluid composition contains an aqueous buffer solution.In some embodiments, the first fluid composition includes an aqueous buffer solution, and the second fluid composition does not include a buffer solution.

[0396] In some embodiments, the first fluid composition and the second fluid composition contain different salts. In some embodiments, the first fluid composition and the second fluid composition contain the same salt. The salts can include, for example, ammonium sulfate, sodium sulfate, sodium chloride, potassium chloride, ammonium acetate, and the like.

[0397] In some embodiments, the first fluid composition includes a first pH and the second fluid composition includes a second pH. In some embodiments, the first pH and the second pH are the same. In some embodiments, the first pH and the second pH are different. In some embodiments, the first fluid composition, the second fluid composition, or both include a pH between 2 and 4. In some embodiments, the first fluid composition, the second fluid composition, or both include a pH between 5 and 7. In some embodiments, the first fluid composition, the second fluid composition, or both include a pH between 9 and 10. In some embodiments, the first fluid composition, the second fluid composition, or both include a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the first fluid composition, the second fluid composition, or both include a pH of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the method may further include diluting a sample to create the first composition, the second composition, or both. In some embodiments, the diluting step includes adding a buffer. In some embodiments, the buffer includes a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the buffer includes a pH of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the sample and the buffer include different pHs. In some embodiments, the sample and the buffer include substantially the same pH. In some embodiments, the second composition has a pH between 5 and 9. In some embodiments, the second composition has a pH between 6 and 8. In some embodiments, the second composition has a pH between 8 and 11. In some embodiments, the second composition has a pH between 9 and 10. In some embodiments, the second composition does not include a buffer. In some embodiments, the second composition includes a buffer different from a buffer configured to maintain the pH of the composition between 8 and 11. In some embodiments, the second composition includes the same buffer as the composition. In some embodiments, both the composition and the second composition include a Tris buffer having a pH between 9 and 10.In some embodiments, the step of incubating the composition and the step of incubating the second composition are performed simultaneously. In some embodiments, the composition comprises a buffer. In some embodiments, the buffer is configured to maintain the pH of the composition between 8 and 11. In some embodiments, the buffer is configured to maintain the pH of the composition between 9 and 10. In some embodiments, the buffer is configured to maintain the pH of the composition at about 9.5. In some embodiments, the buffer is a Tris buffer. In some embodiments, the buffer is an aqueous buffer.

[0398] The composition and / or properties of the first fluid composition, the second fluid composition, or both can be used as features for identifying a biological state associated with a sample. For example, the features can be used in a machine learning algorithm. The features can include the identity of biomolecules, the particle(s) used to assay the biomolecules, temperature, pH, etc. Thus, various compositions and / or properties can provide additional features for stratifying biological samples to distinguish biological states. In some embodiments, the method further includes identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first fluid composition. In some embodiments, the method further includes identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second fluid composition. In some embodiments, the method further includes identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first washing composition. In some embodiments, the method further includes identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second washing composition. In some embodiments, the method further includes identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first reconstitution composition. In some embodiments, the method further includes identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstitution composition.

[0399] In some embodiments, the present disclosure provides a kit for selectively enriching and assaying a plurality of biomolecules in a fluid composition. In some embodiments, the kit includes a first reagent having a first pH. In some embodiments, the kit includes a second reagent having a second pH. In some embodiments, the kit includes a first surface configured to selectively enrich a first plurality of biomolecule types in a first fluid composition including the first surface and the first reagent. In some embodiments, the kit includes a second surface configured to selectively enrich a second plurality of biomolecule types in a second fluid composition including the second surface and the second reagent. In some embodiments, the first fluid composition includes the first reagent. In some embodiments, the second fluid composition includes the second reagent. In some embodiments, the first pH is different from the second pH.

[0400] In some embodiments, the present disclosure provides a kit including a first composition including a first particle and a second particle. The first particle may have a first functionalized surface chemistry structure different from a second functionalized surface chemistry structure of the second particle. The first particle and the second particle may both have a negative charge. The first composition may be solid. The kit may further include a second composition including a third particle and a fourth particle. The third particle may have a third functionalized surface chemistry structure different from a fourth functionalized surface chemistry structure of the fourth particle. The third particle and the fourth particle may both have a positive charge. The third composition may be solid.

[0401] The kit may include a buffer having a pH between 9 and 10. The kit may include a Tris buffer having a pH between 9 and 10. The first composition and the second composition may both be lyophilized. In some embodiments, the first particles are magnetic particles and include an outer layer surface-functionalized with silanol. In some embodiments, the second particles are magnetic particles and include an outer layer surface-functionalized with carboxylic acid. In some embodiments, the third particles are magnetic particles and include an outer layer surface-functionalized with amine. In some embodiments, the fourth particles are magnetic particles and include an outer layer surface-functionalized with amine. In some embodiments, the outer layer of the third particles includes a polymer. In some embodiments, the first composition has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles. In some embodiments, the second composition has a ratio of about 10 to about 15 parts by weight of the third particles to about 1 part by weight of the fourth particles. In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is about 3:2 to about 2:3. In some embodiments, the ratio of the average diameter of the third particles to the average diameter of the fourth particles is about 3:2 to about 2:3. In some embodiments, each of the first particles, the second particles, the third particles, and the fourth particles is a superparamagnetic iron oxide nanoparticle. In some embodiments, the first composition has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles. In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is about 3:2 to about 2:3. In some embodiments, the first particles and the second particles are superparamagnetic iron oxide nanoparticles.

[0402] In some embodiments, the present disclosure provides a composition for selectively enriching and assaying a plurality of biomolecules in a fluid composition. In some embodiments, the composition comprises a suspension. In some embodiments, the suspension comprises a first particle. In some embodiments, the first particle comprises a first paramagnetic moiety. In some embodiments, the first particle comprises a first surface chemistry. In some embodiments, the suspension comprises a second particle. In some embodiments, the second particle comprises a second paramagnetic moiety. In some embodiments, the second particle comprises a second surface chemistry. In some embodiments, the second surface chemistry is different from the first surface chemistry. In some embodiments, the suspension comprises a plurality of biomolecules adsorbed to the first particle. In some embodiments, the suspension comprises a plurality of biomolecules adsorbed to the second particle.

[0403] In some embodiments, the present disclosure provides a suspension. In some embodiments, the suspension comprises first particles. In some embodiments, the first particles comprise an outer layer surface-functionalized with a negative charge. In some embodiments, the first particles comprise a first outer layer surface-functionalized with a positive charge. In some embodiments, the suspension comprises second particles comprising a second outer layer surface-functionalized with a negative charge. In some embodiments, the second particles comprise a second outer layer surface-functionalized with a positive charge. In some embodiments, both the first particles and the second particles have a negative charge. In some embodiments, both the first particles and the second particles have a positive charge. In some embodiments, the biomolecule adsorbs to the outer layer of the first particles. In some embodiments, the second outer layer is different from the first outer layer. In some embodiments, the biomolecule adsorbs to the second outer layer of the second particles. In some embodiments, the biomolecule adsorbs to the first outer layer of the first particles and the second outer layer of the particles. In some embodiments, the first particles comprise a first functionalized surface chemical structure. In some embodiments, the second particles comprise a second functionalized surface chemical structure. In some embodiments, the first functionalized surface chemical structure is different from the second functionalized surface chemical structure of the second particles. In some embodiments, the first outer layer comprises silanol. In some embodiments, the second outer layer comprises carboxylic acid. In some embodiments, the first outer layer and the second outer layer comprise an amine group. In some embodiments, the first particles are magnetic particles and comprise an outer layer surface-functionalized with an amine. In some embodiments, the second particles are magnetic particles and comprise an outer layer surface-functionalized with an amine. In some embodiments, the second outer layer is different from the first outer layer. In some embodiments, the biomolecule comprises at least 100 different peptides or proteins. In some embodiments, the first particles and optionally the second particles are magnetic.

[0404] In some embodiments, the suspension comprises a buffer solution. In some embodiments, the buffer solution is configured to maintain the pH of the suspension between 9 and 10. In some embodiments, the buffer solution is a Tris buffer solution. In some embodiments, the buffer solution is configured to maintain the pH between about 8 and about 11. In some embodiments, the buffer solution is configured to maintain a pH between 9 and 10. In some embodiments, the buffer solution is a Tris buffer solution having a pH between 9 and 10. In some embodiments, the first particles are magnetic particles and comprise an outer layer surface-functionalized with silanol, and the second particles are magnetic particles and comprise an outer layer surface-functionalized with carboxylic acid.

[0405] In some embodiments, the outer layer of the first particles comprises a polymer. In some embodiments, the suspension has a ratio of about 10 to about 15 parts by weight of the first particles to about 1 part by weight of the second particles. In some embodiments, the ratio of the average diameter of the first particles to the average diameter of the second particles is about 3:2 to about 2:3. In some embodiments, the first particles and the second particles are superparamagnetic iron oxide nanoparticles.

[0406] In some embodiments, the present disclosure provides a system for analyzing a plurality of biological samples. In some embodiments, the system includes a plurality of compartments including a first compartment and a second compartment. In some embodiments, the system includes a plurality of reagent storages including a first reagent and a second reagent. In some embodiments, the first reagent includes a first pH. In some embodiments, the second reagent includes a second pH. In some embodiments, the first pH and the second pH are different. In some embodiments, the system includes a plurality of substrates including a first substrate and a second substrate. In some embodiments, the first substrate includes a first surface chemistry. In some embodiments, the second substrate includes a second surface chemistry. In some embodiments, the system includes one or more transfer devices operably connected to the plurality of compartments, the plurality of reagent storages, the plurality of substrates, or any combination thereof. In some embodiments, the system includes at least one processor and a computer including instructions executable by the at least one processor to perform steps of analyzing a plurality of biological samples. In some embodiments, the instructions can include using one or more transfer devices to generate a first fluid composition within a first compartment including a first substrate, a first reagent, and a first plurality of biomolecules. In some embodiments, the first plurality of biomolecules adsorb to the first substrate. In some embodiments, the instructions can include using one or more transfer devices to generate a second fluid composition within a second compartment including a second substrate, a second reagent, and a second plurality of biomolecules. In some embodiments, the second plurality of biomolecules adsorb to the second substrate. In some embodiments, the instructions can include using one or more transfer devices to prepare the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry.

[0407] In some aspects, the present disclosure provides a system for analyzing a plurality of biological samples. In some embodiments, the system includes compartments. In some embodiments, the system includes a reagent storage unit containing reagents. In some embodiments, the system includes a plurality of substrates including a first substrate having a first surface chemistry and a second substrate having a second surface chemistry. In some embodiments, the first surface chemistry is different from the second surface chemistry. In some embodiments, the system includes one or more transfer devices operably connected to the compartments, the reagent storage unit, the plurality of substrates, or any combination thereof. In some embodiments, the system includes at least one processor and a computer including instructions executable by the at least one processor to perform the step of analyzing a plurality of biological samples. In some embodiments, the instructions may include the step of using one or more transfer devices to generate a fluid composition within a compartment containing a plurality of substrates, reagents, and a plurality of biomolecules, wherein the plurality of biomolecules adsorb to the substrates. In some embodiments, the instructions may include the step of using one or more transfer devices to prepare a plurality of biomolecules for mass spectrometry.

[0408] biological sample The sample may be of various amounts. In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. In some embodiments, the sample contains at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per 1 mL of the sample. In some embodiments, the sample contains at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per 1 mL of the sample. In some embodiments, the sample contains biomolecules derived from up to about 1000, 100, 10, or 1 cell. In some embodiments, the sample contains biomolecules derived from at least about 1000, 100, 10, or 1 cell. In some embodiments, the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters. In some embodiments, the sample contains at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters.

[0409] The composition and / or properties of the first fluid composition and the second fluid composition can be designed such that the dynamic range and / or number of peptides, proteins, or groups of proteins detected is increased using PROTEOGRAPH™ with different types of samples. In some embodiments, the sample includes a complex biological sample. In some embodiments, the complex biological sample can include plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal mucus, ear discharge, gastric juice, pancreatic juice, trabecular bone fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a cotton swab, bronchial aspirate fluid, flowing solids, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof. In some embodiments, the biological sample includes plasma.

[0410] In some embodiments, the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both include one or more polyamino acids. A polyamino acid can refer to any biomolecule that contains two or more amino acids. The amino acids may be the same or different. The amino acids may be covalently bonded. The covalent bond may be a peptide bond. The polyamino acid may include a peptide, a protein, or both.

[0411] In some embodiments, the biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal mucus, ear discharge, gastric juice, pancreatic juice, trabecular bone fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a cotton swab, bronchial aspirate fluid, fluid solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof. In some embodiments, the biological sample is plasma or serum. In some embodiments, the biological sample is cell culture medium. In some embodiments, the composition includes 60% by volume or less of the biological sample. In some embodiments, the composition includes 25% by volume or less of the biological sample. In some embodiments, the composition includes 15% by volume or less of the biological sample. In some embodiments, the composition includes at least 1% by volume of the biological sample. In some embodiments, the composition includes at least 4% by volume of the biological sample. In some embodiments, the composition includes at least 10% by volume of the biological sample. In some embodiments, the composition includes at least 20% by volume of the biological sample.

[0412] The systems and methods of the present disclosure for assaying a biological sample. In some cases, the biological sample may contain cells or may be cell-free. In some cases, the biological sample may include a biological fluid such as blood, serum, plasma, urine, or cerebrospinal fluid (CSF). In some cases, the biological fluid may be a flowing solid, such as a tissue homogenate, or a fluid extracted from a biological sample. The biological sample may be, for example, a tissue sample or a fine needle aspiration (FNA) sample. The biological sample may be a cell culture sample. For example, the biological fluid may be a fluidized cell culture extract. In some cases, the biological sample may be obtained from a subject. In some cases, the subject may be human or non-human. In some cases, the subject may be a plant, a fungus, or an archaebacterium. In some cases, the biological sample may contain a plurality of proteins or proteomic data, which can be analyzed by adsorbing or binding the proteins to the surface of various types of sensor elements (e.g., particles) in a panel and then digesting the protein corona.

[0413] In some cases, the biological sample may include plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid from swabbings, bronchial aspirant, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof. In some cases, the biological sample may include a plurality of biological samples (e.g., pooled plasma from multiple subjects, or multiple tissue samples from a single subject). In some cases, the biological sample may include a single type of biological fluid or biomaterial from a single source.

[0414] In some cases, the biological sample can be diluted or pretreated. In some cases, the biological sample may be depleted before or after contact with the surfaces disclosed herein (e.g., the biological sample contains serum). In some cases, the biological sample may be subjected to physical treatment (e.g., homogenization or sonication) or chemical treatment before or after contact with the surfaces disclosed herein. In some cases, the biological sample can be diluted before or after contact with the surfaces disclosed herein. In some cases, the diluent medium can contain a buffer or salt, or can be purified water (e.g., distilled water). In some cases, the biological sample can be provided in multiple compartments, where each compartment can be subjected to a different degree of dilution. In some cases, the biological sample may comprise or may undergo at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold, or 1000-fold dilution. In some cases, the biological sample can be diluted using a buffer that modifies the pH of the biological sample. In some cases, the pH can be modified to about 2, 3, 4, 5, 6, 7, 8, 9, 10. In some cases, the pH can be modified to 9-10. In some cases, the pH is modified by diluting with a pH 9.5 Tris buffer. In some cases, the biological sample can be divided into portions, the portions adjusted to different pHs, and then treated using the methods disclosed herein.

[0415] In some cases, a biological sample can contain multiple biomolecules. In some cases, the multiple biomolecules can include polyamino acids. In some cases, the polyamino acids can include peptides, proteins, or combinations thereof. In some cases, the multiple biomolecules can include nucleic acids, carbohydrates, polyamino acids, or any combination thereof. A biological sample can include members of any class of biomolecules, where "class" can refer to any named category (e.g., protein, nucleic acid, carbohydrate) that defines a group of biomolecules having common characteristics.

[0416] Proteome analysis As used herein, "proteome analysis", "protein analysis", etc. can refer to any system or method for analyzing proteins in a sample, including the systems and methods disclosed herein. The systems and methods of the present disclosure for assaying using one or more surfaces. In some cases, the surface can include the surface of a material with a large surface area, such as nanoparticles, particles, or porous materials. As used herein, "surface" can refer to a surface for assaying polyamino acids. It should be understood that in some cases, when the particle composition, physical properties, or its use is described herein, the surface of the particle can include the same composition, the same physical properties, or the same use thereof. Similarly, when the surface composition, physical properties, or its use is described herein, it should be understood that the particle can include a surface having the same composition, the same physical properties, or the same use thereof.

[0417] Materials for the particles and surfaces can include metals, polymers, magnetic materials, and lipids. In some cases, the magnetic particles can be iron oxide particles. Examples of metallic materials can include any one or any combination of gold, silver, copper, nickel, cobalt, palladium, platinum, iridium, osmium, rhodium, ruthenium, rhenium, vanadium, chromium, manganese, niobium, molybdenum, tungsten, tantalum, iron, cadmium, or any alloys thereof. In some cases, the particles disclosed herein can be magnetic particles such as superparamagnetic iron oxide nanoparticles (SPIONs). In some cases, the magnetic particles can be ferromagnetic particles, ferrimagnetic particles, paramagnetic particles, superparamagnetic particles, or any combination thereof (e.g., the particles can include ferromagnetic materials and ferrimagnetic materials).

[0418] The present disclosure describes a panel of particles or surfaces. In some cases, the panel can include more than one distinct surface type. The panels described herein can vary the number and diversity of surface types within a single panel. For example, the surfaces within the panel can be varied based on size, polydispersity, shape and morphology, surface charge, surface chemical structure and functionalization, and substrate. In some cases, the panel can be incubated with a sample being analyzed for polyamino acids, polyamino acid concentration, nucleic acids, nucleic acid concentration, or any combination thereof. In some cases, polyamino acids in the sample are adsorbed onto distinct surfaces to form one or more adsorbed layers of biomolecules. The identity and concentration of the biomolecules in the one or more adsorbed layers can depend on the physical properties of the distinct surfaces and the physical properties of the biomolecules. Thus, each surface type within the panel can have biomolecules adsorbed differently due to different sets of biomolecules, different concentrations of a particular biomolecule, or a combination thereof. Each surface type within the panel can have mutually exclusive adsorbed biomolecules or overlapping adsorbed biomolecules.

[0419] In some cases, the panels disclosed herein can be used to identify the number of distinct biomolecules disclosed herein over a wide dynamic range in a given biological sample. For example, the panel can enrich for a subset of biomolecules in the sample, thereby enabling identification over a wide dynamic range where the biomolecules are present in the sample (e.g., a plasma sample). In some cases, the enrichment can be selective; for example, it can enrich for biomolecules within the subset but not, and / or can deplete, biomolecules outside the subset. In some cases, the subset can include proteins having different post-translational modifications. For example, the first particle type within the particle panel can enrich for a protein or group of proteins having a first post-translational modification, the second particle type within the particle panel can enrich for the same protein or group of proteins having a second post-translational modification, and the third particle type within the particle panel can enrich for the same protein or group of proteins lacking post-translational modification. In some cases, a panel comprising any number of distinct particle types disclosed herein enriches and identifies a single protein or group of proteins by binding to different domains, sequences, or epitopes of the protein or group of proteins. For example, the first particle type within the particle panel can enrich for a protein or group of proteins by binding to a first domain of the protein or group of proteins, and the second particle type within the particle panel can enrich for the same protein or group of proteins by binding to a second domain of the protein or group of proteins. In some cases, a panel comprising any number of distinct particle types disclosed herein can enrich and identify biomolecules over a dynamic range of at least 5, 6, 7, 8, 9, 10, 15, or 20 orders of magnitude. In some cases, a panel comprising any number of distinct particle types disclosed herein can enrich and identify biomolecules over a dynamic range of up to 5, 6, 7, 8, 9, 10, 15, or 20 orders of magnitude.

[0420] The panel can have more than one surface type. Increasing the number of surface types within the panel can be one way to increase the number of proteins that can be identified in a given sample.

[0421] The particle or surface can include a polymer. The polymer can constitute a core material (e.g., the core of the particle can include the particle), a layer (e.g., the particle can include a layer of polymer disposed between its core and its shell), a shell material (e.g., the surface of the particle can be coated with a polymer), or any combination thereof. Examples of polymers include polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), or a copolymer of two or more polymers (e.g., a copolymer of polyalkylene glycol (e.g., PEG) and polyester (e.g., PLGA), etc.), any one of them, or any combination thereof. The polymer can include crosslinking. The plurality of polymers within the particle may be phase-separated or may include a certain degree of phase separation.

[0422] Examples of lipids that can be used to form the particles or surfaces of the present disclosure include cationic, anionic, and neutral charged lipids. For example, the particles and / or surfaces can be dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleyolphosphatidylglycerol (POPG), lecithin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidyl-ethanolamine (POPE) palmitoyloleyolphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidyl-ethanolamine (POPE),Any one of 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, cholesterol, and any combination thereof, or it may be composed of any combination thereof.

[0423] The particle panel can include a combination of particles having silica and polymer surfaces. For example, the particle panel can include particles coated with an outer layer of silica and particles coated with an outer layer of poly(dimethylaminopropylmethacrylamide) (PDMAPMA). A particle panel consistent with the present disclosure can also include two or more particles selected from the group consisting of silica-coated particles, N-(3-trimethoxysilylpropyl)diethylenetriamine-coated particles, PDMAPMA-coated particles, carboxyl-functionalized polyacrylic acid-coated particles, amino surface-functionalized particles, polystyrene carboxyl-functionalized particles, and dextran-coated particles. A particle panel consistent with the present disclosure can also include two or more particles selected from the group consisting of carboxylic acid-functionalized particles without surfactants, carboxyl-functionalized polystyrene particles, silica-coated particles, silica particles, dextran-coated particles, oleic acid-coated particles, boronated nanopowder-coated particles, PDMAPMA-coated particles, poly(glycidyl-benzylamine methacrylate)-coated particles, and poly(N-[3-(dimethylamino)propyl]methacrylamide-co-[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, P(DMAPMA-co-SBMA)-coated particles. A particle panel consistent with the present disclosure can include silica-coated particles, N-(3-trimethoxysilylpropyl)diethylenetriamine-coated particles, poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated particles, phosphosugar-functionalized polystyrene particles, amine-functionalized polystyrene particles, polystyrene carboxyl-functionalized particles, ubiquitin-functionalized polystyrene particles, dextran-coated particles, or any combination thereof. In some cases, the particle panel can include silica-coated particles, amine-functionalized particles, amine-functionalized polymer-coated particles, and carboxylic acid-functionalized particles.

[0424] Particle panels consistent with the present disclosure can include silica-functionalized particles, amine-functionalized particles, silicon alkoxide-functionalized particles, carboxylic acid-functionalized particles, and benzyl or phenyl-functionalized particles. Particle panels consistent with the present disclosure can include silica-functionalized particles, amine-functionalized particles, silicon alkoxide-functionalized particles, polystyrene-functionalized particles, and saccharide-functionalized particles. Particle panels consistent with the present disclosure can include silica-functionalized particles, N-(3-trimethoxysilylpropyl)diethylenetriamine-functionalized particles, PDMAPMA-functionalized particles, dextran-functionalized particles, and polystyrene carboxyl-functionalized particles. Particle panels consistent with the present disclosure can include five particles including silica-functionalized particles, amine-functionalized particles, and silicon alkoxide-functionalized particles.

[0425] Separate surfaces or separate particles of the present disclosure may have one or more different physicochemical properties. The one or more physicochemical properties are selected from the group consisting of composition, size, surface charge, hydrophobicity, hydrophilicity, roughness, density, surface functionalization, surface topography, surface curvature, porosity, core material, shell material, shape, and any combination thereof. Surface functionalization can include polymer functionalization, small molecule functionalization, or any combination thereof. Small molecule functionalization can include aminopropyl functionalization, amine functionalization, boronic acid functionalization, carboxylic acid functionalization, alkyl group functionalization, N-succinimidyl ester functionalization, monosaccharide functionalization, phosphosugar functionalization, sulfated sugar functionalization, ethylene glycol functionalization, streptavidin functionalization, methyl ether functionalization, trimethoxysilylpropyl functionalization, silica functionalization, triethoxylpropylaminosilane functionalization, thiol functionalization, PCP functionalization, citric acid functionalization, lipoic acid functionalization, ethyleneimine functionalization. The particle panel can include a plurality of particles having a plurality of small molecule functionalizations selected from the group consisting of silica functionalization, trimethoxysilylpropyl functionalization, dimethylaminopropyl functionalization, phosphosugar functionalization, amine functionalization, and carboxyl functionalization.

[0426] Small molecule functionalization may include polar functional groups. Non-limiting examples of polar functional groups include carboxyl groups, hydroxyl groups, thiol groups, cyano groups, nitro groups, ammonium groups, imidazolium groups, sulfonium groups, pyridinium groups, pyrrolidinium groups, phosphonium groups, or any combination thereof. In some embodiments, the functional group is an acidic functional group (e.g., sulfonic acid group, carboxyl group, etc.), a basic functional group, a carbamoyl group, a hydroxyl group, an aldehyde group, etc. In some cases, the polar functional group may include a primary amine group, a secondary amine group, a tertiary amine group, a quaternary amine group, a cyclic secondary amine group, a primary amide group, a secondary amide group, a tertiary amide group, an imine group, a pyridyl group, a pyrimidine group, a pyrrolidinium group, an imidazole group, a guanidine group, a guanidinium group, or any combination thereof.

[0427] Small molecule functionalization may include ionic or ionizable functional groups. Non-limiting examples of ionic or ionizable functional groups include ammonium groups, imidazolium groups, sulfonium groups, pyridinium groups, pyrrolidinium groups, phosphonium groups. Small molecule functionalization may include polymerizable functional groups. Non-limiting examples of polymerizable functional groups include vinyl groups and (meth)acrylic groups. In some embodiments, the functional group is pyrrolidyl acrylate, acrylic acid, methacrylic acid, acrylamide, 2-(dimethylamino)ethyl methacrylate, hydroxyethyl methacrylate, etc.

[0428] Surface functionalization can include a charge. For example, the particles can be functionalized to have a net neutral surface charge, a net positive surface charge, a net negative surface charge, or a zwitterionic surface. The surface charge can be a determinant of the types of biomolecules that collect on the particle. Thus, optimizing a particle panel can include selecting particles with different surface charges, thereby increasing not only the number of different proteins that collect on the particle panel, but also increasing the likelihood that the biological state of the sample can be identified. The particle panel can include positively charged particles and negatively charged particles. The particle panel can include positively charged particles and neutral particles. The particle panel can include positively charged particles and zwitterionic particles. The particle panel can include neutral particles and negatively charged particles. The particle panel can include neutral particles and zwitterionic particles. The particle panel can include negatively charged particles and zwitterionic particles. The particle panel can include positively charged particles, negatively charged particles, and neutral particles. The particle panel can include positively charged particles, negatively charged particles, and zwitterionic particles. The particle panel can include positively charged particles, neutral particles, and zwitterionic particles. The particle panel can include negatively charged particles, neutral particles, and zwitterionic particles. In some cases, positively charged particles can have a zeta potential greater than 0 mV, greater than 5 mV, greater than 10 mV, greater than 15 mV, greater than 20 mV, greater than 25 mV, greater than 50 mV, or greater than 100 mV. In some cases, negatively charged particles can have a zeta potential less than 0 mV, less than -5 mV, less than -10 mV, less than -15 mV, less than -20 mV, less than -25 mV, less than -50 mV, or less than -100 mV.

[0429] Particles can include a single surface, such as a particular small molecule, or multiple surface functionalizations, such as multiple different small molecules. Surface functionalization can affect the composition of the biomolecular corona of the particle. Such surface functionalization can include small molecule functionalization or polymer functionalization. The surface functionalization can be coupled to the particle material, such as a polymer, metal, metal oxide, inorganic oxide (e.g., silicon dioxide), or another surface functionalization.

[0430] Surface functionalization can include small molecule functionalization, polymer functionalization, or a combination of two or more such functionalizations. In some cases, polymer functionalization can include biopolymers such as proteins or polynucleotides (e.g., 100mer DNA molecules). Polymer functionalization can include proteins, polynucleotides, or polysaccharides, or can be of a size equivalent to any of the species of the above classes. In some cases, surface functionalization can include ionizable moieties. In some cases, surface functionalization can include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pKas. In some cases, surface functionalization can include up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pKas. In some cases, small molecule functionalization can include organic small molecules such as alcohols (e.g., octanol), amines, alkanes, alkenes, alkynes, heterocycles (e.g., piperidinyl groups), heteroaromatic groups, thiols, carboxylic acids, carbonyls, amides, esters, thioesters, carbonates, thiocarbonates, carbamates, thiocarbamates, ureas, thioureas, halogens, sulfates, phosphates, monosaccharides, disaccharides, lipids, or any combination thereof. For example, small molecule functionalization can include sugar phosphates, sugar acids, or sulfated sugars.

[0431] In some cases, polymer functionalization can involve the attachment of a specific form to the particles. In some cases, the polymer can be tethered to the particles via a linker. In some cases, the linker can hold the polymer near the particles, thereby restricting the movement and reorientation of the polymer with respect to the particles, or can extend the polymer away from the particles. In some cases, the linker can be rigid (e.g., a polyolefin linker) or flexible (e.g., a nucleic acid linker). In some cases, the linker can be at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nm in length. In some cases, the linker can be up to about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nm in length. Thus, surface functionalization with respect to the particles can be protruded beyond the initial corona associated with the particles. In some cases, the surface functionalization can also be positioned directly below or within the biomolecular corona formed on the particle surface. In some cases, the polymer can also be tethered to a specific location, e.g., the C-terminus of a protein, or to several possible sites. For example, a peptide can be attached to the particles covalently via any of the lysine residues exposed on its surface.

[0432] In some cases, particles can be contacted with a biological sample (e.g., a biological fluid) to form a biomolecular corona. In some cases, the biomolecular corona can include at least two biomolecules that do not share a common binding motif. The particles and the biomolecular corona can be separated from the biological sample, for example, by centrifugation, magnetic separation, filtration, or gravitational separation. The particle type and the biomolecular corona can be separated from the biological sample using several separation techniques. Non-limiting examples of separation techniques include magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density or gradient-based centrifugation, gravitational separation, or any combination thereof. Protein corona analysis can be performed on the separated particles and biomolecular corona. Protein corona analysis can include identifying one or more proteins in the biomolecular corona, for example, by mass spectrometry. In some cases, a single particle type can be contacted with a biological sample. In some cases, multiple particle types can be contacted with a biological sample. In some cases, multiple particle types can be combined and contacted with a biological sample in a single sample volume. In some cases, multiple particle types can be sequentially contacted with a biological sample and, after separation from the biological sample, subsequent particle types can be contacted with the biological sample. In some cases, the biomolecules adsorbed on the particles can have a compressed (e.g., smaller) dynamic range compared to a given original biological sample.

[0433] In some cases, using the particles of the present disclosure, a continuous investigation of a sample can be carried out by incubating a first particle type with the sample to form a biomolecular corona on the first particle type, separating the first particle type, incubating a second particle type with the sample to form a biomolecular corona on the second particle type, separating the second particle type, and repeating the investigation (by incubating with the sample) and separation for any number of particle types. In some cases, the biomolecular corona on each particle type used in the continuous investigation of the sample can be analyzed by protein corona analysis. The biomolecular content of the supernatant can be analyzed after a continuous investigation using one or more particle types.

[0434] In some cases, the methods of the present disclosure can identify unique biomolecules (e.g., proteins) of multiple species in a biological sample (e.g., a biological fluid). In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb up to 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 groups of unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb up to 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 groups of unique biomolecules. In some cases, several different types of surfaces can be used separately or in combination to identify multiple proteins in a particular biological sample. In other words, the surfaces can be multiplexed to bind and identify multiple biomolecules in a biological sample.

[0435] In some cases, by the methods of the present disclosure, a large number of unique proteoforms in a biological sample can be identified. In some cases, the method can identify at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique proteoforms. In some cases, the method can identify up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique proteoforms. In some cases, the surfaces disclosed herein are incubated with a biological sample to adsorb at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique proteoforms. In some cases, the surfaces disclosed herein are incubated with a biological sample to adsorb up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 unique proteoforms. In some cases, several different types of surfaces can be used separately or in combination to identify a large number of proteins in a particular biological sample. In other words, the surfaces can be multiplexed to bind and identify a large number of biomolecules in a biological sample.

[0436] The biomolecules collected on the particles can be subjected to further analysis. In some cases, the method may include collecting a biomolecular corona or a subset of biomolecules derived from the biomolecular corona. In some cases, the collected biomolecular corona or the collected subset of biomolecules derived from the biomolecular corona can be subjected to further particle-based analysis (e.g., particle adsorption). In some cases, the collected biomolecular corona or the collected subset of biomolecules derived from the biomolecular corona can be purified or fractionated (e.g., by chromatography methods). In some cases, the collected biomolecular corona or the collected subset of biomolecules derived from the biomolecular corona can be analyzed (e.g., by mass spectrometry).

[0437] In some cases, the panels disclosed herein can be used to identify a number of proteins, peptides, protein groups, or protein classes using the protein analysis workflows described herein (e.g., protein corona analysis workflows). In some cases, the panels disclosed herein are used to identify at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 unique proteins. In some cases, the panels disclosed herein are used to identify up to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 unique proteins. In some cases, the panels disclosed herein are used to identify at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 protein groups. In some cases, the panels disclosed herein are used to identify up to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 protein groups.In some cases, using the panels disclosed herein, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 peptides can be identified. In some cases, using the panels disclosed herein, up to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 peptides can be identified. In some cases, the peptide can be a tryptic peptide. In some cases, the peptide can be a semi-tryptic peptide. In some cases, protein analysis can include contacting a sample with a separate surface type (e.g., a particle panel), forming a layer of biomolecules adsorbed on the separate surface type, and identifying the biomolecules in the layer of adsorbed biomolecules (e.g., by mass spectrometry). The signature intensity disclosed herein can refer to the intensity of an individual spike ("signature") seen in a plot of mass-to-charge ratio and intensity from performing mass spectrometry on a sample. In some cases, these signatures can correspond to peptides and / or fragments of proteins that are variably ionized. In some cases, using the data analysis methods described herein, signature intensities can be sorted into protein groups. In some cases, a protein group can refer to two or more proteins identified by a shared peptide sequence. In some cases, a protein group can refer to one protein identified using a unique identification sequence.For example, in a sample, if a peptide sequence common between two proteins (Protein 1: XYZZX and Protein 2: XYZYZ) is assayed, the protein group can be a "XYZ protein group" having two members (Protein 1 and Protein 2). In some cases, if the peptide sequence is specific to only one protein (Protein 1), the protein group can be a "ZZX" protein group having one member (Protein 1). In some cases, each protein group can be supported by more than one peptide sequence. In some cases, the proteins detected or identified according to the present disclosure can refer to distinct proteins detected in the sample (e.g., distinct from other proteins detected using mass spectrometry). In some cases, the analysis of proteins present in distinct coronas corresponding to distinct surface types in a panel gives rise to a number of feature intensities. In some cases, this number decreases when the feature intensities are processed into distinct peptides, further decreases when the distinct peptides are processed into distinct proteins, and further decreases when the peptides are grouped into protein groups (two or more proteins sharing distinct peptide sequences).

[0438] In some cases, the methods disclosed herein include isolating one or more particle types from one sample or from more than one sample (e.g., biological samples or samples that are serially assayed). The particle types can be rapidly isolated or separated from the sample using a magnet. Additionally, multiple spatially isolated samples can be processed in parallel. In some cases, the methods disclosed herein provide for the isolation or separation of particle types from unbound proteins in a sample. In some cases, the particle types can be separated by various means including, but not limited to, magnetic separation, centrifugation, filtration, or gravitational separation. In some cases, a particle panel can be incubated with multiple spatially isolated samples, where the spatially isolated samples are each in a well of a well plate (e.g., a 96-well plate). In some cases, by placing the entire plate on a magnet, the particles in each well of the well plate can be separated from unbound proteins present in the spatially isolated samples. In some cases, this simultaneously pulls down the superparamagnetic particles within the particle panel. In some cases, the supernatant of each sample can be removed to remove unbound proteins. In some cases, these steps (incubate, pull down) can be repeated to effectively wash the particles and thus remove any remaining background unbound proteins that may be present in the sample.

[0439] In some cases, the systems and methods disclosed herein can also elucidate protein classes or interactions between protein classes. In some cases, protein classes include sets of proteins that share a common function (e.g., amine oxidases or proteins involved in angiogenesis); proteins that share a common physiological localization, cellular localization, or intracellular localization (e.g., peroxisomal proteins or membrane proteins); proteins that share a common cofactor (e.g., heme or flavin proteins); proteins corresponding to a particular biological state (e.g., proteins associated with hypoxia); proteins containing a particular structural motif (e.g., cupin fold); functionally related proteins (e.g., part of the same metabolic pathway); or proteins having post-translational modifications (e.g., ubiquitinated proteins or citrullinated proteins). In some cases, a protein class can contain at least 2, 5, 10, 20, 40, 60, 80, 100, 150, 200, or more proteins.

[0440] In some cases, proteomic data from biological samples can be identified, measured, and quantified using several different analytical techniques. For example, SDS-PAGE or any gel-based separation technique can be used to generate proteomic data. In some cases, immunoassays such as ELISA can also be used to identify, measure, and quantify peptides and proteins. In some cases, mass spectrometry, high performance liquid chromatography, LC-MS / MS, Edman degradation, immunoaffinity techniques, and other protein separation techniques can be used to identify, measure, and quantify proteomic data.

[0441] In some cases, the assay may include protein collection of the particles, protein digestion, and mass spectrometry (e.g., MS, LC-MS, LC-MS / MS). In some cases, the digestion may include chemical digestion, e.g., digestion with cyanogen bromide or 2-nitro-5-thiocyanatobenzoic acid (NTCB). In some cases, the digestion may include enzymatic digestion, e.g., digestion with trypsin or pepsin. In some cases, the digestion may include enzymatic digestion with multiple proteases. In some cases, the digestion may include a protease selected from the group consisting of trypsin, chymotrypsin, Glu C, Lys C, elastase, subtilisin, proteinase K, thrombin, factor X, Arg C, papain, Asp N, thermolysin, pepsin, aspartyl protease, cathepsin D, zinc metalloprotease, glycoprotein endopeptidase, prolidase, aminopeptidase, prenyl protease, caspase, kex2 endoprotease, or any combination thereof. In some cases, the digestion can cleave peptides at random positions. In some cases, the digestion can cleave peptides at specific positions (e.g., methionine) or sequences (e.g., glutamate-histidine-glutamate). In some cases, the digestion may enable the identification of similar proteins. For example, in one assay, 8 distinct proteins can be broken down as a single protein group using a first digestion method and as 8 separate proteins with distinct signals using a second digestion method. In some cases, the digestion can generate an average peptide fragment length of 8 to 15 amino acids. In some cases, the digestion can generate an average peptide fragment length of 12 to 18 amino acids. In some cases, the digestion can generate an average peptide fragment length of 15 to 25 amino acids. In some cases, the digestion can generate an average peptide fragment length of 20 to 30 amino acids. In some cases, the digestion can generate an average peptide fragment length of 30 to 50 amino acids.

[0442] In some cases, proteins or peptides can be prepared for mass spectrometry. In some cases, proteins or peptides can be treated with an alkylating agent. For example, proteins or peptides can be treated with N-ethylmaleimide and iodoacetamide. In some cases, proteins or peptides can be treated with a reducing agent. For example, proteins or peptides are treated with dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In some cases, proteins or peptides are digested, alkylated, reduced, and then analyzed. In some cases, proteins or peptides are digested, alkylated, reduced, and then analyzed using mass spectrometry.

[0443] In some cases, proteomics data can be rapidly generated and analyzed by an assay. In some cases, in the methods of the present disclosure, starting from an input biological sample (e.g., a buccal or nasal smear, plasma, or tissue), proteomics data can be generated and analyzed in less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 12 hours, less than about 16 hours, less than about 20 hours, less than about 24 hours, or less than about 48 hours. In some cases, the analysis can include identifying a protein group. In some cases, the analysis can include identifying a protein class. In some cases, the analysis can include quantifying the abundance of a biomolecule, peptide, protein, protein group, or protein class. In some cases, the analysis can include identifying the ratio of the abundances of two biomolecules, peptides, proteins, protein groups, or protein classes. In some cases, the analysis can include identifying a biological state.

[0444] Examples of the types of particles of the present disclosure include carboxylate (citrate) superparamagnetic iron oxide nanoparticles (SPIONs), SPIONs coated with phenol-formaldehyde, SPIONs coated with silica, SPIONs coated with polystyrene, SPIONs coated with carboxylated poly(styrene-co-methacrylic acid), SPIONs coated with N-(3-trimethoxysilylpropyl)diethylenetriamine, SPIONs coated with poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), SPIONs coated with 1,2,4,5-benzenetetracarboxylic acid, SPIONs coated with poly(vinylbenzyltrimethylammonium chloride) (PVBTMAC), carboxylate, SPIONs coated with PAA, SPIONs coated with poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), carboxylate microparticles, polystyrene carboxyl-functionalized particles, particles coated with carboxylic acid, silica particles, carboxylic acid particles having a diameter of about 150 nm, amino surface microparticles having a diameter of about 0.4 - 0.6 μm, silica amino-functionalized microparticles having a diameter of about 0.1 - 0.39 μm, Jeffamine surface particles having a diameter of about 0.1 - 0.39 μm, polystyrene microparticles having a diameter of about 2.0 - 2.9 μm, silica particles, carboxylated particles having a unique coating with a diameter of about 50 nm, particles coated with a dextran-based coating having a diameter of about 0.13 μm, or particles coated with silica silanol having a low acidity. In some cases, the particles may lack a functionalized specific binding moiety for specific binding to their surface. In some cases, the particles may lack a functionalized protein for specific binding onto their surface. In some cases, the surface-functionalized particles do not contain an antibody or T cell receptor, chimeric antigen receptor, receptor protein, or variant or fragment thereof. In some cases, the ratio of surface area to mass can be a determinant of the properties of the particles. The particles disclosed herein have a ratio of surface area to mass of 3 - 30 cm 2 / mg, 5 - 50 cm 2 / mg, 10 - 60 cm 2 / mg, 15 - 70 cm 2 / mg, 20 - 80 cm 2 / mg, 30 - 100 cm 2 / mg, 35 - 120 cm 2 / mg, 40 - 130 cm 2 / mg, 45 - 150 cm 2 / mg, 50 - 160 cm 2 / mg, 60 - 180 cm 2 / mg, 70 - 200 cm 2 / mg, 80 - 220 cm 2 / mg, 90 - 240 cm 2 / mg, 100 - 270 cm 2 / mg, 120 - 300 cm 2 / mg, 200 - 500 cm 2 / mg, 10 - 300 cm 2 / mg, 1 - 3000 cm 2 / mg, 20 - 150 cm 2 / mg, 25 - 120 cm 2 / mg, or 40 - 85 cm 2 / mg may be. Small particles (e.g., having a diameter of 50 nm or less) may have a significantly higher ratio of surface area to mass in some cases due to the higher - order dependence of the diameter on mass rather than surface area in part. In some cases (e.g., for small particles), the ratio of the surface area to the mass of the particles is 200 - 1000 cm 2 / mg, 500 - 2000 cm 2 / mg, 1000 - 4000 cm 2 / mg, 2000 - 8000 cm 2 / mg, or 4000 - 10000 cm 2 / mg may be. In some cases (e.g., for large particles), the ratio of the surface area to the mass of the particles is 1 - 3 cm 2 / mg, 0.5 - 2 cm 2 / mg, 0.25 - 1.5 cm 2 / mg, or 0.1 - 1 cm 2It may be in mg. The particles can include a wide range of physical properties. Examples of the physical properties of the particles can include composition, size, surface charge, hydrophobicity, hydrophilicity, amphiphilicity, surface functionality, surface topography, surface curvature, porosity, core material, shell material, shape, zeta potential, and any combination thereof. The particles can have a core-shell structure. In some cases, the core material can include metal, polymer, magnetic material, paramagnetic substance, oxide, and / or lipid. In some cases, the shell material can include metal, polymer, magnetic material, oxide, and / or lipid.

[0445] In some cases, the particles can include nanoparticles. In some cases, the particles can include microparticles. In some cases, the first particle, the second particle, or both particles in the particle panel are nanoparticles. In some cases, the first particle, the second particle, or both particles in the particle panel are microparticles. In some cases, the first particle can be a nanoparticle and the second particle can be a microparticle.

[0446] In some cases, the particles can include a diameter of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nm. In some cases, the particles can include a maximum diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nm. In some cases, the particles can include a diameter of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 µm. In some cases, the particles can include a maximum diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 µm.

[0447] In some cases, the first size of the first particle is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle. In some cases, the first size of the first particle is at most about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particle. In some cases, the size of the first particle is within ±40% of the size of the second particle, the size of the first particle is within ±30% of the size of the second particle, the size of the first particle is within ±25% of the size of the second particle, the size of the first particle is within ±20% of the size of the second particle, the size of the first particle is within ±15% of the size of the second particle, or the size of the first particle is within ±10% of the size of the second particle. In some cases, the first size is the first diameter and the second size is the second diameter. In some cases, the first size is the first average size and the second size is the second average size. In some cases, the first average size and the second average size are the size average value or the size median.

[0448] In some cases, the particle panel includes at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 parts of a first particle for about 1 part of a second particle. In some cases, the particle panel includes up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 parts of a first particle for about 1 part of a second particle. In some cases, the particle panel includes about 15 parts of a first particle for about 6 parts of a second particle. In some cases, parts are parts by weight, parts by volume, or parts by surface area. In some cases, parts are parts by weight. In some cases, the particle panel includes about 5 to about 20 parts by weight of a first particle for about 1 part by weight of a second particle. In some cases, the particle panel includes about 10 to about 15 parts by weight of a first particle for about 1 part by weight of a second particle.

[0449] Proteomics information In some cases, proteomics information or data can refer to information regarding a substance containing peptide and / or protein components. In some cases, proteomics information can include primary structure information, secondary structure information, tertiary structure information, or quaternary structure information regarding a peptide or protein. In some cases, proteomics information can include information regarding protein-ligand interactions, where the ligand can include any one of various biomolecules and substances found in a living organism, such as nucleotides, nucleic acids, amino acids, peptides, proteins, monosaccharides, polysaccharides, lipids, phospholipids, hormones, or any combination thereof.

[0450] In some cases, proteomics information can include information regarding a single cell, tissue, organ, tissue and / or organ system (e.g., cardiovascular system, respiratory system, digestive system, or nervous system, etc.), or an entire multicellular organism. In some cases, proteomics information can include information regarding an individual (e.g., an individual human or an individual bacterium), or a population of individuals (e.g., humans diagnosed with cancer or a bacterial colony). Proteomics information can include information from various forms of life, including forms of life from Archaea, Bacteria, Eukarya, Protozoa, Chromista, Plantae, Fungi, or Animalia. In some cases, proteomics information can include information from viruses.

[0451] In some cases, proteomics information can include information related to exons and introns in the genome of that organism. In some cases, proteomics information can include information regarding variations in the primary structure, secondary structure, tertiary structure, or quaternary structure of peptides and / or proteins. In some cases, proteomics information can include information regarding variations in exon expression, including alternative splicing variations, structural variations, or both. In some cases, proteomics information can include information regarding the conformation information, post-translational modification information, chemical modification information (e.g., phosphorylation), cofactor (e.g., salt or other regulatory chemical) related information, or substrate related information of peptides and / or proteins.

[0452] In some cases, proteomics information can include information related to various proteoforms in a sample. In some cases, proteomics information can include information related to peptide variants, protein variants, or both. In some cases, proteomics information can include information related to splicing variants, allelic variants, post-translational modification variants, or any combination thereof.

[0453] In some cases, a splicing variant (also sometimes referred to as an "alternative splicing" variant, "differential splicing" variant, or "alternative RNA splicing" variant) can refer to a protein expressed by an alternative splicing process. In some cases, one or more splicing variants can be expressed by different combinations of exons from a set of exons by an alternative splicing process. In some cases, the combinations can include different sequences of exons as compared to another combination. In some cases, the combinations can include different subsets of exons as compared to another combination. In some cases, a splicing variant can include a rearranged amino acid sequence of another splicing variant.

[0454] In some cases, an allelic variant can refer to a protein expressed from a gene that contains a mutation as compared to a reference gene. In some cases, the reference gene can be a gene of a cell, an individual, or a population of individuals. In some cases, the mutation can be a base substitution, base deletion, or base insertion in the gene sequence of the gene as compared to a genetic reference of the reference gene. In some cases, an allelic variant can include an amino acid substitution in the amino acid sequence of another allelic variant.

[0455] In some cases, a post-translational modification can refer to a protein that has been modified after expression. The protein can be modified by various enzymes. In some cases, the enzyme that can modify the protein can be a kinase, protease, ligase, phosphatase, transferase, phosphotransferase, or any other enzyme that performs any one of the modifications disclosed herein.

[0456] In some cases, the peptide variant or protein variant may include post-translational modifications. In some cases, post-translational modifications include acylation, alkylation, prenylation, flavination, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, SUMOylation, NEDDylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hyposine formation, beta-lysine addition, acetylation, formylation, methylation, amidation, amide bond formation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester formation, phosphoramidate formation, adenylation, uridinylation, propionylation, pyroglutamate formation, glutathionylation, sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, glycation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, citrullination, deamidation, eliminylation, disulfide bond formation, proteolytic cleavage, isoaspartic acid formation, racemization, protein splicing, chaperone-mediated folding, or any combination thereof.

[0457] In some cases, proteomics information can be encoded as digital information. In some cases, proteomics information can include one or more elements representing the proteomics information. In some cases, the elements can represent primary structure information, secondary structure information, tertiary structure information, or quaternary structure information regarding a peptide or a protein. In some cases, the elements can represent protein-ligand interactions for a peptide or a protein. In some cases, the elements can represent the source of a peptide or a protein (e.g., a specific cell, tissue, organ, organism, individual, or population of individuals). In some cases, the elements can represent the type of proteoform. In some cases, the elements can be a number, a vector, an array, or any other data type presented herein.

[0458] Non-specific binding The surface can bind to biomolecules by non-selective adsorption (e.g., adsorption of biomolecules or a group of biomolecules when a biological sample containing particles and the biomolecules or the group of biomolecules is contacted, and this adsorption is non-selective randomly depending on factors including the physicochemical properties of the particles) or non-specific binding. Non-specific binding can refer to a class of binding interactions excluding specific binding. Examples of specific binding can include protein-ligand binding interactions, antigen-antibody binding interactions, nucleic acid hybridization, or binding interactions between a template molecule and a target molecule, where the template molecule promotes binding to a target molecule containing a complementary sequence or a complementary 3D structure and is disadvantageous for binding to a non-target molecule not containing either the complementary sequence or the complementary 3D structure, and can include binding interactions that result in a sequence or 3D structure.

[0459] Non-specific binding can include one or a combination of a wide variety of chemical and physical interactions and effects. Non-specific binding can include electromagnetic forces, such as electrostatic interactions, London dispersion forces, van der Waals interactions, or dipole-dipole interactions (e.g., both between permanent dipoles and between induced dipoles). Non-specific binding can be mediated by covalent bonds such as disulfide bridges. Non-specific binding can be mediated by hydrogen bonds. Non-specific binding can include the hydrophobic effect (e.g., the hydrophobic effect), where one object is repelled from the solvent environment and pushed against the boundary of the solvent, such as the surface of another object. Non-specific binding can include depletion interactions or entropy effects such as the thermal energy rising above the critical solution temperature (e.g., the lower critical solution temperature). Non-specific binding can include kinetic effects where one binding molecule can have faster binding kinetics than another binding molecule.

[0460] Non-specific binding can include multiple non-specific binding affinities for multiple targets (e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000 different targets adsorbed to a single particle). The multiple targets can have similar non-specific binding affinities that fall within a range of about 1, 2, or 3 orders of magnitude (e.g., as measured by non-specific binding free energy, equilibrium constant, competitive adsorption, etc.). This can be in contrast to specific binding, which can include a higher binding affinity for a given target molecule than for non-target molecules.

[0461] Biomolecules can adsorb onto a surface by non-specific binding at various densities on the surface. In some cases, the biomolecule or protein can adsorb at a density of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 fg / mm2. In some cases, the biomolecule or protein can adsorb at a density of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 pg / mm2. In some cases, the biomolecule or protein can adsorb at a density of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mm2. In some cases, the biomolecule or protein can adsorb at a density of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg / mm2. In some cases, the biomolecule or protein can adsorb at a density of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg / mm2. In some cases, the biomolecule or protein can adsorb at a maximum density of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 fg / mm2.In some cases, biomolecules or proteins can adsorb at a density of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 pg / mm2. In some cases, biomolecules or proteins can adsorb at a density of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ng / mm2. In some cases, biomolecules or proteins can adsorb at a density of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg / mm2. In some cases, biomolecules or proteins can adsorb at a density of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg / mm2.

[0462] The adsorbed biomolecules can include various types of proteins. In some cases, the adsorbed proteins can include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 types of proteins. In some cases, the adsorbed proteins can include up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 types of proteins.

[0463] In some cases, the proteins in a biological sample can include a concentration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 digits. In some cases, the proteins in a biological sample can include a concentration of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 digits.

[0464] Composition improvement assay In some cases, the methods of the present disclosure can include the use of a composition improvement assay. In some cases, the non-targeted assay can be a composition improvement assay. In some cases, a composition improvement assay can improve access to a subset of biomolecules in a biological sample. In some cases, a composition improvement assay can improve the detection of a subset of biomolecules in a biological sample. In some cases, a composition improvement assay can improve the identification of a subset of biomolecules in a biological sample. In some cases, the subset of biomolecules can be low-abundance biomolecules. In some cases, the subset of biomolecules can be rare biomolecules. In some cases, a composition improvement assay can be used to compress the dynamic range of a biological sample. In some cases, the dynamic range can be compressed by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 digits.

[0465] In some cases, a composition improvement assay may include providing one or more of one or more surface regions that include one or more surface types. In some cases, a composition improvement assay may include contacting a biological sample with one or more surface regions to obtain a set of biomolecules adsorbed on the one or more surface regions. In some cases, a composition improvement assay may include desorbing at least a portion of the set of adsorbed biomolecules from the one or more surface regions to obtain a set of polyamino acids. In some cases, a composition improvement assay may include contacting a biological sample with one or more surface regions to capture a set of biomolecules on the one or more surface regions. In some cases, a composition improvement assay may include releasing at least a portion of the set of biomolecules from the one or more surface regions to obtain a set of polyamino acids. In some cases, the one or more surface regions are disposed on a single continuous surface. In some cases, the one or more surface regions are disposed on one or more individual surfaces. In some cases, the one or more individual surfaces are the surfaces of one or more particles. In some cases, the one or more particles may include nanoparticles. In some cases, the one or more particles may include microparticles. In some cases, the one or more particles may include porous particles. In some cases, the one or more particles may include bifunctional particles, trifunctional particles, or N-functional particles.

[0466] In some cases, a composition improvement assay may include providing a plurality of surface regions that include a plurality of surface types. In some cases, a composition improvement assay may include contacting a biological sample with the plurality of surface regions to obtain a set of biomolecules adsorbed to the plurality of surface regions. In some cases, a composition improvement assay may include desorbing at least a portion of the set of adsorbed biomolecules from the plurality of surface regions to obtain a set of polyamino acids. In some cases, a composition improvement assay may include contacting a biological sample with the plurality of surface regions to capture a set of biomolecules on the plurality of surface regions. In some cases, a composition improvement assay may include releasing at least a portion of the set of biomolecules from the plurality of surface regions to obtain a set of polyamino acids. In some cases, the plurality of surface regions are disposed on a single continuous surface. In some cases, the plurality of surface regions are disposed on a plurality of individual surfaces. In some cases, the plurality of individual surfaces are the surfaces of a plurality of particles. In some cases, the plurality of particles may include nanoparticles. In some cases, the plurality of particles may include microparticles. In some cases, the plurality of particles may include porous particles. In some cases, the plurality of particles may include bifunctional particles, trifunctional particles, or N-functional particles.

Example

[0467] The following examples are provided to further illustrate some embodiments of the present disclosure and are not intended to limit the scope of the present disclosure. From the illustrative nature, it is understood that other procedures, method systems, or techniques known to those skilled in the art can be used instead.

[0468] (Example 1) Protein profiling of biological fluids using the Proteograph™ assay with alternative pH conditions for corona incubation for different nanoparticles A biological fluid sample can be incubated with different nanoparticles (NPs) in a solution containing water and / or a biological pH to form a protein corona on the NPs. In this example, provided is the use of alternative fluid compositions of different pHs with different NPs to improve the binding between NPs and proteins in a biological fluid sample. In some cases, the alternative fluid compositions can be used to improve the depth of proteome coverage.

[0469] As shown in FIGS. 2A - 2B, plasma samples with various pH buffers were mixed at the same volume ratio. The mixture was added to dry NP pellets for incubation. After completion of the incubation, the supernatant was removed from the mixture to isolate the NPs. The corona of the NPs was washed with a pH - adjusted wash buffer to wash off loosely - bound proteins from the surface of the corona. The NPs were washed with the washing composition shown in FIG. 2C. Thereafter, digestion and cleanup steps were performed on the corona proteins. The proteins were analyzed using LC - MS / MS, using a PharmaFluidics column, in data - dependent acquisition (DDA) mode, at the maximum injection mass per condition. Various environments were investigated for corona formation using different NPs to reach a deeper proteome depth.

[0470] As shown in FIG. 3, the data shows that the use of some pH buffers resulted in an increase of 30 - 40 percent in the protein group count for some NPs. For NP2 under corona conditions using pH 9.5 Tris buffer, an increase of about 42% in the number of protein groups was observed, and for NP5 under the same conditions, an increase of 48% in the number of protein groups was observed.

[0471] As shown in FIG. 4, the data shows that the use of some pH buffers increased the peptide yield. When using citrate buffer at pH 3, the peptide yield increased for all the nanoparticles tested.

[0472] As shown in Fig. 5, it is shown that the false cleavage ratio increased due to the use of some pH buffers in the data. When using the citrate buffer at pH 3, the false cleavage ratio increased for all the nanoparticles tested.

[0473] As shown in Figs. 6A - 6D, for NP4, between pH 7 (water) and high pH (when using Tris buffer, pH = 9.5), a low Jaccard index (JI) was observed even though the number of protein groups was similar between these two conditions. The functional annotation of the protein groups shown in Fig. 7A, and the range of physicochemical properties of the proteins shown in Figs. 7B - 7D also varied significantly. The protein / NP ratio can be adjusted to further improve the proteomics depth coverage (for example, by utilizing the Vroman effect).

[0474] (Example 2) Protein profiling of biological fluids using the Proteograph (trademark) assay with multiplexed nanoparticles Nanoparticles can be incubated separately with biological samples, for example, in separate wells. To improve the throughput of the analysis of biological samples, it may be advantageous to incubate a given biological sample in a smaller number of wells with multiplexed nanoparticles. In this example, a multiplexing approach is provided that mixes NPs with the same charge but different physicochemical properties to improve the depth of proteome coverage and assay throughput. In the multiplexing approach, various NPs can be incubated under different pH conditions.

[0475] Possible combinations of NPs with the same charge (e.g., all positive zeta potential or all negative zeta potential measured by DLS), and different ratios of NPs with the same charge were explored. After multiplexing combinations of NPs with the same charge, biological samples were incubated with the NPs. The performance of the multiplexed NPs was compared with the performance of single NPs.

[0476] As shown in FIG. 22, the results showed that multiplexing multiple NPs into a single well increased the number of identified protein groups for many sample types. As shown in FIG. 25, by further incorporating high pH into this design, in a 2-well particle panel, the number of identified protein groups increased by approximately 30% compared to a 5-particle panel performed in 5 wells. As a result, the mass spectrometry time per sample was also reduced by 60%. The high-performance 4-well or 5-well particle panels were under mixed buffer conditions containing pH 6, water, and pH 9.5.

[0477] FIG. 22 shows a comparison of the number of identified protein groups between multiplex assays and singleplex assays according to some embodiments. When NP1:NP4 were used at a mass ratio of about 6 to 15, an improvement in the number of protein groups was observed.

[0478] FIG. 23 shows a comparison of the number of identified protein groups between various multiplex assays using different Vroman conditions according to some embodiments. The combinations had a number of identified protein groups between -5% and +8% compared to a 5-particle reference particle panel. When NP4 and NP1 were combined, stable performance was observed for all samples (shown by the dotted frame).

[0479] FIG. 24 shows a comparison of the number of identified protein groups between various multiplex assays according to some embodiments. The performance of multiplexed NPs using standard pH conditions was better than that of single NPs (comparison of the dotted frame and the solid frame).

[0480] FIG. 25 shows a comparison of the number of identified protein groups between various multiplex assays using different pH conditions according to some embodiments. NP5 and NP2 were multiplexed at pH 9.5, while NP4 and NP1 were multiplexed at standard pH. An average improvement of +9% was observed for all sample types compared to the reference 5-particle panel.

[0481] (Example 3) Protein profiling of biological fluids using Proteograph™ with low sample volumes Plasma samples were diluted 1:1 with (i) water, (ii) 1 M pH 9.5 Tris buffer, (iii) pH 7.4 TE buffer, or (iv) phosphate buffered saline. These diluted plasma samples and undiluted control plasma samples were processed in triplicate using Proteograph™ and analyzed using LC-MS / MS. The total number of protein groups identified for each sample was as follows: control - 1382.7 ± 15.3; water - 1156.7 ± 9.0; pH 9.5 Tris buffer - 1321.0 ± 29.1; pH 7.4 TE - 1084 ± 2.8; and PBS - 1188 ± 5.7. For all dilutions, the total number of protein groups detected in the processed plasma samples decreased, but a significantly higher protein group count was obtained for the pH 9.5 Tris buffer compared to the other dilutions.

[0482] (Example 4) Comparison of sample dilutions in protein profiling of biological fluids using the Proteograph™ workflow Pooled human plasma and mouse serum samples of various dilution volumes were analyzed in triplicate using the Proteograph™ v1.2 workflow. The sample volumes tested were 250 μL, 125 μL, 50 μL, 25 μL, and 10 μL. For sample volumes less than 250 μL, they were diluted with 1 M pH 9.5 Tris buffer to obtain a total volume of 250 μL. For example, 50 μL of pooled plasma sample was diluted with 200 μL of 1 M pH 9.5 Tris buffer. The final volumes were processed in triplicate using the Proteograph™ v1.2 workflow. Briefly, 40 μL of volume was dispensed into five separate wells and combined with 40 μL of various nanoparticles suspended in water in each well. The combined mixtures were then incubated to form a biomolecular corona, after which they were washed, digested, and prepared for analysis by mass spectrometry. The processed peptide samples were analyzed using LC-MS / MS in DIA mode.

[0483] The number of protein groups and peptides identified in each sample is presented in Table 1 below: [Table 1]

[0484] From the results, the number of identified protein groups and peptides decreased with dilution. However, even when analyzing the 1:24 dilution of a 10 μL sample volume, it was shown that the majority of protein groups and peptides were identified compared to the undiluted sample. The coefficient of variation for each dilution was below 25%.

[0485] In an undiluted pooled human plasma sample (i.e., a plasma sample volume of 250 μL), approximately 1060 protein groups that were also annotated in the Human Plasma Proteome Project (HPPP) were identified. These protein groups were sorted using the abundance data from HPPP and grouped into quartiles, where the first quartile contains the proteins with the highest abundance and the fourth quartile contains the proteins with the lowest abundance. The number of proteins not identified for each dilution within each quartile compared to the undiluted 250 μL sample volume is shown in Table 2 below: [Table 2]

[0486] From the results, it was shown that dilution decreased the number of identified protein groups within each quartile, and the decrease was greater for protein groups with lower abundances. Even when using a 1:24 dilution for a 10 μL plasma sample, the majority of protein groups were identified within the fourth quartile compared to the undiluted sample.

[0487] For each of the diluted plasma samples, the Jaccard coefficient was determined relative to undiluted 250 μL. This is an indicator of the similarity of the identified protein groups between samples. The Jaccard coefficients between samples were 125 μL - 0.93 relative to 250 μL; 50 μL - 0.87 relative to 250 μL; 25 μL - 0.83 relative to 250 μL; and 10 μL - 0.70 relative to 250 μL. These results indicate that similar protein groups can be identified even when the samples are diluted.

[0488] (Example 5) System for selectively enriching and assaying biomolecules In this example, an automated system is used to provide various consumable materials (e.g., nanoparticle formulations, solvents, reagents, etc.) to perform an assay, use a mass spectrometer to generate assay results, then use data analysis software to analyze the results and display the results to the user, exemplifying the use of an automated system (instrument) in a pipeline that includes these steps.

[0489] An example of a method in an automated system including a computer-readable medium containing machine-executable code is described below. (1) A user (i.e., an operator) prepares a sample (e.g., by thawing a frozen sample), prepares a reagent (e.g., diluting a reagent), and prepares particles (e.g., reconstituting lyophilized particles into a multiplexed particle suspension). The prepared sample, reagent, and particles are loaded into the automated system. Thereafter, the experimental steps are automatically performed by the automated system: (2) Initialization of the device is performed within 5 minutes (Chassis, MPE2, Hamilton Heater Shaker (HHS), Inheco CPAC), (3) Pipetting of the sample onto the assay plate is performed within 5 minutes, (4) Pipetting of the particles onto the assay plate is performed within 15 minutes, (5) Incubation at 37 °C is performed within 60 minutes, (6) Washing of the assay plate is performed within 30 minutes, (7) Addition of the lysis buffer, reduction buffer, and alkylation buffer onto the assay plate is performed within 10 minutes, (8) Incubation at 95 °C in the HHS is performed within 10 minutes, (9) Cooling of the assay plate at room temperature is performed within 20 minutes, (10) Addition of the trypsin / LysC enzyme is performed within 8 minutes, (11) Incubation at 37 °C using the HHS is performed within 180 minutes, (12) Addition of the stop solution is performed within 3 minutes, (13) Pull-down of the particles is performed within 5 minutes, (14) Treatment of the sample using the SPE plate in the MPE2 is performed within 8 minutes, (15) Treatment of the sample using Wash A with the SPE plate in the MPE2 is performed within 8 minutes, (16) Treatment of the sample using Wash B-1 with the SPE plate in the MPE2 is performed within 8 minutes, (17) Treatment of the sample using Wash B-2 with the SPE plate in the MPE2 is performed within 8 minutes, (18) Elution of the sample using the SPE plate in the MPE2 is performed within 5 minutes. (19) Then, the user can clean up the automated system after the experiment. The total duration of the experiment is about 7 hours.

[0490] The above series of experimental steps may include additional steps, some steps may be excluded, and there may be variations in each step. These variations can be implemented such that the user can select which variation to use. For example, in step (1), the user can dilute the sample (e.g., up to 20 times the original volume for a plasma sample), select different volumes for the assay (e.g., any volume from 40 μL to 100 μL), thaw the sample to a specific temperature (e.g., room temperature or 4 °C), choose to use singleplex or multiplex nanoparticles (e.g., 2, 3, 4, 5, or any number of particles per compartment), or perform an interference step on the sample (e.g., hemolysis / lipid enrichment). In some cases, depending on the physicochemical properties of the particles, the background of biomolecules other than proteins can change the protein corona. In some cases, the background of biomolecules can also form part of the biomolecule corona. In some cases, the interference step can include titrating different concentrations of a particular biomolecule (e.g., lipid) at different concentrations.

[0491] Regarding any of the incubation steps, the duration of the incubation can be varied (e.g., 5 minutes or overnight), the pH of the solution being incubated can be varied (e.g., pH 3.8, 5.0, or 7.4), the ionic strength of the solution being incubated can be varied (e.g., 0, 50, or 150 mM), and the shaking speed of the solution being incubated can be varied (e.g., 0, 150, or 300 RPM).

[0492] Regarding any of the washing steps, there may be variations such that some or all of the components in the solution may or may not be resuspended. Some or all of the components in the solution can be separated, for example, by applying a magnetic field to capture magnetic particles.

[0493] For the dissolution, reduction, or alkylation steps, variations may exist where stepwise denaturation can be performed. The temperature of the solution can be varied (e.g., 50 °C or 95 °C). There may be a step of digesting the protein or peptide by using trypsin at various concentrations (1×, 2× concentrations of the standard amount of trypsin) over various durations (e.g., 3 hours or overnight). In some cases, the standard amount of trypsin can be from about 1 / 10 to about 1 / 100 of the mass of the protein compared to the mass of the protein. The protein or peptide can be digested stepwise, for example, by using trypsin / LysC.

[0494] Variations may exist for the elution step. The volume of the eluent can be varied (e.g., 75, 150, or 300 μL), clean dry air (CDA) or nitrogen can be supplied at various pressures (any pressure from 0 psi to 50 psi), and different types of solid-phase extraction (SPE) plates can be used (e.g., Thermal Fisher SPE plate, iST, C18, or other substrates).

[0495] In some cases, the automated system can be configured to be run on 8 to 16 samples at a time. Biomolecules in biological samples (i.e., biological fluids) can be measured using five different methods per sample. The measurements can be performed on multiple biological fluids, including plasma, cell extracts, and lysates. The measurements can be performed automatically, completed in 7 to 8 hours, and peptides can be injected for detection in liquid chromatography (LC) or MS. Bias-free measurements enable shortening of the LC / MS time, and these measurements can be independent of the LC / MS detector or method. For example, when using the DIA SWATH (data-independent acquisition) method with Sciex 6600+, the gradient length (between samples) is less than 30 minutes per fraction, and / or when using the DDA (data-dependent acquisition) method with Thermo Orbitrap Lumos, the gradient length is less than 1 hour. DIA SWATH (data-independent acquisition) and DDA (data-independent acquisition) are MS modes, differing in the way peptides are analyzed and the way proteins are computationally reconstructed based on the MS raw data. Since the measurements can be performed on intact proteins, the measurements can reveal protein-protein interactions in experimental data.

[0496] In some cases, the automated system may include a 96-well plate that can accommodate up to 16 samples for investigation by 5 nanoparticles. In some cases, the amount of sample volume required can be less than or equal to 240 μL or less than or equal to 40 μL. In some cases, the reagent can be stored at 4 °C for longer than 9 months or at room temperature for longer than 6 months (greater than 6 months) while maintaining stability. In some cases, the assay can be performed within 7 hours. In some cases, the running time of the MS experiment can be within 120 minutes. In some cases, the MS experiment can be performed with ScanningSWATH. In some cases, ScanningSWATH can refer to a rapid MS acquisition mode with a short gradient that is shortened to a few minutes. In some cases, ScanningSWATH can refer to a rapid MS acquisition mode using a scanning quadrupole. In some cases, Sciex timsTOF rapid IMS-IMS can be used for ScanningSWATH, which may involve ion mobility separation and may also involve prior separation based on the charge / dipole and shape characteristics of the ions. In some cases, the automated system may include analysis tools including visualization (e.g., cluster analysis, PCA) tools or quality control tools, which may be incorporated into a cloud-based computing system. In some cases, the protein detection method implemented in the automated system can show a 5-fold superiority (i.e., superiority in the number of protein groups detected) over a shallow plasma method and a 3-fold superiority over a depleted plasma method. In some cases, the protein detection method implemented in the automated system can have a 5% improvement in accuracy (low CV) over published datasets (e.g., Geyer et al. Mol. Syst. Biol. 13, 942 (2017)).

[0497] List of embodiments The following list of embodiments of the present invention should be considered to disclose various features of the present invention, and these features can be considered specific to the particular embodiment being considered, or can be combined with various other features listed in other embodiments. Thus, the use of a feature is not necessarily limited to that embodiment just because the feature is being considered under one particular embodiment.

[0498] Embodiment 1. A method comprising: (a) selectively enriching a first plurality of types of biomolecules in a first fluid composition; (b) selectively enriching a second plurality of types of biomolecules in a second fluid composition; and (c) performing a downstream assay on the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules, wherein the first fluid composition includes a first pH, the second fluid composition includes a second pH, and the first pH and the second pH are different or the same.

[0499] Embodiment 2. The method of Embodiment 1, wherein the second infinite dilution limit enthalpy or free energy of solvation of a second biomolecule within a second subset of biomolecules is different when the second biomolecule is in the second fluid composition compared to when the second biomolecule is in the first fluid composition.

[0500] Embodiment 3. The method of Embodiment 1 or 2, wherein the first fluid composition includes a first set of strong physical properties that mediate the selective enrichment of the first plurality of types of biomolecules, the second fluid composition includes a second set of strong physical properties that mediate the selective enrichment of the second plurality of types of biomolecules, and the first set of strong physical properties and the second set of strong physical properties are different.

[0501] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the first fluid composition includes a first ratio between a first sample volume and a first surface area of a first surface, the second fluid composition includes a second ratio between a second sample volume and a second surface area of a second surface, and the first ratio and the second ratio are different.

[0502] Embodiment 5. A method according to any one of Embodiments 1 to 4, wherein the first fluid composition and the second fluid composition have different temperatures.

[0503] Embodiment 6. A method according to any one of Embodiments 1 to 5, wherein the first fluid composition has a first ionic strength, the second fluid composition has a second ionic strength, and the first ionic strength and the second ionic strength are different.

[0504] Embodiment 7. A method according to any one of Embodiments 1 to 6, wherein the first fluid composition and the second fluid composition contain different salts.

[0505] Embodiment 8. A method according to any one of Embodiments 1 to 7, wherein the first fluid composition and the second fluid composition contain different solvents.

[0506] Embodiment 9. A method according to any one of Embodiments 1 to 8, wherein the first fluid composition, the second fluid composition, or both contain a buffer solution containing tris(hydroxymethyl)aminomethane.

[0507] Embodiment 10. A method according to any one of Embodiments 1 to 9, wherein the first fluid composition, the second fluid composition, or both contain a buffer solution containing citrate.

[0508] Embodiment 11. A method according to any one of Embodiments 1 to 10, wherein the first fluid composition, the second fluid composition, or both have a pH between 2 and 4.

[0509] Embodiment 12. A method according to any one of Embodiments 1 to 11, wherein the first fluid composition, the second fluid composition, or both have a pH between 5 and 7.

[0510] Embodiment 13. A method according to any one of Embodiments 1 to 12, wherein the first fluid composition, the second fluid composition, or both have a pH between 9 and 10.

[0511] Method according to any one of Embodiments 1 to 13, wherein the first fluid composition, the second fluid composition, or both contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pH.

[0512] Method according to any one of Embodiments 1 to 14, wherein the first fluid composition, the second fluid composition, or both contain a maximum of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pH.

[0513] Method according to any one of Embodiments 1 to 15, further comprising the step of diluting the sample to produce the first composition, the second composition, or both.

[0514] Method according to Embodiment 16, wherein the step of diluting comprises adding a buffer solution.

[0515] Method according to Embodiment 17, wherein the buffer solution contains at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pH.

[0516] Method according to Embodiment 17 or 18, wherein the buffer solution contains a maximum of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 pH.

[0517] Method according to any one of Embodiments 17 to 19, wherein the sample and the buffer solution contain different pH values.

[0518] Method according to any one of Embodiments 16 to 20, wherein the sample contains a maximum of about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules.

[0519] Method according to any one of Embodiments 16 to 21, wherein the sample contains a maximum of about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per mL of the sample.

[0520] Embodiment 23. The method according to any one of Embodiments 16 to 22, wherein the sample contains biomolecules derived from up to about 1000, 100, 10, or 1 cell.

[0521] Embodiment 24. The method according to any one of Embodiments 16 to 23, wherein the sample contains up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters.

[0522] Embodiment 25. The method according to any one of Embodiments 16 to 24, wherein the sample contains a complex biological sample.

[0523] Embodiment 26. The method according to Embodiment 25, wherein the complex biological sample contains plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, ductal lavage fluid, vaginal fluid, nasal discharge, ear discharge, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a cotton swab, bronchial aspirate fluid, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0524] Embodiment 27. The method according to Embodiment 26, wherein the biological sample contains plasma.

[0525] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the step of selectively enriching the first plurality of types of biomolecules therein comprises contacting a first fluid composition with a first surface and adsorbing the first plurality of types of biomolecules onto the first surface.

[0526] Embodiment 29. The method according to any one of Embodiments 1 to 28, wherein the step of selectively enriching the second plurality of types of biomolecules therein comprises contacting a second fluid composition with a second surface and adsorbing the second plurality of types of biomolecules onto the second surface.

[0527] Embodiment 30. The method according to any one of Embodiments 1 to 29, wherein the first surface, the second surface, or both contain particles.

[0528] Embodiment 31. The method of Embodiment 30, wherein the particles are porous particles.

[0529] Embodiment 32. The method of Embodiment 30 or 31, wherein the particles are microparticles.

[0530] Embodiment 33. The method of Embodiment 30 or 31, wherein the particles are nanoparticles.

[0531] Embodiment 34. The method of any one of Embodiments 30 to 33, wherein the particles contain a paramagnetic material.

[0532] Embodiment 35. The method of Embodiment 34, wherein the paramagnetic material is a superparamagnetic material.

[0533] Embodiment 36. The method of Embodiment 34 or 35, wherein the paramagnetic material contains iron oxide.

[0534] Embodiment 37. The step of selectively enriching the first plurality of types of biomolecules among them is to contact a first fluid composition with a first plurality of types of surfaces, and adsorb the first plurality of types of biomolecules onto the first plurality of types of surfaces. The method according to any one of Embodiments 1 to 36.

[0535] Embodiment 38. The step of selectively enriching the second plurality of types of biomolecules among them is to contact a second fluid composition with a second plurality of types of surfaces, and adsorb the second plurality of types of biomolecules onto the second plurality of types of surfaces. The method according to any one of Embodiments 1 to 37.

[0536] Embodiment 39. The method according to any one of Embodiments 1 to 38, wherein the first plurality of types of surfaces, the second plurality of types of surfaces, or both contain charges of the same sign.

[0537] Embodiment 40. The method according to any one of Embodiments 1 to 39, wherein the first plurality of types of surfaces, the second plurality of types of surfaces, or both contain zeta potentials of the same sign.

[0538] Embodiment 41. The method according to any one of Embodiments 1 to 40, wherein the first plurality of surface types, the second plurality of surface types, or both contain acidic functional groups.

[0539] Embodiment 42. The method according to any one of Embodiments 1 to 41, wherein the acidic functional group contains a Bronsted-Lowry acid or a Lewis acid functional group.

[0540] Embodiment 43. The method according to any one of Embodiments 1 to 42, wherein the first plurality of surface types, the second plurality of surface types, or both contain a carboxylic acid group, an acrylic acid group, a methacrylic acid group, an acetal group, a hemiacetal group, a hemiketal group, a sulfonic acid group, a sulfinic acid group, a thiocarboxylic acid group, a phosphonic acid group, a phosphoric acid group, a phosphoric acid diester group, a boronic acid group, a boronic acid ester group, a boric acid group, a boric acid ester group, a silica group, a silanol group, a polymer, or any combination thereof.

[0541] Embodiment 44. The method according to any one of Embodiments 1 to 43, further comprising, before (c), the step of washing the first plurality of biomolecule types with the first washing composition and the step of washing the second plurality of biomolecule types with the second washing composition.

[0542] Embodiment 45. The method of Embodiment 44, wherein the first fluid composition and the first washing composition contain at least one common strong physical property.

[0543] Embodiment 46. The method of Embodiment 44 or 45, wherein the first fluid composition and the first washing composition contain at least one common solvent.

[0544] Embodiment 47. The method according to any one of Embodiments 44 to 46, wherein the first fluid composition and the first washing composition contain at least one different strong physical property.

[0545] Method of any one of Embodiments 44 to 47, wherein the second fluid composition and the second cleaning composition include at least one common strong physical property.

[0546] Method of any one of Embodiments 44 to 48, wherein the second fluid composition and the second cleaning composition include at least one common solvent.

[0547] Method of any one of Embodiments 44 to 49, wherein the second fluid composition and the second cleaning composition include at least one different strong physical property.

[0548] Method of any one of Embodiments 44 to 50, wherein the first cleaning composition and the second cleaning composition include at least one common strong physical property.

[0549] Method of any one of Embodiments 44 to 51, wherein the first cleaning composition and the second cleaning composition are the same.

[0550] Method of any one of Embodiments 44 to 52, wherein the first cleaning composition and the second cleaning composition include at least one different strong physical property.

[0551] Method of any one of Embodiments 44 to 53, wherein the first cleaning composition releases a first plurality of types of biomolecules adsorbed on the first surface from the first surface.

[0552] Method of Embodiment 54, further including the step of purifying the first plurality of types of biomolecules to produce a first purified composition.

[0553] Method of Embodiment 55, wherein the step of purifying includes drying the plurality of types of biomolecules to remove the first cleaning composition.

[0554] Step of reconstructing the first purified composition using the first reconstruction composition, further comprising the step of producing the first reconstructed composition, the method of Embodiment 56.

[0555] Embodiment 58. The method according to any one of Embodiments 44 to 57, wherein the second washing composition releases a second plurality of types of biomolecules deposited on the second surface from the second surface.

[0556] Embodiment 59. The method of Embodiment 58, further comprising the step of purifying the second plurality of types of biomolecules to produce a second purified composition.

[0557] Embodiment 60. The method of Embodiment 59, wherein the step of purifying comprises drying the second plurality of types of biomolecules to remove the second washing composition.

[0558] Embodiment 61. The method of Embodiment 60, further comprising the step of reconstructing the second purified composition using the second reconstruction composition to produce a second reconstructed composition.

[0559] Embodiment 62. The method according to any one of Embodiments 1 to 61, wherein the downstream assay comprises mass spectrometry.

[0560] Embodiment 63. The method of Embodiment 62, wherein the mass spectrometry comprises LC-MS / MS.

[0561] Embodiment 64. The method according to any one of Embodiments 1 to 63, wherein the downstream assay comprises protein sequencing.

[0562] Embodiment 65. The downstream assay comprises contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both with a pair of antibodies capable of binding to at least one type of biomolecule among the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, the pair of antibodies comprising a complementary single-stranded nucleic acid sequence bound thereto such that when the pair of antibodies binds to at least one type of biomolecule, a complementary nucleic acid hybridizes to form a double-stranded nucleic acid, the double-stranded nucleic acid forms a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction, and a detectable signal is generated. The method according to any one of Embodiments 1 to 64.

[0563] Embodiment 66. The downstream assay comprises contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both with one or more aptamers capable of binding to at least one type of biomolecule among the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both, the one or more aptamers being bound to a surface via a cleavable linker. The method according to any one of Embodiments 1 to 65.

[0564] Embodiment 67. The method according to Embodiment 66, wherein the surface is a particle surface.

[0565] Embodiment 68. The method according to Embodiment 67, wherein the cleavable linker is a linker cleavable by light.

[0566] Embodiment 69. The method according to Embodiment 68, further comprising contacting a first plurality of types of biomolecules, a second plurality of types of biomolecules, or both with a polymeric competitor configured to reduce dissociation of a complex composed of one or more aptamers and the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both in a fluid composition.

[0567] Embodiment 70. The method of Embodiment 69, further configured such that the polymeric competitor binds to a type of biomolecule different from the first plurality of types of biomolecules, the second plurality of types of biomolecules, or both.

[0568] Embodiment 71. The method of Embodiment 70, wherein the polymeric competitor is a polyanionic polymer.

[0569] Embodiment 72. The method of any one of Embodiments 1 - 71, wherein the downstream assay comprises nucleic acid sequencing.

[0570] Embodiment 73. The method of any one of Embodiments 1 - 72, wherein (a) and (b) are performed on one machine.

[0571] Embodiment 74. The method of any one of Embodiments 1 - 72, wherein (a) and (b) are performed on different machines.

[0572] Embodiment 75. The method of any one of Embodiments 1 - 74, wherein (a) and (b) are performed in parallel.

[0573] Embodiment 76. The method of any one of Embodiments 1 - 74, wherein (a) and (b) are performed sequentially.

[0574] Embodiment 77. The method of any one of Embodiments 1 - 76, wherein the first fluid composition and the second fluid composition are part of the same sample.

[0575] Embodiment 78. The method of any one of Embodiments 1 - 76, wherein the first fluid composition and the second fluid composition are derived from different samples.

[0576] Embodiment 79. The method of any one of Embodiments 1 - 78, wherein the step of selectively enriching is performed in parallel with respect to the first fluid composition and the second fluid composition.

[0577] Embodiment 80. The method according to any one of Embodiments 1 to 78, wherein the step of selectively enriching is carried out continuously for the first fluid composition and the second fluid composition.

[0578] Embodiment 81. The method according to any one of Embodiments 1 to 80, wherein the step of selectively enriching is carried out at a rate at which at least 100, 1000, 10000, 100000, or 1000000 biomolecules are enriched per hour.

[0579] Embodiment 82. The method according to any one of Embodiments 1 to 81, wherein the step of selectively enriching is carried out at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are enriched per hour.

[0580] Embodiment 83. The method according to any one of Embodiments 1 to 82, wherein the contacting is carried out in parallel for the first fluid composition and the second fluid composition.

[0581] Embodiment 84. The method according to any one of Embodiments 1 to 82, wherein the contacting is carried out continuously for the first fluid composition and the second fluid composition.

[0582] Embodiment 85. The method according to any one of Embodiments 1 to 84, wherein the contacting is carried out at a rate at which at least 100, 1000, 10000, 100000, or 1000000 biomolecules are deposited per hour.

[0583] Embodiment 86. The method according to any one of Embodiments 1 to 85, wherein the contacting is carried out at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are deposited per hour.

[0584] Embodiment 87. The method according to any one of Embodiments 1 to 86, wherein the washing step is carried out in parallel for the first fluid composition and the second fluid composition.

[0585] Embodiment 88. The method according to any one of Embodiments 1 to 86, wherein the washing step is carried out successively on the first fluid composition and the second fluid composition.

[0586] Embodiment 89. The method according to any one of Embodiments 1 to 88, wherein the washing step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 biomolecules are washed per hour.

[0587] Embodiment 90. The method according to any one of Embodiments 1 to 89, wherein the washing step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are washed per hour.

[0588] Embodiment 91. The method according to any one of Embodiments 1 to 90, wherein the purification step is carried out in parallel on the first fluid composition and the second fluid composition.

[0589] Embodiment 92. The method according to any one of Embodiments 1 to 90, wherein the purification step is carried out successively on the first fluid composition and the second fluid composition.

[0590] Embodiment 93. The method according to any one of Embodiments 1 to 92, wherein the purification step is carried out at a rate such that at least 100, 1000, 10000, 100000, or 1000000 are purified per hour.

[0591] Embodiment 94. The method according to any one of Embodiments 1 to 92, wherein the purification step is carried out at a rate such that at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are purified per hour.

[0592] Embodiment 95. The method according to any one of Embodiments 1 to 94, wherein the reconstitution step is carried out in parallel on the first fluid composition and the second fluid composition.

[0593] Embodiment 96. The method according to any one of Embodiments 1 to 94, wherein the step of reconstituting is performed continuously on the first fluid composition and the second fluid composition.

[0594] Embodiment 97. The method according to any one of Embodiments 1 to 96, wherein the step of reconstituting is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 being reconstituted per hour.

[0595] Embodiment 98. The method according to any one of Embodiments 1 to 97, wherein the step of reconstituting is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules being reconstituted per hour.

[0596] Embodiment 99. The method according to any one of Embodiments 1 to 98, wherein the downstream assay is performed in parallel on the first fluid composition and the second fluid composition.

[0597] Embodiment 100. The method according to any one of Embodiments 1 to 98, wherein the downstream assay is performed continuously on the first fluid composition and the second fluid composition.

[0598] Embodiment 101. The method according to any one of Embodiments 1 to 100, wherein the downstream assay is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples being assayed per hour.

[0599] Embodiment 102. The method according to any one of Embodiments 1 to 101, wherein the downstream assay is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules being assayed per hour.

[0600] Method according to any one of Embodiments 1 to 102, wherein the downstream assay is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 biomolecules identified per hour.

[0601] Method according to any one of Embodiments 1 to 103, wherein the downstream assay is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 protein groups identified per hour.

[0602] Method according to any one of Embodiments 1 to 104, which is performed in parallel on a first fluid composition and a second fluid composition.

[0603] Method according to any one of Embodiments 1 to 105, which is performed sequentially on a first fluid composition and a second fluid composition.

[0604] Method according to any one of Embodiments 1 to 106, which is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0605] Method according to any one of Embodiments 1 to 107, which is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules assayed per hour.

[0606] Method according to any one of Embodiments 1 to 108, wherein the downstream assay is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 biomolecules identified per hour.

[0607] Method according to any one of Embodiments 1 to 109, wherein the downstream assay is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 protein groups identified per hour.

[0608] Embodiment 111. A method according to any one of Embodiments 1 to 110, wherein when the first fluid composition and the second fluid composition are HeLa cell extracts, at least about 100, 1000, 10000, 100000, or 1000000 biomolecules are identified in the downstream assay.

[0609] Embodiment 112. A method according to any one of Embodiments 1 to 111, wherein when the downstream assay is performed before the step of selectively enriching (a) and (b) for the first fluid composition and the second fluid composition, the first plurality of biomolecule types and the second plurality of biomolecule types together include at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0610] Embodiment 113. A method according to any one of Embodiments 1 to 112, wherein the first plurality of biomolecule types are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the abundance of the first biomolecule in the first fluid composition is greater than that of the second biomolecule.

[0611] Embodiment 114. A method according to any one of Embodiments 1 to 113, wherein the second plurality of biomolecule types are such that at least the first biomolecule is depleted and the second biomolecule is enriched, and the abundance of the first biomolecule in the second fluid composition is greater than that of the second biomolecule.

[0612] Embodiment 115. The first possibility that a biomolecule with a low abundance in the first plurality of biomolecule types or the second plurality of biomolecule types is identified in the downstream assay is higher than the second possibility that a biomolecule with a low abundance in the first fluid composition or the second fluid composition is identified in the downstream assay, and the biomolecule with a low abundance is less than about 1 mass percent, about 10 -1 mass percent, about 10 -2 mass percent, about 10 -3 mass percent, about 10 -4 mass percent, about 10 -5 mass percent, about 10 -6Less than a mass percentage, about 10 -7 Less than a mass percentage, about 10 -8 Less than a mass percentage, about 10 -9 Less than a mass percentage, or about 10 -10 A method according to any one of embodiments 1 to 114, comprising a first set of biomolecules or a second set of biomolecules less than a mass percentage.

[0613] Embodiment 116. The first possibility is at least 2, 5, 10, 10 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 times higher, the method of embodiment 115.

[0614] Embodiment 117. One or more biomolecules in one or both of a first plurality of biomolecule types and a second plurality of biomolecule types downstream are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent, a method according to any one of embodiments 1 to 116.

[0615] Embodiment 118. One or more biomolecules in one or both of a first plurality of biomolecule types and a second plurality of biomolecule types downstream are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent, a method according to any one of embodiments 1 to 117.

[0616] Embodiment 119. One or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 peptides, the method of embodiment 117 or 118.

[0617] Embodiment 120. A method according to any one of embodiments 117 - 119, wherein one or more biomolecules comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins.

[0618] Embodiment 121. A method according to any one of embodiments 1 - 120, wherein the first plurality of biomolecule types, the second plurality of biomolecule types, or both comprise one or more polyamino acids.

[0619] Embodiment 122. A method according to any one of embodiments 1 - 121, further comprising identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first fluid composition.

[0620] Embodiment 123. A method according to any one of embodiments 1 - 122, further comprising identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second fluid composition.

[0621] Embodiment 124. A method according to any one of embodiments 44 - 123, further comprising identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first washing composition.

[0622] Embodiment 125. A method according to any one of embodiments 44 - 124, further comprising identifying a biological state associated with the second fluid composition based at least in part on one or more physical properties of the second washing composition.

[0623] Embodiment 126. A method according to any one of embodiments 57 - 125, further comprising identifying a biological state associated with the first fluid composition based at least in part on one or more physical properties of the first reconstitution composition.

[0624] Embodiment 127. The method according to any one of Embodiments 61 to 126, further comprising the step of identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstitution composition.

[0625] Embodiment 128. The method according to any one of Embodiments 1 to 127, wherein the first surface is disposed in a first lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers.

[0626] Embodiment 129. The method according to any one of Embodiments 1 to 128, wherein the second surface is disposed in a second lyophilized composition comprising (i) at least one nanoparticle and (ii) one or more modifiers.

[0627] Embodiment 130. The method according to Embodiment 128 or 129, wherein the one or more modifiers comprise a pH adjuster, an ionic strength adjuster, a viscosity adjuster, or any combination thereof.

[0628] Embodiment 131. The method according to any one of Embodiments 1 to 130, wherein the first pH is different from the second pH.

[0629] Embodiment 132. The method according to any one of Embodiments 1 to 130, wherein the first pH is the same as the second pH.

[0630] Step 133. (a) selectively enriching a first plurality of types of biomolecules in a first fluid composition; (b) selectively enriching a second plurality of types of biomolecules in a second fluid composition; and (c) performing an assay downstream of the types of biomolecules of the first plurality of types of biomolecules and the second plurality of types of biomolecules, wherein the first infinite dilution limit enthalpy or free energy of solvation of a first biomolecule among the first plurality of types of biomolecules is different when the first biomolecule is in the first fluid composition compared to when the biomolecule is in the second fluid composition, such that the first fluid composition is different from the second fluid composition.

[0631] Kit of Step 134. (a) a first reagent having a first pH; (b) a second reagent having a second pH; (c) a first surface configured to selectively enrich a first plurality of types of biomolecules in a first fluid composition including the first surface and the first reagent; and (d) a second surface configured to selectively enrich a second plurality of types of biomolecules in a second fluid composition including the second surface and the second reagent, wherein the first fluid composition includes the first reagent, the second fluid composition includes the second reagent, and the first pH and the second pH are different or the same.

[0632] Composition of Step 135. A composition comprising a suspension, the suspension including: i. a first particle including (i) a first paramagnetic moiety and (ii) a first surface chemistry; ii. a second particle including (i) a second paramagnetic moiety and (ii) a second surface chemistry; and iii. a plurality of biomolecules adsorbed to the first particle and the second particle, wherein the second surface chemistry is different from the first surface chemistry.

[0633] Composition of Step 136. The composition of Step 135, wherein the first particle and the second particle include charges of the same sign.

[0634] Composition of Step 137. The composition of Step 135 or 136, wherein the first particle and the second particle include zeta potentials of the same sign.

[0635] Embodiment 138. The composition of Embodiment 136 or 137, where the same sign is a negative sign.

[0636] Embodiment 139. The composition of any one of Embodiments 135 to 138, where the first surface chemical structure, the second surface chemical structure, or both contain acidic functional groups.

[0637] Embodiment 140. The composition of Embodiment 139, where the acidic functional group contains a Bronsted-Lowry acid or a Lewis acid functional group.

[0638] Embodiment 141. The composition of any one of Embodiments 135 to 140, where the first surface chemical structure, the second first surface chemical structure, or both contain a carboxylic acid group, an acrylic acid group, a methacrylic acid group, an acetal group, a hemiacetal group, a hemiketal group, a sulfonic acid group, a sulfinic acid group, a thiocarboxylic acid group, a phosphonic acid group, a phosphoric acid group, a phosphoric acid diester group, a boronic acid group, a boronic acid ester group, a boric acid group, a boric acid ester group, a silica group, a silanol group, a polymer, or any combination thereof.

[0639] Embodiment 142. The composition of any one of Embodiments 135 to 141, where the suspension is stable for at least about 1, 5, 10, 15, 30, or 60 minutes.

[0640] Embodiment 143. The composition of any one of Embodiments 135 to 142, where the time constant for destabilization of the suspension is at least about 1, 5, 10, 15, 30, or 60 minutes.

[0641] Embodiment 144. The composition of any one of Embodiments 135 to 143, where the average aggregation number of the first and second particles in the suspension is at most about 1000, 100, or 10.

[0642] Embodiment 145. The composition of any one of Embodiments 135 to 144, where the suspension contains at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles per about 1 part of the second particles.

[0643] Embodiment 146. A composition according to any one of Embodiments 135 to 145, wherein the suspension comprises up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles per about 1 part of the second particles.

[0644] Embodiment 147. A composition according to any one of Embodiments 135 to 146, wherein the suspension comprises about 15 parts of the first particles per about 6 parts of the second particles.

[0645] Embodiment 148. The composition of Embodiment 147, wherein the parts are parts by weight, parts by volume, or parts by surface area.

[0646] Embodiment 149. A composition according to any one of Embodiments 135 to 148, wherein the suspension comprises at least about 3, 4, 5, 6, 7, 8, 9, or 10 distinct particles.

[0647] Embodiment 150. A composition according to any one of Embodiments 135 to 149, wherein the first particles, the second particles, or both are nanoparticles.

[0648] Embodiment 151. A composition according to any one of Embodiments 135 to 150, wherein the first particles, the second particles, or both are microparticles.

[0649] Embodiment 152. A composition according to any one of Embodiments 135 to 151, wherein the first particles, the second particles, or both are porous.

[0650] Embodiment 153. A composition according to any one of Embodiments 135 to 152, wherein the first size of the first particles is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particles.

[0651] Composition of Embodiment 154. The composition of Embodiment 153, wherein the first size of the first particles is at most about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the second size of the second particles.

[0652] Composition of Embodiment 155. The composition according to any one of Embodiments 153 to 154, wherein the first size is the first diameter and the second size is the second diameter.

[0653] Composition of Embodiment 156. The composition according to any one of Embodiments 153 to 155, wherein the first size is the first average size and the second size is the second average size.

[0654] Composition of Embodiment 157. The composition of Embodiment 156, wherein the first average size and the second average size are the size average value or the size median.

[0655] Composition of Embodiment 158. The composition according to any one of Embodiments 135 to 152, wherein the ratio of the average diameter of the first particles to the average diameter of the second particles is from about 3:2 to about 2:3.

[0656] Composition of Embodiment 159. The composition according to any one of Embodiments 135 to 158, wherein the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules.

[0657] Composition of Embodiment 160. The composition according to any one of Embodiments 135 to 159, wherein the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per mL.

[0658] Composition of Embodiment 161. The composition according to any one of Embodiments 135 to 160, wherein the plurality of biomolecules comprises biomolecules derived from at most about 1000, 100, 10, or 1 cell.

[0659] Step of assaying a plurality of biomolecules in the composition according to any one of Embodiments 135 to 161, the step including identifying one or more of the plurality of biomolecules.

[0660] Embodiment 163. The method according to Embodiment 162, wherein the step of assaying includes performing mass spectrometry.

[0661] Embodiment 164. The method according to Embodiment 163, wherein the mass spectrometry includes LC-MS / MS.

[0662] Embodiment 165. The method according to Embodiment 162, wherein the step of assaying includes performing protein sequencing.

[0663] Embodiment 166. The step of assaying includes contacting the plurality of biomolecules with a pair of antibodies capable of binding to at least one of the plurality of biomolecules, and the pair of antibodies includes a complementary single-stranded nucleic acid sequence that hybridizes to a nucleic acid complementary to the at least one biomolecule that binds to the pair of antibodies to form a double-stranded nucleic acid, and the double-stranded nucleic acid forms a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction and is configured to generate a detectable signal. The method according to any one of Embodiments 162 to 165.

[0664] Embodiment 167. The step of assaying includes contacting the plurality of biomolecules with one or more aptamers capable of binding to at least one of the plurality of biomolecules, and the one or more aptamers are bound to the surface via a cleavable linker. The method according to any one of Embodiments 162 to 166.

[0665] Embodiment 168. The method according to Embodiment 167, wherein the surface is a particle surface.

[0666] The method of embodiment 167 or 168, wherein the cleavable linker is a photocleavable linker.

[0667] Embodiment 170. The method of any one of embodiments 162 - 169, further comprising contacting a plurality of biomolecules with a polymeric competitor configured to reduce dissociation of a complex composed of one or more aptamers and a biomarker in a fluid composition.

[0668] Embodiment 171. The method of embodiment 170, wherein the polymeric competitor is further configured to bind to a biomolecule different from the biomarker.

[0669] Embodiment 172. The method of embodiment 171, wherein the polymeric competitor is a polyanionic polymer.

[0670] Embodiment 173. The method of any one of embodiments 167 - 172, wherein the step of assaying comprises performing nucleic acid sequencing.

[0671] Embodiment 174. The method of any one of embodiments 162 - 173, wherein the sample comprises a plurality of biomolecules.

[0672] Embodiment 175. The method of embodiment 174, wherein the sample is a complex biological sample.

[0673] Embodiment 176. The method of embodiment 175, wherein the complex biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate fluid, breast duct lavage fluid, vaginal fluid, nasal mucus, ear discharge, gastric juice, pancreatic juice, trabecular bone fluid, lung lavage fluid, sweat, gingival crevicular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid swabbed with a cotton swab, bronchial aspirate fluid, flowing solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0674] Embodiment 177. The method of embodiment 176, wherein the biological sample comprises plasma.

[0675] Embodiment 178. The method according to any one of Embodiments 162 to 177, wherein a plurality of biomolecules contain one or more polyamino acids.

[0676] Embodiment 179. The method according to any one of Embodiments 162 to 178, wherein the step of assaying is performed at a rate at which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100 samples are assayed per hour.

[0677] Embodiment 180. The method according to any one of Embodiments 162 to 179, wherein the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecules are assayed per hour.

[0678] Embodiment 181. The method according to any one of Embodiments 162 to 180, wherein the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 biomolecules are identified per hour.

[0679] Embodiment 182. The method according to any one of Embodiments 162 to 181, wherein the step of assaying is performed at a rate at which at least 1, 10, 100, 1000, 10000, 100000, or 1000000 protein groups are identified per hour.

[0680] Embodiment 183. The method according to any one of Embodiments 162 to 182, wherein when the suspension contains a HeLa cell extract, at least about 100, 1000, 10000, or 100000 biomolecules are identified in the step of assaying.

[0681] Embodiment 184. The method according to any one of Embodiments 162 to 183, wherein when the step of assaying is performed on a plurality of biomolecules in a biological sample in the absence of the first particle and the second particle, the plurality of biomolecules contain at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0682] Embodiment 185. The first possibility that a biomolecule with a low abundance among a plurality of biomolecules is identified in the assay step is higher than the second possibility that a biomolecule with a low abundance in a biological sample is identified in the assay step in the absence of the first and second particles. The biomolecule with a low abundance is less than about 1 mass percent, about 10 -1 mass percent, about 10 -2 mass percent, about 10 -3 mass percent, about 10 -4 mass percent, about 10 -5 mass percent, about 10 -6 mass percent, about 10 -7 mass percent, about 10 -8 mass percent, about 10 -9 mass percent, or about 10 -10 mass percent, and constitutes any one of the methods of Embodiments 162 to 184.

[0683] Embodiment 186. The first possibility is at least 2, 5, 10, 10 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 times higher than the second possibility, and constitutes any one of the methods of Embodiments 162 to 185.

[0684] Embodiment 187. One or more biomolecules among a plurality of biomolecules are identified with a coefficient of variation of at most about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent in the assay step, and constitutes any one of the methods of Embodiments 162 to 186.

[0685] A method according to any one of embodiments 162-187, wherein in the step of assaying, one or more biomolecules among a plurality of biomolecules are identified with a coefficient of variation of at least about 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent.

[0686] Embodiment 189. A system for analyzing a plurality of biological samples, comprising: (a) a plurality of compartments including a first compartment and a second compartment; (b) a plurality of reagent storage units including a first reagent having a first pH and a second reagent having a second pH, wherein the first pH and the second pH are different or the same; (c) a plurality of substrates including a first substrate having a first surface chemistry and a second substrate having a second surface chemistry; (d) one or more transfer devices operably connected to the plurality of compartments, the plurality of reagent storage units, and the plurality of substrates; (e) at least one processor, and a computer including instructions executable by the at least one processor to perform steps including: i. using one or more transfer devices to generate a first fluid composition within a first compartment including the first substrate, the first reagent, and a first plurality of biomolecules, wherein the first plurality of biomolecules adsorb to the first substrate; ii. using one or more transfer devices to generate a second fluid composition within a second compartment including the second substrate, the second reagent, and a second plurality of biomolecules, wherein the second plurality of biomolecules adsorb to the second substrate; and iii. using one or more transfer devices to prepare the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry.

[0687] Embodiment 190. A system for analyzing a plurality of biological samples, comprising: (a) a compartment; (b) a reagent storage unit containing reagents; (c) a plurality of substrates including a first substrate having a first surface chemical structure and a second substrate having a second surface chemical structure, wherein the first surface chemical structure and the second surface chemical structure are different; (d) one or more transfer devices operably connected to the compartment, the reagent storage unit, and the plurality of substrates; and (e) at least one processor, and i. a step of using one or more transfer devices to generate a fluid composition within the compartment containing the plurality of substrates, reagents, and a plurality of biomolecules, wherein the plurality of biomolecules adsorb to the substrates; and ii. a step of using one or more transfer devices to prepare the plurality of biomolecules for mass spectrometry, and a computer including instructions executable by the at least one processor for performing the steps.

[0688] Embodiment 191. (a) Forming a first suspension comprising a first plurality of particles and a first portion of a biological sample, wherein the first plurality of particles includes a first particle and a second particle, the first particle has a first functionalized surface chemical structure different from the second functionalized surface chemical structure of the second particle, and the first particle and the second particle both have a negative charge; (b) forming a second suspension comprising a second plurality of particles and a second portion of a biological sample, wherein the second plurality of particles includes a third particle and a fourth particle, the third particle has a third functionalized surface chemical structure different from the fourth functionalized surface chemical structure of the fourth particle, and the third particle and the fourth particle both have a positive charge; (c) enriching the biomolecules adsorbed to the first plurality of particles and the biomolecules adsorbed to the second plurality of particles; and (d) performing a downstream assay on the enriched biomolecules.

[0689] Embodiment 192. The method of Embodiment 191, wherein the first suspension and the second suspension each have a pH between 9 and 10.

[0690] Method of Embodiment 192, wherein the first suspension and the second suspension each contain a Tris buffer.

[0691] Method of Embodiment 191, wherein the first suspension has a pH between 9 and 10.

[0692] Method of Embodiment 194, wherein the first suspension contains a Tris buffer.

[0693] Method of Embodiment 191, 194, or 195, wherein the second suspension has a pH between 6 and 8.

[0694] Method of any one of Embodiments 191 - 196, wherein the first particles are magnetic particles and include an outer layer surface-functionalized with silanol, and the second particles are magnetic particles and include an outer layer surface-functionalized with carboxylic acid.

[0695] Method of any one of Embodiments 191 - 197, wherein the third particles are magnetic particles and include an outer layer surface-functionalized with amine, and the fourth particles are magnetic particles and include an outer layer surface-functionalized with amine.

[0696] Method of any one of Embodiments 191 - 198, wherein the outer layer of the third particles contains a polymer.

[0697] Method of any one of Embodiments 191 - 199, wherein the first plurality of particles has a ratio of about 10 - about 15 parts by weight of the first particles to about 1 part by weight of the second particles.

[0698] Method of any one of Embodiments 191 - 200, wherein the second plurality of particles has a ratio of about 10 - about 15 parts by weight of the third particles to about 1 part by weight of the fourth particles.

[0699] The method according to any one of Embodiments 191 to 201, wherein the ratio of the average diameter of the first particles to the average diameter of the second particles is from about 3:2 to about 2:3.

[0700] Embodiment 203. The method according to any one of Embodiments 191 to 202, wherein the ratio of the average diameter of the third particles to the average diameter of the fourth particles is from about 3:2 to about 2:3.

[0701] Embodiment 204. The method according to any one of Embodiments 191 to 203, wherein each of the first particles, the second particles, the third particles, and the fourth particles is a superparamagnetic iron oxide nanoparticle.

[0702] Embodiment 205. A kit comprising: (a) a first composition comprising first particles and second particles, wherein the first particles have a first functionalized surface chemical structure different from the second functionalized surface chemical structure of the second particles, both the first particles and the second particles have a negative charge, and the first composition is a solid; and (b) a second composition comprising third particles and fourth particles, wherein the third particles have a third functionalized surface chemical structure different from the fourth functionalized surface chemical structure of the fourth particles, both the third particles and the fourth particles have a positive charge, and the second composition is a solid.

[0703] Embodiment 206. The kit according to Embodiment 205, further comprising a buffer having a pH between 9 and 10.

[0704] Embodiment 207. The kit according to Embodiment 205, further comprising a Tris buffer having a pH between 9 and 10.

[0705] Embodiment 208. The kit according to Embodiment 205, wherein both the first composition and the second composition are lyophilized.

[0706] Embodiment 209. The kit according to any one of Embodiments 205 to 208, wherein the first particles are magnetic particles and include an outer layer surface-functionalized with silanol, and the second particles are magnetic particles and include an outer layer surface-functionalized with carboxylic acid.

[0707] Embodiment 210. A kit according to any one of Embodiments 205 to 209, wherein the third particle is a magnetic particle and includes an outer layer surface-functionalized with an amine, and the fourth particle is a magnetic particle and includes an outer layer surface-functionalized with an amine.

[0708] Embodiment 211. A kit according to any one of Embodiments 205 to 210, wherein the outer layer of the third particle includes a polymer.

[0709] Embodiment 212. A kit according to any one of Embodiments 205 to 211, wherein the first composition has a ratio of about 10 to about 15 parts by weight of the first particle to about 1 part by weight of the second particle.

[0710] Embodiment 213. A kit according to any one of Embodiments 205 to 212, wherein the second composition has a ratio of about 10 to about 15 parts by weight of the third particle to about 1 part by weight of the fourth particle.

[0711] Embodiment 214. A kit according to any one of Embodiments 205 to 213, wherein the ratio of the average diameter of the first particle to the average diameter of the second particle is about 3:2 to about 2:3.

[0712] Embodiment 215. A kit according to any one of Embodiment...

Claims

1. (a) A step of incubating a composition in contact with one or more surfaces, wherein the composition comprises a biological sample and a buffer configured to maintain the pH of the composition between 8 and 11, and biomolecules derived from the biological sample adsorb to the one or more surfaces to form a biomolecular corona, (b) A step of enriching the biomolecules adsorbed on one or more surfaces, (c) A step of assaying the enriched biomolecules, the step of identifying one or more of the enriched biomolecules derived from the biological sample. A method that includes this.

2. The method according to claim 1, wherein one or more surfaces contain particles, and the particles are fine particles or nanoparticles.

3. The method according to claim 2, wherein the particles include a paramagnetic material.

4. The method according to claim 3, wherein the particles include an iron oxide core.

5. The method according to claim 4, wherein the iron oxide core contains superparamagnetic iron oxide.

6. The method according to claim 2, wherein the particles include a polymer outer layer.

7. The method according to claim 2, wherein the particles have an average diameter of less than 500 nm.

8. The method according to claim 2, wherein the particles have an average diameter of less than 200 nm.

9. The method according to claim 2, wherein the particles include a carboxylic acid-functionalized outer layer.

10. The method according to claim 2, wherein the particles contain an acidic functional group.

11. The method according to claim 2, wherein the particles include a carboxylic acid group, an acrylic acid group, a methacrylic acid group, an acetal group, a hemiacetal group, a hemiketal group, a sulfonic acid group, a sulfinic acid group, a thiocarboxylic acid group, a phosphonic acid group, a phosphate group, a phosphate diester group, a boronic acid group, a boronic acid ester group, a boric acid group, a boric acid ester group, a silica group, a silanol group, a thiol group, a polymer, or any combination thereof.

12. The method according to claim 2, wherein the particles have a negative zeta potential.

13. The method according to any one of claims 1 to 12, wherein the buffer solution is configured to maintain the pH of the composition between 9 and 10.

14. The method according to any one of claims 1 to 12, wherein the buffer solution comprises tris(hydroxymethyl)aminomethane, glycine, phosphate, or N-cyclohexyl-3-aminopropanesulfonic acid.

15. The method according to claim 14, wherein the buffer solution comprises tris(hydroxymethyl)aminomethane.

16. The method according to any one of claims 1 to 12, further comprising the step of diluting the biological sample with the buffer solution to form the composition.

17. The method according to any one of claims 1 to 12, wherein the biomolecule comprises one or more proteins.

18. The method according to any one of claims 1 to 12, wherein the biological sample comprises plasma.

19. The method according to any one of claims 1 to 12, wherein one or more surfaces have a zeta potential of less than -5 mV, less than -10 mV, less than -15 mV, or less than -20 mV.

20. The method according to any one of claims 1 to 12, wherein the enriching step includes washing the one or more surfaces with a cleaning composition having a pH of at least 8.

21. The method according to any one of claims 1 to 12, wherein the assay step includes mass spectrometry.

22. The method according to claim 21, wherein the mass spectrometry includes LC-MS / MS.

23. The method according to claim 17, further comprising the step of digesting the protein adsorbed on one or more surfaces before the assay step.

24. The method according to claim 17, further comprising the step of treating the protein adsorbed on one or more surfaces with a reducing agent, an alkylating agent, or both, prior to the assay step.

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