Systems and methods for biomolecule assays

Abstract

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

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 352,591, filed on June 15, 2022, U.S. Provisional Application No. 63 / 379,275, filed on October 12, 2022, and U.S. Provisional Application No. 63 / 491,012, filed on March 17, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Biological samples such as biological fluids contain a variety of proteins, whose presence, processing, and relative abundance may indicate biological states. Highly abundant proteins and other proteins may mask the signal relative to other proteins in the assay. Sample preparation (such as dilution) may further mask the relative signal in the assay. Summary of the invention

[0004] In some aspects, the present disclosure provides a method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in a second fluid composition; and (c) performing a downstream assay on the first plurality of biomolecule types and the second plurality of biomolecule types, wherein the first fluid composition comprises a first pH and the second fluid composition comprises a second pH, wherein the first pH and the second pH are different or the same.

[0005] In some embodiments, the second infinite dilution limiting solvation enthalpy or solvation free energy of the second biomolecules in the second subset of biomolecules is different when the second biomolecules are in the second fluid composition than when the second biomolecules are in the first fluid composition.

[0006] In some embodiments, the first fluid composition comprises a first set of intensity physical properties that mediate selective enrichment of a first plurality of biomolecule types, and the second fluid composition comprises a second set of intensity physical properties that mediate selective enrichment of a second plurality of biomolecule types, wherein the first set of intensity physical properties and the second set of intensity physical properties are different.

[0007] In some embodiments, the first fluid composition comprises a first ratio between a first sample volume and a first surface area of ​​a first surface, and the second fluid composition comprises a second ratio between a second sample volume and a second surface area of ​​a second surface, wherein the first ratio and the second ratio are different.

[0008] In some embodiments, the first fluid composition and the second fluid composition comprise different temperatures.

[0009] In some embodiments, the first fluid composition comprises a first ionic strength and the second fluid composition comprises a second ionic strength, wherein the first ionic strength and the second ionic strength are different.

[0010] In some embodiments, the first fluid composition and the second fluid composition comprise different salts.

[0011] In some embodiments, the first fluid composition and the second fluid composition comprise different solvents.

[0012] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a buffer comprising tris(hydroxymethyl)aminomethane.

[0013] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a buffer comprising citrate.

[0014] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a pH between 2 and 4.

[0015] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a pH between 5 and 7.

[0016] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a pH between 9 and 10.

[0017] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0018] In some embodiments, the first fluid composition, the second fluid composition, or both comprise a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0019] 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.

[0020] In some embodiments, the first fluid composition and the second fluid composition have a pH between 9 and 10.

[0021] In some embodiments, the method further comprises diluting the sample to prepare the first composition, the second composition, or both.

[0022] In some embodiments, diluting comprises adding a buffer.

[0023] In some embodiments, the buffer comprises a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0024] In some embodiments, the buffer comprises a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0025] In some embodiments, the sample and the buffer comprise different pHs.

[0026] In some embodiments, the sample comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of the biomolecule.

[0027] In some embodiments, the sample comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules per mL of sample.

[0028] In some embodiments, the sample comprises biomolecules from at most about 1000, 100, 10, or 1 cell.

[0029] In some embodiments, the sample contains at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters.

[0030] In some embodiments, the sample comprises a complex biological sample.

[0031] In some embodiments, the complex biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal secretions, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, stool, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration samples, tissue homogenates, lymph fluid, cell culture samples, or any combination thereof.

[0032] In some embodiments, the biological sample comprises plasma.

[0033] In some embodiments, selectively enriching the first plurality of biomolecule types comprises contacting the first fluid composition with a first surface to adsorb the first plurality of biomolecule types on the first surface.

[0034] In some embodiments, selectively enriching the second plurality of biomolecule types comprises contacting the second fluid composition with a second surface to adsorb the second plurality of biomolecule types on the second surface.

[0035] In some embodiments, the first surface, the second surface, or both comprise particles.

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

[0037] In some embodiments, the particles are microparticles.

[0038] In some embodiments, the particles are nanoparticles.

[0039] In some embodiments, the particles comprise a magnetic material.

[0040] In some embodiments, the particles comprise a paramagnetic material.

[0041] In some embodiments, the paramagnetic material is a superparamagnetic material.

[0042] In some embodiments, the paramagnetic material comprises iron oxide.

[0043] In some embodiments, selectively enriching the first plurality of biomolecule types comprises contacting the first fluid composition with a first plurality of surface types to adsorb the first plurality of biomolecule types on the first plurality of surface types.

[0044] In some embodiments, selectively enriching the second plurality of biomolecule types comprises contacting the second fluid composition with a second plurality of surface types to adsorb the second plurality of biomolecule types on the second plurality of surface types.

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

[0046] In some embodiments, the second plurality of surface types have the same charge.

[0047] In some embodiments, the first plurality of surface types have the same charge and the second plurality of surface types have the same charge.

[0048] In some embodiments, the charge of the first plurality of surface types is opposite to the charge of the second plurality of surface types.

[0049] In some embodiments, the first plurality of surface types have the same sign of zeta potential.

[0050] In some embodiments, the second plurality of surface types have the same sign of zeta potential.

[0051] In some embodiments, the first plurality of surface types and the second plurality of surface types have the same sign of zeta potential.

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

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

[0054] In some embodiments, the first plurality of surface types each have a zeta potential greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0055] In some embodiments, the second plurality of surface types each have a zeta potential greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0056] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both comprise acidic functional groups.

[0057] In some embodiments, the acidic functional group comprises a Bronsted-Lowry acid or a Lewis acid functional group.

[0058] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both comprise carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boronic acid groups, boric acid ester groups, diborinic acid groups, diboric acid ester groups, silica groups, silanol groups, thiol groups, polymers, or any combination thereof.

[0059] In some embodiments, the first plurality of surface types includes a first surface comprising a carboxylate-functionalized surface and a second surface comprising a silanol-functionalized surface.

[0060] In some embodiments, the first plurality of surface types, the second plurality of surface types, or both comprise 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, pyrimidinyl groups, pyrrolidinium groups, imidazolyl groups, guanidine groups, guanidinium groups, carbamoyl groups, ammonium groups, pyridinium groups, or any combination thereof.

[0061] In some embodiments, the first plurality of surface types each comprises an amine group.

[0062] In some embodiments, the method further comprises washing the first plurality of biomolecule types with a first wash composition and washing the second plurality of biomolecule types with a second wash composition.

[0063] In some embodiments, the first fluid composition and the first wash composition comprise at least one strength physical property in common.

[0064] In some embodiments, the first fluid composition and the first wash composition comprise at least one common solvent.

[0065] In some embodiments, the first fluid composition and the first wash composition comprise at least one different intensive physical property.

[0066] In some embodiments, the second fluid composition and the second wash composition comprise at least one strength physical property in common.

[0067] In some embodiments, the second fluid composition and the second wash composition comprise at least one common solvent.

[0068] In some embodiments, the second fluid composition and the second wash composition comprise at least one different intensive physical property.

[0069] In some embodiments, the first washing composition and the second washing composition comprise at least one strength physical property in common.

[0070] In some embodiments, the first cleaning composition and the second cleaning composition are the same.

[0071] In some embodiments, the first washing composition and the second washing composition comprise at least one different strong physical property.

[0072] In some embodiments, the first washing composition releases the first plurality of biomolecule types adsorbed on the first surface from the first surface.

[0073] In some embodiments, the method further comprises purifying the first plurality of biomolecule types to produce a first purified composition.

[0074] In some embodiments, purifying includes drying the plurality of biomolecule types to remove the first wash composition.

[0075] In some embodiments, the method further comprises reconstituted the first purified composition with the first reconstituted composition to produce a first reconstituted composition.

[0076] In some embodiments, the second washing composition releases a second plurality of biomolecule types deposited on the second surface from the second surface.

[0077] In some embodiments, the method further comprises purifying a second plurality of biomolecule types to produce a second purified composition.

[0078] In some embodiments, purifying includes drying the second plurality of biomolecule types to remove the second wash composition.

[0079] In some embodiments, the method further comprises reconstituting the second purified composition with a second reconstituted composition to produce a second reconstituted composition.

[0080] In some embodiments, downstream assays include mass spectrometry.

[0081] In some embodiments, mass spectrometry comprises LC-MS / MS.

[0082] In some embodiments, downstream assays include protein sequencing.

[0083] In some embodiments, a downstream assay comprises contacting a first plurality of biomolecule types, a second plurality of biomolecule types, or both with an antibody pair capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the antibody pair comprises complementary single-stranded nucleic acid sequences attached thereto such that when the antibody pair binds to the at least one biomolecule type, the complementary nucleic acids hybridize to form a double-stranded nucleic acid, wherein the double-stranded nucleic acid is configured to form a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction to produce a detectable signal.

[0084] In some embodiments, the downstream assay comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with one or more aptamers capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the one or more aptamers are coupled to the surface via a cleavable linker.

[0085] In some embodiments, the surface is a particle surface.

[0086] In some embodiments, the cleavable linker is photocleavable.

[0087] In some embodiments, the method further comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a macromolecular competitor configured to reduce dissociation of a complex comprising one or more aptamers and the first plurality of biomolecule types, the second plurality of biomolecule types, or both in the fluid composition.

[0088] In some embodiments, the macromolecular competitor is further configured to bind to a biomolecule type that is different from the first plurality of biomolecule types, the second plurality of biomolecule types, or both.

[0089] In some embodiments, the macromolecular competitor is a polyanionic macromolecule.

[0090] In some embodiments, downstream assays include nucleic acid sequencing.

[0091] In some embodiments, (a) and (b) are performed on one machine.

[0092] In some embodiments, (a) and (b) are performed on different machines.

[0093] In some embodiments, (a) and (b) are performed in parallel.

[0094] In some embodiments, (a) and (b) are performed serially.

[0095] In some embodiments, the first fluid composition and the second fluid composition are part of the same sample.

[0096] In some embodiments, the first fluid composition and the second fluid composition are from different samples.

[0097] In some embodiments, the first fluid composition and the second fluid composition are selectively enriched in parallel.

[0098] In some embodiments, the first fluid composition and the second fluid composition are selectively enriched serially.

[0099] In some embodiments, the selective enrichment is performed at a rate of enrichment of at least 100, 1000, 10000, 100000, or 1000000 biomolecules per hour.

[0100] In some embodiments, the selective enrichment is performed at a rate of enrichment of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule per hour.

[0101] In some embodiments, the first fluid composition and the second fluid composition are contacted in parallel.

[0102] In some embodiments, the first fluid composition and the second fluid composition are contacted serially.

[0103] In some embodiments, contacting is performed at a rate to deposit at least 100, 1000, 10,000, 100,000, or 1,000,000 biomolecules per hour.

[0104] In some embodiments, contacting is performed at a rate to deposit at least 1, 10, 100, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 μg of the biomolecule per hour.

[0105] In some embodiments, the first fluid composition and the second fluid composition are washed in parallel.

[0106] In some embodiments, the first fluid composition and the second fluid composition are washed serially.

[0107] In some embodiments, washing is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 biomolecules per hour.

[0108] In some embodiments, washing is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule per hour.

[0109] In some embodiments, the first fluid composition and the second fluid composition are purified in parallel.

[0110] In some embodiments, the first fluid composition and the second fluid composition are purified serially.

[0111] In some embodiments, purification is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 purified per hour.

[0112] In some embodiments, purification is performed at a rate of purifying at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of biomolecule per hour.

[0113] In some embodiments, the first fluid composition and the second fluid composition are reconstituted in parallel.

[0114] In some embodiments, the first fluid composition and the second fluid composition are reconstituted serially.

[0115] In some embodiments, reconstitution is performed at a rate of at least 100, 1000, 10000, 100000, or 1000000 reconstitutions per hour.

[0116] In some embodiments, reconstitution is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of the biomolecule per hour.

[0117] In some embodiments, downstream assays are performed on the first fluid composition and the second fluid composition in parallel.

[0118] In some embodiments, the downstream assay is performed serially on the first fluid composition and the second fluid composition.

[0119] In some embodiments, downstream assays are performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0120] In some embodiments, the downstream assay is performed at a rate of assaying at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of a biomolecule per hour.

[0121] In some embodiments, downstream assays are performed at a rate of identifying at least 100, 1000, 10,000, 100,000, or 1,000,000 biomolecules per hour.

[0122] In some embodiments, the downstream assays are performed at a rate of identifying at least 100, 1000, 10000, 100000, or 1000000 protein groups per hour.

[0123] In some embodiments, the method is performed in parallel on the first fluid composition and the second fluid composition.

[0124] In some embodiments, the method is performed serially on the first fluid composition and the second fluid composition.

[0125] In some embodiments, the methods are performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0126] In some embodiments, the methods are performed at a rate of assaying at least 1, 10, 100, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 μg of a biomolecule per hour.

[0127] In some embodiments, the methods are performed at a rate of identifying at least 100, 1000, 10000, 100000, or 1000000 biomolecules per hour.

[0128] In some embodiments, the methods are performed at a rate of identifying at least 100, 1000, 10000, 100000, or 1000000 protein groups per hour.

[0129] In some embodiments, when the first fluid composition and the second fluid composition are HeLa cell extracts, the downstream assay identifies at least about 100, 1000, 10000, 100000, or 1000000 biomolecules.

[0130] In some embodiments, when the first fluid composition and the second fluid composition are subjected to downstream assays prior to the selective enrichment in (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together comprise at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0131] In some embodiments, the first plurality of biomolecule types is depleted of at least a first biomolecule and enriched for a second biomolecule, wherein the first biomolecule is more abundant than the second biomolecule in the first fluid composition.

[0132] In some embodiments, the second plurality of biomolecule types is depleted of at least a first biomolecule and enriched for a second biomolecule, wherein the first biomolecule is more abundant than the second biomolecule in the second fluid composition.

[0133] In some embodiments, a first likelihood that a downstream assay identifies a low-abundance biomolecule in the first plurality of biomolecule types or the second plurality of biomolecule types is greater than a second likelihood that a downstream assay identifies a low-abundance biomolecule in the first fluid composition or the second fluid composition, wherein the low-abundance biomolecule comprises less than about 1, 10, or 10 percent by mass of the first set of biomolecules or the second set of biomolecules. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 .

[0134] In some embodiments, the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 times.

[0135] In some embodiments, the downstream identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0136] In some embodiments, the downstream identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0137] In some embodiments, the one or more biomolecules include 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.

[0138] In some embodiments, the one or more biomolecules include at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins.

[0139] In some embodiments, the first plurality of biomolecule types, the second plurality of biomolecule types, or both include one or more polyamino acids.

[0140] 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.

[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 fluid composition.

[0142] 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 wash composition.

[0143] 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 wash composition.

[0144] 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 reconstituted composition.

[0145] In some embodiments, the method further comprises identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstituted composition.

[0146] In some embodiments, the first surface is disposed in a first lyophilized composition comprising (i) at least one nanoparticle, and (ii) one or more modulators.

[0147] In some embodiments, the second surface is disposed in a second lyophilized composition comprising (i) at least one nanoparticle, and (ii) one or more modulators.

[0148] In some embodiments, the one or more modifiers include a pH modifier, an ionic strength modifier, a viscosity modifier, or any combination thereof.

[0149] 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.

[0150] In some aspects, the present disclosure provides a method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in a second fluid composition; and (c) performing a downstream assay on the biomolecule types of the first plurality of biomolecule types and the second plurality of biomolecule types, wherein the first fluid composition and the second fluid composition are different such that a first infinite dilution limiting solvation enthalpy or solvation free energy of a first biomolecule in the first plurality of biomolecule types is different when the first biomolecule is in the first fluid composition than when the biomolecule is in the second fluid composition.

[0151] In some aspects, the present disclosure provides a kit comprising: a first reagent comprising a first pH; a second reagent comprising a second pH; a first surface configured to selectively enrich a first plurality of biomolecule types 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 biomolecule types in a second fluid composition comprising the second surface and the second reagent, wherein the first fluid composition comprises the first reagent and the second fluid composition comprises the second reagent, wherein the first pH and the second pH are different or the same.

[0152] In some aspects, the present disclosure provides a composition comprising: a suspension comprising: first particles comprising (i) a first paramagnetic portion and (ii) a first surface chemical; second particles comprising (i) a second paramagnetic portion and (ii) a second surface chemical, wherein the second surface chemical is different from the first surface chemical; and a plurality of biomolecules adsorbed on the first particles and the second particles.

[0153] In some embodiments, the first particle and the second particle have the same charge.

[0154] In some embodiments, the first particle and the second particle have the same sign of zeta potential.

[0155] In some embodiments, the same sign is a minus sign.

[0156] In some embodiments, the same sign is a positive sign.

[0157] 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.

[0158] In some embodiments, the first particle and the second particle each have a zeta potential greater than 5 mV, greater than 10 mV, greater than 15 mV, or greater than 20 mV.

[0159] In some embodiments, the first particle, the second particle, or both comprise acidic functional groups.

[0160] In some embodiments, the acidic functional group comprises a Bronsted-Lowry acid or a Lewis acid functional group.

[0161] In some embodiments, the first particles, the second particles, or both comprise carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boric acid groups, boric acid ester groups, dihydrocarbylboronic acid groups, dihydrocarbylboronic ester groups, silica groups, silanol groups, thiol groups, polymers, or any combination thereof.

[0162] In some embodiments, the first particle, the second particle, or both comprise carboxylate functionalization, silanol functionalization, or both.

[0163] 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 pyrimidyl group, a pyrrolidinium group, an imidazolyl group, a guanidine group, a guanidinium group, a carbamoyl group, an ammonium group, a pyridinium group, or any combination thereof.

[0164] In some embodiments, the first particle, the second particle, or both comprise amine groups.

[0165] In some embodiments, the first surface chemistry, the second surface chemistry, or both comprise acidic functional groups.

[0166] In some embodiments, the acidic functional group comprises a Bronsted-Lowry acid or a Lewis acid functional group.

[0167] In some embodiments, the first surface chemistry, the second surface chemistry, or both comprise carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boronic acid groups, borate ester groups, dihydrocarbylboronic acid groups, dihydrocarbylboronic ester groups, silica groups, silanol groups, polymers, or any combination thereof.

[0168] In some embodiments, the suspension is stable for at least about 1, 5, 10, 15, 30, or 60 minutes.

[0169] In some embodiments, the time constant for destabilization of the suspension is at least about 1, 5, 10, 15, 30, or 60 minutes.

[0170] In some embodiments, the average aggregation number of the first particles and the second particles in the suspension is at most about 1000, 100, or 10.

[0171] In some embodiments, the suspension comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts first particles to about 1 part second particles.

[0172] In some embodiments, the suspension comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts first particles to about 1 part second particles.

[0173] In some embodiments, the suspension comprises about 15 parts first particles to about 6 parts second particles.

[0174] In some embodiments, parts are parts by weight, parts by volume, or parts by surface area.

[0175] In some embodiments, parts are parts by weight.

[0176] In some embodiments, the suspension comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 different particles.

[0177] In some embodiments, the suspension comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different particles.

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

[0179] In some embodiments, the first particle, the second particle, or both are microparticles.

[0180] In some embodiments, the first particle, the second particle, or both are porous.

[0181] In some embodiments, the first dimension of the first particle is at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the second dimension of the second particle.

[0182] In some embodiments, the first dimension of the first particle is at most about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the second dimension of the second particle.

[0183] 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.

[0184] In some embodiments, the first dimension is a first diameter and the second dimension is a second diameter.

[0185] In some embodiments, the first size is a first average size and the second size is a second average size.

[0186] In some embodiments, the first average size and the second average size are average sizes or median sizes.

[0187] 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.

[0188] In some embodiments, the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules.

[0189] In some embodiments, the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules / mL.

[0190] In some embodiments, the plurality of biomolecules comprises biomolecules from at most about 1000, 100, 10, or 1 cell.

[0191] In some aspects, the present disclosure provides a method comprising: assaying a plurality of biomolecules in a composition disclosed herein to identify one or more biomolecules in the plurality of biomolecules.

[0192] In some embodiments, determining comprises performing mass spectrometry.

[0193] In some embodiments, mass spectrometry comprises LC-MS / MS.

[0194] In some embodiments, determining comprises performing protein sequencing.

[0195] In some embodiments, the assay comprises contacting a plurality of biomolecules with an antibody pair capable of binding to at least one biomolecule of the plurality of biomolecules, wherein the antibody pair comprises complementary single-stranded nucleic acid sequences attached thereto such that when the antibody pair binds to the at least one biomolecule, the complementary nucleic acids hybridize to form a double-stranded nucleic acid, wherein the double-stranded nucleic acid is configured to form a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction to produce a detectable signal.

[0196] In some embodiments, the assay comprises contacting a plurality of biomolecules with one or more aptamers capable of binding to at least one biomolecule in the plurality of biomolecules, wherein the one or more aptamers are coupled to the surface via a cleavable linker.

[0197] In some embodiments, the surface is a particle surface.

[0198] In some embodiments, the cleavable linker is photocleavable.

[0199] In some embodiments, the method further comprises contacting the plurality of biomolecules with a macromolecular competitor configured to reduce dissociation of a complex comprising the one or more aptamers and the biomarker in the fluid composition.

[0200] In some embodiments, the macromolecular competitor is further configured to bind to a biomolecule other than the biomarker.

[0201] In some embodiments, the macromolecular competitor is a polyanionic macromolecule.

[0202] In some embodiments, determining comprises performing nucleic acid sequencing.

[0203] In some embodiments, the sample comprises a plurality of biomolecules.

[0204] In some embodiments, the sample is a complex biological sample.

[0205] In some embodiments, the complex biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal secretions, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, stool, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration samples, tissue homogenates, lymph fluid, cell culture samples, or any combination thereof.

[0206] In some embodiments, the biological sample comprises plasma.

[0207] In some embodiments, the plurality of biomolecules comprises one or more polyamino acids.

[0208] In some embodiments, the assays are performed at a rate of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100 samples per hour.

[0209] In some embodiments, the assay is performed at a rate of at least 1, 10, 100, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 μg of the biomolecule per hour.

[0210] In some embodiments, the assay is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000, or 1000000 biomolecules per hour.

[0211] In some embodiments, the assay is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000, or 1000000 protein groups per hour.

[0212] In some embodiments, when the suspension comprises a HeLa cell extract, the assay identifies at least about 100, 1000, 10000, or 100000 biomolecules.

[0213] In some embodiments, when the plurality of biomolecules in the biological sample are assayed in the absence of the first particle and the second particle, the plurality of biomolecules comprises at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0214] In some embodiments, determining a first likelihood of identifying a low-abundance biomolecule in the plurality of biomolecules is greater than determining a second likelihood of identifying the low-abundance biomolecule in the biological sample in the absence of the first particle and the second particle, wherein the low-abundance biomolecule comprises less than about 1, 10, or 10 percent by mass of the first or second set of biomolecules. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 .

[0215] In some embodiments, the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 times.

[0216] In some embodiments, an assay identifies one or more biomolecules in a plurality of biomolecules with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0217] In some embodiments, an assay identifies one or more biomolecules in a plurality of biomolecules with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0218] In some aspects, the present disclosure provides a system for analyzing multiple biological samples, comprising: (a) multiple partitions, including a first partition and a second partition; (b) multiple reagent reservoirs, including a first reagent comprising a first pH and a second reagent comprising a second pH, wherein the first pH and the second pH are different or the same; (c) multiple substrates, including a first substrate comprising a first surface chemical and a second substrate comprising a second surface chemical; (d) one or more transfer devices, which are operably connected to the multiple partitions, the multiple reagent reservoirs and the multiple substrates; and (e) a computer, which includes at least one processor and instructions executable by the at least one processor to perform steps including the following: (i) using one or more transfer devices to generate a first fluid composition in the first partition, the first fluid composition comprising a first substrate, a first reagent and a first plurality of biomolecules, wherein the first plurality of biomolecules are adsorbed on the first substrate; (ii) using one or more transfer devices to generate a second fluid composition in the second partition, the second fluid composition comprising a second substrate, a second reagent and a second plurality of biomolecules, wherein the second plurality of biomolecules are adsorbed on 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.

[0219] In some aspects, the present disclosure provides a system for analyzing multiple biological samples, comprising: (a) partitions; (b) a reagent reservoir containing reagents; (c) multiple substrates, comprising a first substrate containing a first surface chemical and a second substrate containing a second surface chemical, wherein the first surface chemical and the second surface chemical are different; (d) one or more transfer devices, which are operably connected to the partitions, the reagent reservoir and the multiple substrates; and (e) a computer, which includes at least one processor and instructions executable by the at least one processor to perform the following steps: (i) using one or more transfer devices to generate a fluid composition in the partition, the fluid composition comprising multiple substrates, reagents and multiple biomolecules, wherein the multiple biomolecules are adsorbed on the substrates; and (ii) using one or more transfer devices to prepare the multiple biomolecules for mass spectrometry.

[0220] In some aspects, the present disclosure provides a method comprising: (a) forming a first suspension comprising a first plurality of particles using a first portion of a biological sample, wherein the first plurality of particles comprises first particles and second particles, wherein the first particles have a first functionalized surface chemistry that is different from a second functionalized surface chemistry of the second particles, and wherein both the first particles and the second particles have a negative charge; (b) forming a second suspension comprising a second plurality of particles using a second portion of the biological sample, wherein the second plurality of particles comprises third particles and fourth particles, wherein the third particles have a third functionalized surface chemistry that is different from a fourth functionalized surface chemistry of the fourth particles, and wherein both the third particles and the fourth particles have a positive charge; (c) enriching biomolecules that have been adsorbed to the first plurality of particles and the second plurality of particles; and (d) performing downstream assays on the enriched biomolecules.

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

[0222] In some embodiments, the first suspension and the second suspension each comprise Tris buffer.

[0223] In some embodiments, the first suspension has a pH between 9 and 10.

[0224] In some embodiments, the first suspension comprises Tris buffer.

[0225] In some embodiments, the second suspension has a pH between 6 and 8.

[0226] In some embodiments, the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0227] In some embodiments, the third particle is a magnetic particle and comprises an outer layer having an amine surface functionalization, and the fourth particle is a magnetic particle and comprises an outer layer having an amine surface functionalization.

[0228] In some embodiments, the outer layer of the third particle comprises a polymer.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In some embodiments, 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.

[0233] In some embodiments, each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle.

[0234] 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 chemistry different from the second functionalized surface chemistry of the second particle, and wherein the first particle and the second particle both have a negative charge, wherein 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 chemistry different from the fourth functionalized surface chemistry of the fourth particle, and wherein the third particle and the fourth particle both have a positive charge, wherein the second composition is a solid.

[0235] In some embodiments, the kit further comprises a buffer having a pH between 9 and 10.

[0236] In some embodiments, the kit further comprises a Tris buffer having a pH between 9 and 10.

[0237] In some embodiments, both the first composition and the second composition in the kit are lyophilized.

[0238] In some embodiments, a first particle in the kit is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and a second particle in the kit is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0239] In some embodiments, a third particle in the kit is a magnetic particle and comprises an outer layer having an amine surface functionalization, and a fourth particle in the kit is a magnetic particle and comprises an outer layer having an amine surface functionalization.

[0240] In some embodiments, the outer layer of the third particle in the kit comprises a polymer.

[0241] In some embodiments, the first composition in the kit 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.

[0242] In some embodiments, the second composition in the kit 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.

[0243] 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.

[0244] 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.

[0245] In some embodiments, each of the first particle, the second particle, the third particle, and the fourth particle in the kit is a superparamagnetic iron oxide nanoparticle.

[0246] In some aspects, the present disclosure provides a suspension comprising: (a) a first particle comprising an outer layer having a negatively charged surface functionalization; (b) a biological sample; and (c) a buffer configured to maintain a pH of the suspension between 9 and 10, wherein the biomolecule is adsorbed to the outer layer of the first particle.

[0247] In some aspects, the present disclosure provides a suspension comprising: (a) a first particle comprising a first outer layer having a negatively charged surface functionalization; (b) a second particle comprising a second outer layer having a negatively charged surface functionalization, 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 the biomolecules are adsorbed 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 comprises a silanol and the second outer layer comprises a carboxylate.

[0249] In some aspects, the present disclosure provides a suspension comprising: (a) a first particle comprising a first outer layer having a positively charged surface functionalization; (b) a second particle comprising a second outer layer having a positively charged surface functionalization, wherein the second outer layer is different from the first outer layer; (c) a biological sample; and (d) a buffer configured to maintain a pH of the suspension between 9 and 10, wherein the biomolecules are adsorbed to the first outer layer of the first particle and the second outer layer of the particle.

[0250] In some embodiments, the first outer layer and the second outer layer comprise amine groups.

[0251] In some embodiments, the biomolecules comprise at least 100 different peptides or proteins.

[0252] In some embodiments, the first particles and optional second particles are magnetic.

[0253] In some embodiments, the buffer is Tris buffer.

[0254] In some aspects, the present disclosure provides a method comprising: (a) 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, wherein biomolecules from the biological sample are adsorbed onto the one or more surfaces; (b) enriching the biomolecules that have been adsorbed to the surface; (c) measuring the enriched biomolecules to identify one or more of the enriched biomolecules from the biological sample.

[0255] In some embodiments, the buffer is configured to maintain the pH of the composition between 9 and 10.

[0256] In some embodiments, the buffer is configured to maintain the pH of the composition at about 9.5.

[0257] In some embodiments, the buffer is Tris buffer.

[0258] In some embodiments, the biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal secretions, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, stool, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0259] In some embodiments, the biological sample is plasma or serum.

[0260] In some embodiments, the biological sample is a cell culture medium.

[0261] In some embodiments, the composition comprises no more than 60% biological sample by volume.

[0262] In some embodiments, the composition comprises no more than 25% biological sample by volume.

[0263] In some embodiments, the composition comprises no more than 15% biological sample by volume.

[0264] In some embodiments, the composition comprises at least 1% by volume of the biological sample.

[0265] In some embodiments, the composition comprises at least 4% biological sample by volume.

[0266] In some embodiments, the composition comprises at least 10% by volume of the biological sample.

[0267] In some embodiments, the composition comprises at least 20% by volume of the biological sample.

[0268] In some embodiments, the assay comprises SDS-PAGE, gel-based separation techniques, immunoassays, ELISA, high performance liquid chromatography, mass spectrometry, Edman degradation, or immunoaffinity techniques.

[0269] In some embodiments, determining comprises mass spectrometry.

[0270] In some embodiments, the assay comprises LC-MS / MS.

[0271] In some embodiments, determining comprises quantifying a biomolecule.

[0272] In some embodiments, the assay comprises identifying one or more proteins in a biological sample.

[0273] In some embodiments, at least 100 different proteins are identified in the biological sample.

[0274] In some embodiments, at least 250 different proteins are identified in the biological sample.

[0275] In some embodiments, at least 500 different proteins are identified in the biological sample.

[0276] In some embodiments, one or more surfaces are contained on particles that are dispersed in the composition during the incubation period.

[0277] In some embodiments, the nanoparticles have an average diameter of less than 500 nanometers.

[0278] In some embodiments, the particles are magnetic.

[0279] In some embodiments, the particle comprises an outer layer of poly(dimethylaminopropylmethacrylamide) (PDMAPMA).

[0280] In some embodiments, the particles comprise an outer layer of silica.

[0281] In some embodiments, the particle comprises a carboxylate-functionalized outer layer.

[0282] In some embodiments, the particle comprises an amine-functionalized outer layer.

[0283] In some embodiments, the particle comprises a polymer outer layer.

[0284] In some embodiments, the particle comprises an outer layer of polyacrylamide.

[0285] In some embodiments, the particles comprise an acrylate polymer.

[0286] In some embodiments, the method further comprises: (a) incubating a second composition in contact with the one or more second surfaces, wherein the second composition comprises a biological sample, wherein biomolecules from the biological sample are adsorbed onto the one or more second surfaces; (b) enriching the biomolecules that have been adsorbed to the one or more second surfaces; and (c) assaying the enriched biomolecules to identify one or more of the enriched biomolecules from the one or more second surfaces in the biological sample.

[0287] In some embodiments, the second composition has a pH between 5 and 9.

[0288] In some embodiments, the second composition has a pH between 6 and 8.

[0289] In some embodiments, the second composition has a pH between 8 and 11.

[0290] In some embodiments, the second composition has a pH between 9 and 10.

[0291] In some embodiments, the second composition does not include a buffer.

[0292] In some embodiments, the second composition comprises a buffer different than the buffer configured to maintain the pH of the composition between 8 and 11.

[0293] In some embodiments, the second composition comprises the same buffer as the composition.

[0294] In some embodiments, incubation of the composition and incubation of the second composition occur simultaneously.

[0295] In some embodiments, the assay identifies enriched biomolecules with a coefficient of variation of at most 30%.

[0296] In some embodiments, the assay identifies enriched biomolecules with a coefficient of variation of at most 25%.

[0297] In some embodiments, the method further comprises digesting the protein adsorbed to the first surface prior to assaying the biomolecule.

[0298] In some embodiments, the method further comprises treating the protein within the biomolecule corona with a reducing agent prior to assaying the biomolecule.

[0299] In some embodiments, the method further comprises treating the protein within the biomolecule corona with an alkylating agent prior to assaying the biomolecule.

[0300] 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, 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 negative charge; (c) a biological sample; and (d) a buffer configured to maintain a pH of about 8 to about 11.

[0301] In some embodiments, the buffer is configured to maintain the pH between 9 and 10.

[0302] In some embodiments, the buffer is Tris buffer at a pH between 9 and 10.

[0303] In some embodiments, the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0304] 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.

[0305] 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.

[0306] In some embodiments, the first particle and the second particle are superparamagnetic iron oxide nanoparticles.

[0307] 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, 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; and (c) a biological sample.

[0308] In some embodiments, the first particle is a magnetic particle and comprises an outer layer having an amine surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having an amine surface functionalization.

[0309] In some embodiments, the outer layer of the first particle comprises a polymer.

[0310] 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.

[0311] 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.

[0312] In some embodiments, the first particle and the second particle are superparamagnetic iron oxide nanoparticles.

[0313] In some embodiments, the biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal secretions, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, stool, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0314] In some embodiments, the biological sample comprises plasma or serum.

[0315] In some embodiments, the biological sample is a cell-free biological sample.

[0316] Incorporation by reference

[0317] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0318] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings which illustrate illustrative embodiments utilizing the principles of the present disclosure, in which:

[0319] Figure 1 Methods for performing the assay according to some embodiments are shown.

[0320] Figure 2A The composition of a pH adjusted biological sample according to some embodiments is shown.

[0321] Figure 2B A method for performing PROTEOGRAPH on a biological sample using pH adjustment is shown according to some embodiments. TM 96-well plate planning for the assay.

[0322] Figure 2C The composition of a pH adjusted wash buffer according to some embodiments is shown.

[0323] Figure 3 According to some embodiments, the use of PROTEOGRAPHTM The number of protein groups detected in biological samples adjusted with different particles and various pH was determined.

[0324] Figure 4 According to some embodiments, the use of PROTEOGRAPH TM The mass of peptides detected in biological samples adjusted with different particles and various pH was determined.

[0325] Figure 5 According to some embodiments, the use of PROTEOGRAPH TM The mis-cleavage rate of proteins detected in biological samples adjusted with different particles and various pH was determined.

[0326] Fig. 6A According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteome identified using pH adjusted buffer versus water.

[0327] Figure 6B-6D According to some embodiments, the use of PROTEOGRAPH TM Comparison of the concentration of identified proteomes measured with pH adjusted buffer versus water.

[0328] Fig. 7A According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0329] Figure 7B-7D According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0330] Fig. 8A According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteome identified using pH adjusted buffer versus water.

[0331] Figure 8B-Figure 8D According to some embodiments, the use of PROTEOGRAPH TM Comparison of the concentration of identified proteomes measured with pH adjusted buffer versus water.

[0332] Fig.9A According to some embodiments, the use of PROTEOGRAPH TMComparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0333] Figure 9B-9D According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0334] Fig. 10A According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteome identified using pH adjusted buffer versus water.

[0335] Figure 10B-10D According to some embodiments, the use of PROTEOGRAPH TM Comparison of the concentration of identified proteomes measured with pH adjusted buffer versus water.

[0336] Fig.11A According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0337] Figure 11B-Figure 11D According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0338] Fig.12 Median proteome counts for a panel of particles are shown according to some embodiments. A two-particle panel used pH 9.5 for one particle and pH 7 for the other particle.

[0339] Fig.13A According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteome identified using pH adjusted buffer versus water.

[0340] Figure 13B-Figure 13D According to some embodiments, the use of PROTEOGRAPH TM Comparison of the concentration of identified proteomes measured with pH adjusted buffer versus water.

[0341] Fig.14A According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0342] Figure 14B-Figure 14D According to some embodiments, the use of PROTEOGRAPH TM Comparison of functional annotation of the proteome identified using pH-adjusted buffer versus water.

[0343] Fig.15 According to some embodiments, the use of PROTEOGRAPH TM The number of protein groups identified at different pH using the particle set was determined. A 17% increase in the number of protein groups identified was observed using high pH.

[0344] Fig.16 Shown are particle sets with mixed buffer conditions according to some embodiments.

[0345] Figures 17A-17F According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteomes identified using the particles at different pH.

[0346] Figure 18A-18F According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteomes identified using the particles at different pH.

[0347] Figures 19A-19F According to some embodiments, the use of PROTEOGRAPH TM Statistical comparison of the proteomes identified using the particles at different pH.

[0348] Fig. 20 A method for crown formation according to some embodiments is shown.

[0349] Figure 21A-21B A method for performing PROTEOGRAPH on a biological sample using pH adjustment is shown according to some embodiments. TM 96-well plate planning for the assay.

[0350] Fig. 22 Shown is a comparison of the number of protein groups identified between a multiplex assay compared to a singleplex assay according to some embodiments.

[0351] Fig.23 Shown is a comparison of the number of protein groups identified between various multiplexed assays using different Vroman conditions according to some embodiments.

[0352] Fig.24Shown is a comparison of the number of protein groups identified between various multiplexed assays according to some embodiments.

[0353] Fig.25 Shown is a comparison of the number of protein groups identified between various multiplexed assays using different pH conditions according to some embodiments.

[0354] Figure 26A-Figure 26I Surfaces according to some embodiments are shown. The surface can be functionalized in one or more regions to capture biomolecules. The surface can include one or more holes or depressions for capturing biomolecules. For example, the functionalized surface can be arranged in a 96-well plate or a 384-well plate. The surface can be arranged on one or more particles. In some embodiments, one or more particles can be arranged in one or more holes or depressions. The surface can be arranged on a plurality of particles that are filled in a channel or arranged in a porous material in a channel. The surface can be arranged on the inner surface of a channel. The surface can include 1, 2, 3, 4 or any number of different surface areas. In some cases, the surface can be arranged on a particle. In some cases, the particle can be a porous particle. DETAILED DESCRIPTION

[0355] Although the human genome contains approximately 20,000 genes, some researchers estimate that the human proteome contains more than 1 million proteins expressed from these genes. Many different protein variants can be expressed from a repertoire of various transcriptional, translational, and post-translational mechanisms (e.g., alternative splicing forms, allelic variations, and protein modifications) that produce proteins that differ from those containing the canonical sequences expressed from these genes. Of the large number of proteins estimated to be present in the human proteome, only a small fraction has thus been meaningfully identified and / or quantified in humans.

[0356] Some challenges in identifying and quantifying proteins are related to the rarity of certain proteins. For example, human plasma contains protein species over a dynamic range of more than 12 orders of magnitude, with the top few proteins (e.g., albumin, transferrin, complement proteins, apolipoproteins, and alpha-2-macroglobulin) comprising 95% of the mass of proteins in plasma, and most protein species comprising the remaining 5%. Some protein species exist in the nanogram / milliliter range (e.g., transforming growth factor beta-1-induced transcript 1 protein at ∼10 ng / ml; fructose-bisphosphate aldolase A at ∼20 ng / ml; thioredoxin at ∼18 ng / ml; and L-selectin at ∼92 ng / ml), and some proteins are expected to exist at levels even below this range. Liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS) have developed into ubiquitous detection platforms due to their speed, sensitivity, and breadth of application. LC-MS and LC-MS / MS can be used to identify protein species, however, due to the stochastic nature of the method, only a portion of the ion species generated from a given sample at a certain moment may be selected for acquisition of a mass spectrum. Therefore, the presence of highly abundant species compared to rare species can produce an overwhelming number of signals that make the rare species difficult to reach.

[0357] PROTEOGRAPH TM Some aspects of the technology aim to address some of these challenges by “compressing” the dynamic range of protein species in a sample. TM Some aspects of the technology operate based on the non-specific binding of proteins to the surface of nanoparticles to form a protein corona. Without the need for the presence of a specific entity configured to bind to a single specific protein (e.g., as in an immunoassay), non-specific binding can result in a compression of the dynamic range of proteins bound to the surface of the nanoparticle, while capturing multiple proteins. In other words, the relative abundance of proteins in a sample can be modified on the surface of the nanoparticle so that rare proteins are relatively more abundant and high-abundance proteins are relatively less abundant than in the original sample. The proteins can then be separated from the sample and analyzed, for example, by mass spectrometry. The compressed dynamic range can allow rare proteins to contain a higher fraction of ion species, thereby allowing a higher probability of detecting those rare proteins in an MS experiment. Although the above examples are described in terms of proteins, other biomolecule classes (e.g., lipids, sugars, etc.) can be similarly targeted. PROTEOGRAPH TM Other aspects of the technology include PROTEOGRAPH TM Controlled automation of workflows that increases speed / throughput and accuracy / reliability.

[0358] Although PROTEOGRAPH TMThe introduction of technology has increased the number of proteins that can be detected from a sample, but has presented another challenge, which is to find biomarkers and / or therapeutic targets in those proteins. As the number of proteins that can be considered for diagnosis or therapeutic potential increases, the sample size can also be increased in order to effectively screen the relevant proteins. Due to individual differences in biology between people, thousands of proteins can have different levels in plasma samples between two individuals. Therefore, samples from hundreds or thousands of individuals can be experimented with to identify meaningful and systematic signals with clinical relevance. Therefore, some aspects of the present disclosure provide systems, compositions and methods that provide higher depth (e.g., the number of biomolecules detected and the increased dynamic range) and throughput, which can identify the finer details present in the biosignatures of complex biological samples. By increasing the depth, more obscure biosignatures (e.g., those associated with very rare proteins) can become more visible. By increasing the throughput, pseudo-variations in the data (e.g., pseudo-variations associated with known and unknown error sources, i.e., standard deviation / error) can be reduced by increasing the certainty.

[0359] In some aspects, the present disclosure provides a composition of multiple particles having physicochemically distinct characteristics in one volume. Physicochemically distinct particles can each enrich / deplete different portions of the biomolecule spectrum in a biological sample. Thus, the use of multiple physicochemically distinct particles can provide advantages in spectral analysis of biological samples at a higher dynamic range. By multiplexing physicochemically distinct particles together into a single volume (e.g., wells in a 96-well plate), PROTEOGRAPH TM Experiments can be performed at a higher throughput than is said when the particles are each placed in a separate well. As a simple illustration, multiplexing two particles in each well can increase the overall throughput by a factor of two. In some aspects, the present disclosure provides systems and methods that employ such compositions.

[0360] Another aspect of the present disclosure provides a composition comprising particles and a solvent environment customized for the particles. The customized solvent environment may include a certain solvent, salt, pH, etc., which can improve the performance of the PROTEOGRAPH TM Another aspect of the present disclosure provides a composition comprising a multiplexed particle and a solvent environment customized for the multiplexed particle. The customized solvent environment can increase the number of biomolecules identified, but can also improve the stability and / or dispersibility of the multiplexed particle. In some aspects, the present disclosure provides systems and methods using such compositions.

[0361] Another aspect of the present disclosure provides methods and systems for performing fast, scalable, deep and unbiased plasma proteomics. In some cases, the method and system can be used to identify known and / or novel biomarkers of disease. In some cases, the method and system can be used to promote the identification of disease-related protein variants, for example, as disclosed in PCT / US2023 / 060271, which is incorporated herein by reference in its entirety. Important advances in the proteomic panorama of characterizing lung cancer such as non-small cell lung cancer (NSCLC) and squamous cell lung cancer have identified important protein biomarkers. However, relatively few protein variants associated with lung cancer have been identified. 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., <1% false discovery rate (FDR)). However, large-scale LC-MS / MS-based proteomics research may be challenging because a long workflow is required to achieve depth (e.g., extensive detection of proteins in the dynamic range from high abundance to low abundance proteins) and unbiased (e.g., hypothesis-free detection) sampling of clinically relevant biological samples (such as plasma) with a large dynamic range of protein abundance. Although LC-MS and LC-MS / MS methods can provide the ability to infer protein variants, peptide identification in LC-MS / MS-based proteomics data can rely on protein databases, such as UniProt, which can exclude protein variants that may be present in individual proteomes. In some cases, compositions, methods, and systems disclosed herein can be used to observe examples of variable exon use. In some cases, compositions, methods, and systems disclosed herein can be used to identify protein variants produced by alternative splicing. In some cases, compositions, methods, and systems disclosed herein can be used to identify protein variants produced by genetic variation. In some cases, compositions, methods and systems disclosed herein can be used to identify protein variants based at least in part on a custom protein database generated by genotype data (such as whole exome sequencing (WES) data) matched by a subject. In some cases, compositions, methods and systems disclosed herein can be used to discover new protein variants. In some cases, compositions, methods and systems disclosed herein can be used to identify protein variants that cannot be identified using targeting techniques based on protein affinity in other ways. In some cases, compositions, methods and systems disclosed herein can be used to support an enhanced understanding of human health and disease by identifying protein variants.

[0362] In some aspects, the present disclosure provides methods for selectively enriching and measuring multiple biomolecules in a fluid composition. In some embodiments, the method includes selectively enriching a first plurality of biomolecule types in a first fluid composition. In some embodiments, the method includes selectively enriching a second plurality of biomolecule types in a second fluid composition. In some embodiments, the method includes downstream measuring the biomolecule types of the first plurality of biomolecule types and the second plurality of biomolecule types.

[0363] The methods, compositions and systems disclosed herein can, for example, provide improved proteomic analysis of biological samples. In some embodiments, similar or more proteomic groups can be detected in biological samples using fewer mass spectrometry injections and / or shorter chromatographic gradients in LC-MS / MS. For example, two 30-minute gradient injections compared to five 30-minute gradient injections can be used to identify similar proteomic groups. In some embodiments, less sample volume can be used while still obtaining similar proteomic identifications.

[0364] Methods for selective enrichment and determination of biomolecules

[0365] In some embodiments, selectively enriching a first plurality of biomolecule types in a fluid composition comprises contacting the first fluid composition with a first surface to adsorb the first plurality of biomolecule types on the first surface. In some embodiments, selectively enriching a second plurality of biomolecule types in a fluid composition comprises contacting the second fluid composition with a second surface to adsorb the second plurality of biomolecule types on the second surface. In some embodiments, selectively enriching a first plurality of biomolecule types in a fluid composition comprises contacting the first fluid composition with a first plurality of surface types to adsorb the first plurality of biomolecule types on the first plurality of surface types. In some embodiments, selectively enriching a second plurality of biomolecule types in a fluid composition comprises contacting the second fluid composition with a second plurality of surface types to adsorb the second plurality of biomolecule types on the second plurality of surface types.

[0366] In some embodiments, the first multiple surface types and the second multiple surface types include the same charge symbol. In some embodiments, the first multiple surface types and the second multiple surface types include different charge symbols. In some embodiments, the first multiple surface types and the second multiple surface types include the same zeta potential symbol. In some embodiments, the first multiple surface types and the second multiple surface types include different zeta potential symbols. In some embodiments, the first multiple surface types, the second multiple surface types, or both include acidic functional groups. In some embodiments, the acidic functional groups include Bronsted-Lowry acid or Lewis acid functional groups. In some embodiments, the first multiple surface types, the second multiple surface types, or both include carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boric acid groups, boric acid ester groups, dihydrocarbyl boronic acid groups, dihydrocarbyl boronic 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 comprise basic functional groups. In some embodiments, the basic functional groups comprise Bronsted-Lowry acid or Lewis acid functional groups.

[0367] In some embodiments, the first fluid composition is incubated with the first surface at least 30°C for at least 20 minutes to adsorb the first plurality of biomolecule types on the first surface. In some embodiments, the second fluid composition is incubated with the second surface at least 30°C for at least 20 minutes to adsorb the second plurality of biomolecule types on the second surface. In some embodiments, the first fluid composition is incubated with the first surface at least 30°C for at least 20 minutes to adsorb the first plurality of biomolecule types on the first surface, and the second fluid composition is incubated with the second surface at least 30°C for at least 20 minutes to adsorb the second plurality of biomolecule types on the second surface.

[0368] In some embodiments, the first multiple biomolecule type is depleted of at least the first biomolecule and enriched with the second biomolecule, wherein the first biomolecule is more abundant than the second molecule in the first fluid composition. In some embodiments, the second multiple biomolecule type is depleted of at least the first biomolecule and enriched with the second biomolecule, wherein the first biomolecule is more abundant than the second molecule in the second fluid composition. For example, the first fluid composition and the second fluid composition can each comprise the same particle composition, but comprise different pH, solvents, temperatures, pressures, etc., as described herein. Even if the particle composition is identical between the first fluid composition and the second fluid composition, they can enrich and deplete different biomolecules. In some cases, the first particle composition and the second particle composition can be different.

[0369] In some embodiments, the first surface is disposed in a first freeze-dried composition, the first freeze-dried composition comprising (i) at least one nanoparticle, and (ii) one or more modifiers. In some embodiments, the second surface is disposed in a second freeze-dried composition, the second freeze-dried 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, when the freeze-dried composition is reconstituted in a solution, the average zeta potential of the surface is between 85% and 115% of the zeta potential of the same particles dissolved in the same solution without freeze-drying, as determined by zeta potential measurement.

[0370] In some embodiments, when the lyophilized composition is reconstituted in solution, the average zeta potential of the surface is between 90% and 110% of the zeta potential of the same particles dissolved in the same solution without lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the average zeta potential of the surface is between 95% and 105% of the zeta potential of the same particles dissolved in the same solution without lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the average zeta potential standard deviation of the surface is between 85% and 115% of the standard deviation of the zeta potential of the same particles dissolved in the same solution without lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the average zeta potential standard deviation of the surface particles is between 90% and 110% of the standard deviation of the zeta potential of the same particles dissolved in the same solution without lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the standard deviation of the zeta potential of the surface particles is between 95% and 105% of the standard deviation of the zeta potential of the same particles dissolved in the same solution without lyophilization, as determined by zeta potential measurement. In some embodiments, when the lyophilized composition is reconstituted in solution, the average diameter of the surface particles is between 85% and 115% of the average diameter of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the average diameter of the surface particles is between 90% and 110% of the average diameter of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the average diameter of the surface particles is between 95% and 105% of the average diameter of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the average diameter of the surface particles is between 98% and 102% of the average diameter of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstituted in solution, the standard deviation of the diameter of the surface particles is between 85% and 115% of the standard deviation of the diameter of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS).In some embodiments, when the lyophilized composition is reconstructed in solution, the diameter standard deviation of the surface particles is between 90% and 110% of the diameter standard deviation of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstructed in solution, the diameter standard deviation of the surface particles is between 95% and 105% of the diameter standard deviation of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). In some embodiments, when the lyophilized composition is reconstructed in solution, the diameter standard deviation of the surface particles is between 98% and 102% of the diameter standard deviation of the same particles dissolved in the same solution without lyophilization, as determined by dynamic light scattering (DLS). The lyophilized composition can have stable physicochemical properties over different time periods. In some cases, the time period may include 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 may have stable physicochemical properties at various temperatures. In some cases, the temperature may be about room temperature. In some cases, the temperature may be about 37°C. In some cases, the temperature may be about 60°C. In some cases, the temperature may be about -26°C to about 0°C. In some cases, the temperature may be about -10°C to about -5°C. In some cases, the temperature may be about 0°C to 20°C. In some cases, the temperature may be about 0°C to about 10°C. In some cases, the temperature may be about 25°C to about 60°C. In some cases, the temperature may be about 35°C to about 40°C. In some cases, the dry composition or lyophilized composition is stable at about 37°C for at least 40 days. In some cases, the dried composition or lyophilized composition is stable at ambient temperature for at least 11 months.

[0371] In some embodiments, the surface is included on particles dispersed in the composition during incubation. In some embodiments, the particles are nanoparticles. In some embodiments, the nanoparticles have an average diameter of less than 500 nanometers. In some embodiments, the particles are porous particles. In some embodiments, the particles are microparticles. In some embodiments, the particles comprise paramagnetic materials. In some embodiments, the paramagnetic materials are superparamagnetic materials. In some embodiments, the paramagnetic materials include iron oxide, aluminum, platinum, or any combination thereof. In some embodiments, the particles comprise a poly(dimethylaminopropylmethacrylamide) (PDMAPMA) outer layer. In some embodiments, the particles comprise a silica outer layer. In some embodiments, the particles comprise a carboxylate functionalized outer layer. In some embodiments, the particles comprise an amine functionalized outer layer. In some embodiments, the particles comprise a polymer outer layer. In some embodiments, the particles comprise a polyacrylamide outer layer. In some embodiments, the particles comprise an acrylate polymer.

[0372] In some embodiments, the method further comprises digesting the protein adsorbed to the first surface prior to assaying the biomolecule. In some embodiments, the method further comprises treating the protein within the biomolecule corona with a reducing agent prior to assaying the biomolecule. In some embodiments, the method further comprises treating the protein within the biomolecule corona with an alkylating agent prior to assaying the biomolecule.

[0373] In some embodiments, the method further comprises washing the first multiple biomolecule types with the first washing composition, and washing the second multiple biomolecule types with the second washing composition. In some embodiments, the first fluid composition and the first washing composition comprise at least one common strength physical property. In some embodiments, the first fluid composition and the first washing composition comprise at least one different strength physical property. In some embodiments, the first fluid composition and the first washing composition comprise at least one common solvent. In some embodiments, the first fluid composition and the first washing composition comprise at least one different solvent. In some embodiments, the first fluid composition and the first washing composition comprise approximately the same ionic strength. In some embodiments, the first fluid composition and the first washing composition comprise different ionic strengths. In some embodiments, the first fluid composition and the first washing composition comprise at least one common salt. In some embodiments, the first fluid composition and the first washing composition comprise at least one different salt. In some embodiments, the first fluid composition and the first washing composition comprise approximately the same pH. In some embodiments, the first fluid composition and the first washing composition comprise different pH.

[0374] In some embodiments, the second fluid composition and the second washing composition comprise at least one common strength physical property. In some embodiments, the second fluid composition and the second washing composition comprise at least one different strength physical property. In some embodiments, the second fluid composition and the second washing composition comprise at least one common solvent. In some embodiments, the second fluid composition and the second washing composition comprise at least one different solvent. In some embodiments, the second fluid composition and the second washing composition comprise approximately the same ionic strength. In some embodiments, the second fluid composition and the second washing composition comprise different ionic strengths. In some embodiments, the second fluid composition and the second washing composition comprise at least one common salt. In some embodiments, the second fluid composition and the second washing composition comprise at least one different salt.

[0375] In some embodiments, the first washing composition and the second washing composition comprise at least one common strength physical property. In some embodiments, the first washing composition and the second washing composition comprise at least one different strength physical property. In some embodiments, the first washing composition and the second washing composition comprise approximately the same ionic strength. In some embodiments, the first washing composition and the second washing composition comprise different ionic strengths. In some embodiments, the first washing composition and the second washing composition comprise at least one common salt. In some embodiments, the first washing composition and the second washing composition comprise at least one different salt. In some embodiments, the first washing composition and the second washing composition comprise approximately the same pH. In some embodiments, the first washing composition and the second washing composition comprise different pH. In some embodiments, the first washing composition and the second washing composition are the same. In some embodiments, the first washing composition and the second washing composition are different. Various strength properties, solvents, salts and pH have been described elsewhere herein.

[0376] In some embodiments, the first washing composition releases the first plurality of biomolecule types adsorbed on the first surface from the first surface. In some embodiments, the method further includes purifying the first plurality of biomolecule types to produce a first purified composition. In some embodiments, purification includes drying the plurality of biomolecule types to remove the first washing composition. In some embodiments, the method further includes reconstructing the first purified composition with a first reconstructing composition to produce a first reconstructed composition. In some embodiments, the second washing composition releases the second plurality of biomolecule types deposited on the second surface from the second surface. In some embodiments, the method further includes purifying the second plurality of biomolecule types to produce a second purified composition. In some embodiments, purification includes drying the second plurality of biomolecule types to remove the second washing composition. In some embodiments, the method further includes reconstructing the second purified composition with a second reconstructing composition to produce a second reconstructed composition.

[0377] The first or second multiple biomolecule types may include peptides. The peptides may include proteolytic cleavage derivatives of proteins adsorbed on the first or second surface. The peptides may be proteolytically cleaved by proteases (e.g., trypsin or lytic enzymes). Drying may include applying negative pressure (e.g., negative gauge pressure relative to atmospheric pressure), while optionally heating or cooling the dried sample. Drying may be performed to the extent that solvent evaporation is no longer observable, such as by changes in mass. Drying may be performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes. Drying may be performed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 24, 36, or 48 hours. Drying may be performed for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes. Drying can be carried out for up to 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°C, -150°C, -100°C, -50°C, -25°C, 0°C, 25°C, 50°C, 75°C or 100°C. Drying can be carried out at a temperature of up to -200°C, -150°C, -100°C, -50°C, -25°C, 0°C, 25°C, 50°C, 75°C 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 at most 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kPa. Reconstruction can be carried out by adding a buffer. Various buffer compositions have been described elsewhere herein. The first reconstructed composition and the second reconstructed composition can be the same or different. When reconstructed, the first biological sample and the second biological sample can contain peptides at about a 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 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 (mass of biomolecule / buffer volume).

[0378] Various analytical methods for identifying peptide or protein species can be used for determination (e.g., downstream determination). In some embodiments, determination includes SDS-PAGE, gel-based separation techniques, immunoassays, ELISA, high performance liquid chromatography, mass spectrometry, Edman degradation or immunoaffinity techniques. In some embodiments, determination includes mass spectrometry. In some embodiments, determination includes LC-MS / MS. In some embodiments, determination includes quantifying biomolecules. In some embodiments, determination includes identifying one or more proteins in a biological sample. In some embodiments, downstream determination includes mass spectrometry. In some embodiments, downstream determination includes protein sequencing. In some embodiments, downstream determination includes LC-MS / MS. In some embodiments, downstream determination includes immunoassay. In some embodiments, the downstream assay comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with an antibody pair capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the antibody pair comprises a complementary single-stranded nucleic acid sequence attached thereto such that when the antibody pair binds to at least one of the biomolecule types, the complementary nucleic acids hybridize to form a double-stranded nucleic acid, wherein the double-stranded nucleic acid is configured to form a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs, and / or nucleoside analogs to perform an amplification reaction to produce a detectable signal. In some embodiments, the downstream assay comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with one or more aptamers capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the one or more aptamers are coupled to a surface via a cleavable linker. In some embodiments, the cleavable linker is photocleavable. In some embodiments, the surface is a particle surface. In some embodiments, the method further comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a macromolecular competitor configured to reduce dissociation of a complex comprising one or more aptamers and the first plurality of biomolecule types, the second plurality of biomolecule types, or both in the fluid composition. In some embodiments, the macromolecular competitor is further configured to bind to a biomolecule type different from the first plurality of biomolecule types, the second plurality of biomolecule types, or both. In some embodiments, the macromolecular competitor is a polyanionic macromolecule. In some embodiments, the macromolecular competitor is a polycationic macromolecule. In some embodiments, the downstream assay comprises nucleic acid sequencing.

[0379] In some embodiments, the assay is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 100 samples per hour. In some embodiments, the assay is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 100000, or 10000000 μg of biomolecules per hour. In some embodiments, the assay is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000, or 1000000 biomolecules per hour. In some embodiments, the assay is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000, or 1000000 protein groups per hour. The flux of the assay can be measured with a standard such as a HeLa cell extract. For example, in some embodiments, when the suspension comprises a HeLa cell extract, the assay identifies at least about 100, 1000, 10000, or 100000 biomolecules. In some embodiments, when the assay 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 comprises at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0380] In some embodiments, a first likelihood of determining that a low-abundance biomolecule is identified in a plurality of biomolecules is greater than a second likelihood of the determination identifying the low-abundance biomolecule in the biological sample in the absence of the first particle and the second particle, wherein the low-abundance biomolecule comprises less than about 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 In some embodiments, the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10In some embodiments, the assay identifies one or more biomolecules in a plurality of biomolecules with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the assay identifies one or more biomolecules in a plurality of biomolecules with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0381] In some embodiments, the method includes forming a first suspension comprising a first plurality of particles with 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 chemical different from the second functionalized surface chemical of the second particle. In some embodiments, the first particle has the same first functionalized surface chemical as the second functionalized surface chemical of the second particle. In some embodiments, the first particle and the second particle both have a negative charge. In some embodiments, the first particle and the second particle both have a positive charge. In some embodiments, the first particle and the second particle both have a neutral charge of about 0.001. In some embodiments, the first particle and the second particle both have a zwitterionic charge of about 0.001. In some embodiments, the method includes forming a second suspension comprising a second plurality of particles with 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 chemical different from the fourth functionalized surface chemical of the fourth particle. In some embodiments, the third particle has a third functionalized surface chemical the same as the fourth functionalized surface chemical of the fourth particle. In some embodiments, the third particle and the fourth particle have a positive charge. In some embodiments, the third particle and the fourth particle have a negative charge. In some embodiments, the third particle and the fourth particle have an approximately neutral charge. In some embodiments, the third particle and the fourth particle have a zwitterionic charge. In some embodiments, the method includes enriching biomolecules that have been adsorbed to the first plurality of particles. In some embodiments, the method includes enriching biomolecules that have been adsorbed to the second plurality of particles. In some embodiments, the method includes performing a downstream assay on the enriched biomolecules.

[0382] 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 include a Tris buffer. In some embodiments, the first suspension has a pH between 9 and 10. In some embodiments, the first suspension includes 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 composition parameters (e.g., buffer, salt, pH, etc.) have been described elsewhere herein. The suspension described herein may include various compositions described elsewhere herein.

[0383] In some embodiments, the first particle is a magnetic particle. In some embodiments, the first particle comprises an outer layer having a silanol surface functionalization. In some embodiments, the second particle is a magnetic particle. In some embodiments, the second particle comprises an outer layer having a carboxylate surface functionalization. In some embodiments, the third particle is a magnetic particle. In some embodiments, the third particle comprises an outer layer having an amine surface functionalization. In some embodiments, the fourth particle is a magnetic particle. In some embodiments, the fourth particle comprises an outer layer having an amine surface functionalization. In some embodiments, the outer layer of the third particle comprises 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, each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle. Various particle compositions have been described elsewhere herein. The first particle, the second particle, the third particle, and the fourth particle can comprise various particles described herein in various proportions.

[0384] Systems for the selective enrichment and determination of biomolecules

[0385] Can be used in PROTEOGRAPH TMVarious parallel or sequential schemes are used on the 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 processed in series. 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 first fluid composition and the second fluid composition are selectively enriched in parallel. In some embodiments, the first fluid composition and the second fluid composition are selectively enriched in series. In some embodiments, selective enrichment is carried out at a rate of enriching at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biomolecules per hour. In some embodiments, the first fluid composition and the second fluid composition are contacted in parallel. In some embodiments, the first fluid composition and the second fluid composition are contacted in series. In some embodiments, contact is carried out with the speed of depositing at least 100, 1000, 10000, 100000 or 1000000 biomolecules per hour. In some embodiments, contact is carried out with the speed of depositing at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μ g of biomolecules per hour. In some embodiments, the first fluid composition and the second fluid composition are washed in parallel. In some embodiments, the first fluid composition and the second fluid composition are washed serially. In some embodiments, washing is carried out with the speed of washing at least 100, 1000, 10000, 100000 or 1000000 biomolecules per hour. In some embodiments, washing is carried out with the speed of washing at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μ g of biomolecules per hour. In some embodiments, the first fluid composition and the second fluid composition are purified in parallel. In some embodiments, the first fluid composition and the second fluid composition are purified in series. In some embodiments, purification is carried out with a speed of purifying at least 100, 1000, 10000, 100000 or 1000000 per hour. In some embodiments, purification is carried out with a speed of purifying at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μ g of biomolecule per hour. In some embodiments, the first fluid composition and the second fluid composition are reconstructed in parallel.In some embodiments, serially the first fluid composition and the second fluid composition are reconstructed.In some embodiments, reconstruction is carried out with the speed of reconstructing at least 100,1000,10000,100000 or 1000000 per hour.In some embodiments, reconstruction is carried out with the speed of reconstructing at least 1,10,100,1000,10000,100000,1000000 or 10000000 μ g of biomolecule per hour.In some embodiments, parallelly the first fluid composition and the second fluid composition are carried out downstream determination.In some embodiments, serially the first fluid composition and the second fluid composition are carried out downstream determination.In some embodiments, downstream determination is carried out with the speed of measuring at least 1,2,3,4,5,6,7,8,9,10,100 samples per hour. In some embodiments, the downstream assay is carried out at a rate of measuring at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biomolecules per hour. In some embodiments, the downstream assay is carried out at a rate of identifying at least 100, 1000, 10000, 100000 or 1000000 biomolecules per hour. In some embodiments, the downstream assay is carried out at a rate of identifying at least 100, 1000, 10000, 100000 or 1000000 protein groups per hour. In some embodiments, the first fluid composition and the second fluid composition are performed in parallel. In some embodiments, the first fluid composition and the second fluid composition are performed in series. 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 of at least 1, 10, 100, 1000, 10000, 100000, 100000 or 10000000 μg of biomolecules per hour. In some embodiments, the method is performed at a rate of at least 100, 1000, 10000, 100000 or 1000000 biomolecules per hour. In some embodiments, the method is performed at a rate of at least 100, 1000, 10000, 100000 or 1000000 protein groups per hour. Suitable standards can be used to measure the rate of biomolecule identification. In some embodiments, when the first fluid composition and the second fluid composition are HeLa cell extracts, downstream determinations identify at least about 100, 1000, 10000, 100000 or 1000000 biomolecules.

[0386] In some embodiments, the downstream assay identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the downstream assay identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the assay identifies enriched biomolecules with a coefficient of variation of at most 30%. In some embodiments, the assay identifies enriched biomolecules with a coefficient of variation of at most 25%. In some embodiments, the assay identifies enriched biomolecules with a coefficient of variation of at least 0%.

[0387] In some embodiments, when the first fluid composition and the second fluid composition are subjected to downstream assays prior to the selective enrichment in (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together comprise at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges. In some embodiments, when the first fluid composition and the second fluid composition are subjected to downstream assays prior to the selective enrichment in (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together comprise at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 dynamic ranges.

[0388] In some embodiments, a first likelihood that a downstream assay identifies a low-abundance biomolecule in the first plurality of biomolecule types or the second plurality of biomolecule types is greater than a second likelihood that the downstream assay identifies a low-abundance biomolecule in the first fluid composition or the second fluid composition, wherein the low-abundance biomolecule comprises less than about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 790, 80 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 In some embodiments, the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 108 , 10 9 or 10 10 times.

[0389] In some embodiments, at least 250 different biomolecules are identified in the biological sample. In some embodiments, at least 500 different biomolecules are identified in the biological sample. The biomolecules can be proteins. 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, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 100000 peptides. In some embodiments, the one or more biomolecules comprise at most 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, 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. In some embodiments, the one or more biomolecules comprise at most about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins.

[0390] Compositions and kits for selective enrichment and determination of biomolecules

[0391] In some aspects, the present disclosure provides a suspension for measuring biomolecules from a biological sample. The suspension may include a first particle, the first particle including (i) a first paramagnetic portion and (ii) a first surface chemical. The suspension may include a second particle, the second particle including (i) a second paramagnetic portion and (ii) a second surface chemical. The second surface chemical and the first surface chemical may be different. A plurality of biomolecules may be adsorbed on the first particle and the second particle. The suspension may be a complex of physicochemically different particles provided in a continuous phase of a solution (e.g., in a hole).

[0392] The physicochemical properties of the first and second particles may be such that they are colloidally stable or semi-stable in the suspension. The physicochemical properties of the first and second particles may be such that they are dispersed in the suspension. The physicochemical properties of the first and second particles may be such that they can be dispersed in the suspension upon agitation. When the particles are magnetic particles, the agitation may be, for example, magnetic agitation of the particles. Without being bound by a particular theory, the stability of the colloidal suspension may be explained in part by the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The theory states that the stability of the colloidal suspension depends on at least the Bjerrum length, the Debye–Hücke screening length, the charge of the particles, and the size of the particles. It is worth noting that the DLVO theory does not take into account all possible forces that affect the stability of the colloid, for example, depletion forces are not considered in the theory. The Bjerrum length is the distance at which electrostatic interactions and thermal energies 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 it depends on at least the dielectric constant and the ionic strength of the solution. Therefore, 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 particle and the second particle can contain the same charge sign. The charge can be, for example, the zeta potential of the particle. The same charge sign can be negative, positive or approximately 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 the suspension to destabilize is at least about 1, 5, 10, 15, 30 or 60 minutes. The time constant for the suspension to destabilize is up to about 1, 5, 10, 15, 30 or 60 minutes. The average aggregation number of the first particle and the second particle in the suspension can be up to about 1, 10 or 100. The average aggregation number of the first particle and the second particle in the suspension can be up to about 1000, 100 or 10. The suspension can contain at least about 3, 4, 5, 6, 7, 8, 9 or 10 different particles. In some embodiments, the suspension is incubated at a temperature of at least 30°C.

[0393] The first surface chemical substance, the second surface chemical substance or both may comprise an acidic functional group. The acidic functional group comprises a Bronsted-Lowry acid or a Lewis acid functional group. The first surface chemical substance, the second surface chemical substance or both may comprise a carboxylate group, an acrylate group, a methacrylate 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 phosphodiester group, a boric acid group, a boric acid ester group, a dihydrocarbyl boronic acid group, a dihydrocarbyl boric acid ester group, a silica group, a silanol group, a polymer or any combination thereof.

[0394] The suspension may contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles to about 1 part of the second particles. The suspension may contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of the first particles to about 1 part of the second particles. The suspension may contain about 15 parts of the first particles to about 6 parts of the second particles. Parts may be by weight, volume, or surface area. The first size of the first particle can be at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the second size of the second particle. The first size of the first particle can be at most about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times or 1000 times the second size of the second particle. The first size can be the first diameter, and the second size can be the second diameter. The first size can be the first average size, and the second size can be the second average size. The first average size and the second average size can be average sizes or median sizes. The ratio of the average diameter of the first particles to the average diameter of the second particles can be about 3:2 to about 2:3.

[0395] The first particle, the second particle, or both can be nanoparticles. The first particle, the second particle, or both can be microparticles. The first particle, the second particle, or both can be porous. A plurality of biomolecules can include up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. A plurality of biomolecules can include at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules. A plurality of biomolecules can include up to about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules / mL. A plurality of biomolecules can include at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules / mL.

[0396] In some cases, the compositions, methods and systems disclosed herein can provide spatial or temporal differences in biomolecular compositions of small volume samples (e.g., individual cells). Spatial differences in biomolecular compositions can be obtained by sampling biomolecules from different parts of cells (e.g., different compartments in cells) or tissues (e.g., healthy cells and cancer cells in tumors, or cells from the epidermis, dermis and subcutaneous tissue of the skin). Temporal differences in biomolecular compositions can be obtained by sampling biomolecules at different times (e.g., cells before and after treatment with potential therapeutic agents). In some cases, systems and methods disclosed herein can provide differential biomolecular compositions across populations of subjects (e.g., tumor cells from those subjects treated with potential chemotherapeutic agents and tumor cells from those subjects not treated). Various biomolecules (e.g., proteins or nucleic acids) can be targeted to provide differential transcriptomics or proteomics information between samples. Multiple biomolecules can include biomolecules from up to about 1000, 100, 10 or 1 cells. Multiple biomolecules can include biomolecules from at least about 1000, 100, 10 or 1 cells.

[0397] The first fluid composition and the second fluid composition can comprise the same or different intensive properties. Intensive properties can refer to properties that do not depend on the amount of the substance being measured. Intensive properties can be temperature, pressure, density, component concentration, pH, ionic strength, heat capacity, viscosity, surface tension, just to give a few examples. In some embodiments, the first fluid composition comprises a first set of intensive physical properties that mediate selective enrichment of a first plurality of biomolecule types. In some embodiments, the second fluid composition comprises a second set of intensive physical properties that mediate selective enrichment of a second plurality of biomolecule types. In some embodiments, the first set of intensive physical properties and the second set of intensive physical properties are different. In some embodiments, the first set of intensive physical properties and the second set of intensive physical properties are the same.

[0398] In some embodiments, the first fluid composition comprises a first ratio between the first sample volume and the first surface area of ​​the first surface. In some embodiments, the second fluid composition comprises a second ratio between the second sample volume and the second surface area of ​​the second surface. In some embodiments, the first ratio and the second ratio are different. In some embodiments, the first ratio and the second ratio are the same. The ratio can be, for example, at least 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 cm 2 The ratio may be, for example, at most 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5 cm 2 Surface area / μL sample volume.

[0399] In some embodiments, the first fluid composition and the second fluid composition comprise different temperatures. In some embodiments, the first fluid composition and the second fluid composition comprise 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 ℃. The temperature can be at most 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 ℃.

[0400] 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 identical. In some embodiments, the first fluid composition comprises a first ionic concentration, and the second fluid composition comprises a second ionic concentration. In some embodiments, the first ionic concentration and the second ionic concentration are different. In some embodiments, the first ionic concentration and the second ionic concentration are identical. Ionic concentration can 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 7M, and ionic concentration can 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 7M.

[0401] In some embodiments, the first fluid composition and the second fluid composition comprise different solvents. In some embodiments, the first fluid composition and the second fluid composition comprise the same solvent. The solvent can include, for example, acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, tert-butanol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether (diethylene glycol dimethyl ether), 1,2-dimethoxyethane (glyme dimethyl ether, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide, hexamethylphosphortriamide, hexane, methanol, methyl tert-butyl ether, dichloromethane, N-methyl 2-pyrrolidone, 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 include a buffer containing tris (hydroxymethyl) aminomethane. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing citrate. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing glycine. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing phosphate. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing carbonate. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing N- cyclohexyl -3- aminopropanesulfonic acid. In some embodiments, the first fluid composition, the second fluid composition or both include a buffer containing 3- ((3- gallamide propyl) dimethylammonium) -1- propanesulfonate. In some embodiments, the first fluid composition includes an aqueous buffer. In some embodiments, the second fluid composition includes an aqueous buffer. In some embodiments, the first fluid composition includes an aqueous buffer, and the second fluid composition does not include a buffer.

[0402] In some embodiments, the first fluid composition and the second fluid composition comprise different salts. In some embodiments, the first fluid composition and the second fluid composition comprise the same salt. Salt may include, for example, ammonium sulfate, sodium sulfate, sodium chloride, potassium chloride, ammonium acetate, etc.

[0403] In some embodiments, the first fluid composition comprises a first pH, and the second fluid composition comprises 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 are included in a pH between 2 and 4. In some embodiments, the first fluid composition, the second fluid composition, or both are included in a pH between 5 and 7. In some embodiments, the first fluid composition, the second fluid composition, or both are included in a pH between 9 and 10. In some embodiments, the first fluid composition, the second fluid composition, or both include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 pH. In some embodiments, the first fluid composition, the second fluid composition, or both include at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 pH. In some embodiments, the method may further include diluting the sample to prepare the first composition, the second composition, or both. In some embodiments, diluting includes adding a buffer. In some embodiments, the buffer comprises 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 comprises a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the sample and the buffer comprise different pHs. In some embodiments, the sample and the buffer comprise about 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 comprise a buffer. In some embodiments, the second composition comprises a buffer different from a buffer configured to maintain the pH of the composition between 8 and 11. In some embodiments, the second composition comprises the same buffer as the composition. In some embodiments, the composition and the second composition both comprise a Tris buffer at a pH between 9 and 10. In some embodiments, the incubation of the composition and the incubation of the second composition occur 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.

[0404] The composition and / or property of the first fluid composition, the second fluid composition or both can be used as a feature for identifying a biological state associated with a sample. For example, features can be used in a machine learning algorithm. Features can include the identity of a biomolecule, particles for determining biomolecules, temperature, pH, etc. Therefore, various compositions and / or properties can provide additional features to stratify biological samples for distinguishing biological states. In some embodiments, the method further includes identifying the 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 the 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 the 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 the 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 the biological state associated with the first fluid composition based at least in part on one or more physical properties of the first reconstructed composition. In some embodiments, the method further comprises identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstituted composition.

[0405] In some aspects, the present disclosure provides a kit for selectively enriching and measuring multiple biomolecules in a fluid composition. In some embodiments, the kit comprises a first reagent containing a first pH. In some embodiments, the kit comprises a second reagent containing a second pH. In some embodiments, the kit comprises a first surface, which is configured to selectively enrich a first plurality of biomolecule types in a first fluid composition comprising a first surface and a first reagent. In some embodiments, the kit comprises a second surface, which is configured to selectively enrich a second plurality of biomolecule types in a second fluid composition comprising a second surface and a second reagent. In some embodiments, the first fluid composition comprises a first reagent. In some embodiments, the second fluid composition comprises a second reagent. In some embodiments, the first pH and the second pH are different.

[0406] In some aspects, the present disclosure provides a kit comprising a first composition comprising a first particle and a second particle. The first particle may have a first functionalized surface chemical substance different from the second functionalized surface chemical substance of the second particle. The first particle and the second particle may both have a negative charge. The first composition may be a solid. The kit may further comprise a second composition comprising a third particle and a fourth particle. The third particle may have a third functionalized surface chemical substance different from the fourth functionalized surface chemical substance of the fourth particle. The third particle and the fourth particle may both have a positive charge. The third composition may be a solid.

[0407] 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. Both the first composition and the second composition may be lyophilized. In some embodiments, the first particle is a magnetic particle and includes an outer layer having a silanol surface functionalization. In some embodiments, the second particle is a magnetic particle and includes an outer layer having a carboxylate surface functionalization. In some embodiments, the third particle is a magnetic particle and includes an outer layer having an amine surface functionalization. In some embodiments, the fourth particle is a magnetic particle and includes an outer layer having an amine surface functionalization. In some embodiments, the outer layer of the third particle 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 are superparamagnetic iron oxide nanoparticles. 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.

[0408] In some aspects, the present disclosure provides a composition for selectively enriching and measuring multiple 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 portion. In some embodiments, the first particle comprises a first surface chemical. In some embodiments, the suspension comprises a second particle. In some embodiments, the second particle comprises a second paramagnetic portion. In some embodiments, the second particle comprises a second surface chemical. In some embodiments, the second surface chemical is different from the first surface chemical. In some embodiments, the suspension comprises a plurality of biomolecules adsorbed on the first particle. In some embodiments, the suspension comprises a plurality of biomolecules adsorbed on the second particle.

[0409] In some aspects, the present disclosure provides a suspension. In some embodiments, the suspension comprises a first particle. In some embodiments, the first particle comprises an outer layer having a negatively charged surface functionalization. In some embodiments, the first particle comprises a first outer layer having a positively charged surface functionalization. In some embodiments, the suspension comprises a second particle, the second particle comprising a second outer layer having a negatively charged surface functionalization. In some embodiments, the second particle comprises a second outer layer having a positively charged surface functionalization. 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, biomolecules are adsorbed to the outer layer of the first particle. In some embodiments, the second outer layer is different from the first outer layer. In some embodiments, biomolecules are adsorbed to the second outer layer of the second particle. In some embodiments, biomolecules are adsorbed to the first outer layer of the first particle and the second outer layer of the particle. In some embodiments, the first particle comprises a first functionalized surface chemical. In some embodiments, the second particle comprises a second functionalized surface chemical. In some embodiments, the first functionalized surface chemical is different from the second functionalized surface chemical of the second particle. In some embodiments, the first outer layer comprises a silanol. In some embodiments, the second outer layer comprises a carboxylate. In some embodiments, the first outer layer and the second outer layer comprise amine groups. In some embodiments, the first particle is a magnetic particle and comprises an outer layer having an amine surface functionalization. In some embodiments, the second particle is a magnetic particle and comprises an outer layer having an amine surface functionalization. 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 particle and the optional second particle are magnetic.

[0410] In some embodiments, the suspension comprises a buffer. In some embodiments, the buffer is configured to maintain the pH of the suspension between 9 and 10. In some embodiments, the buffer is a Tris buffer. In some embodiments, the buffer is configured to maintain a pH of about 8 to about 11. In some embodiments, the buffer is configured to maintain a pH between 9 and 10. In some embodiments, the buffer is a Tris buffer with a pH between 9 and 10. In some embodiments, the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0411] 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.

[0412] In some aspects, the present disclosure provides a system for analyzing multiple biological samples. In some embodiments, the system includes multiple partitions, the multiple partitions include a first partition and a second partition. In some embodiments, the system includes multiple reagent reservoirs, the multiple reagent reservoirs include 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 multiple substrates, the multiple substrates include a first substrate and a second substrate. In some embodiments, the first substrate includes a first surface chemical substance. In some embodiments, the second substrate includes a second surface chemical substance. In some embodiments, the system includes one or more transfer devices, the one or more transfer devices are operably connected to multiple partitions, multiple reagent reservoirs, multiple substrates or any combination thereof. In some embodiments, the system includes a computer, the computer includes at least one processor and can be executed by at least one processor to perform instructions for analyzing steps of multiple biological samples. In some embodiments, the instructions may include using one or more transfer devices to produce a first fluid composition in a first partition, the first fluid composition includes a first substrate, a first reagent and a first plurality of biomolecules. In some embodiments, the first plurality of biomolecules are adsorbed on the first substrate. In some embodiments, the instructions may include using one or more transfer devices to produce a second fluid composition in the second partition, the second fluid composition comprising a second substrate, a second reagent, and a second plurality of biomolecules. In some embodiments, the second plurality of biomolecules are adsorbed on the second substrate. In some embodiments, the instructions may include using one or more transfer devices to prepare the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry.

[0413] In some aspects, the present disclosure provides a system for analyzing multiple biological samples. In some embodiments, the system includes a partition. In some embodiments, the system includes a reagent storage device comprising a reagent; in some embodiments, the system includes multiple substrates, the multiple substrates including a first substrate comprising a first surface chemical substance and a second substrate comprising a second surface chemical substance. In some embodiments, the first surface chemical substance and the second surface chemical substance are different. In some embodiments, the system includes one or more transfer devices, which are operably connected to a partition, a reagent storage device, a plurality of substrates, or any combination thereof. In some embodiments, the system includes a computer, the computer includes at least one processor and instructions that can be executed by at least one processor to perform steps for analyzing multiple biological samples. In some embodiments, the instructions may include using one or more transfer devices to produce a fluid composition in a partition, the fluid composition comprising multiple substrates, reagents and multiple biomolecules, wherein multiple biomolecules are adsorbed on a substrate. In some embodiments, the instructions may include using one or more transfer devices to prepare multiple biomolecules for mass spectrometry.

[0414] Biological samples

[0415] In some embodiments, the amount of the sample can be different. In some embodiments, the sample comprises at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules. In some embodiments, the sample comprises at least about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules. In some embodiments, the sample comprises at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules / mL sample. In some embodiments, the sample comprises at least about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules / mL sample. In some embodiments, the sample comprises biomolecules from at most about 1000, 100, 10 or 1 cells. In some embodiments, the sample comprises biomolecules from at least about 1000, 100, 10 or 1 cells. In some embodiments, the sample comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters. In some embodiments, the sample comprises at least about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 microliters.

[0416] The composition and / or properties of the first fluid composition and the second fluid composition can be designed to increase the use of PROTEOGRAPH for different types of samples. TMThe dynamic range and / or number of peptides, proteins or proteomes detected. In some embodiments, the sample comprises a complex biological sample. The complex biological sample may include plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirates, ductal lavage fluid, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcus fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid from swabs, bronchial aspirates, fluidized solids, fine needle aspiration samples, tissue homogenates, lymph fluid, cell culture samples or any combination thereof. In some embodiments, the biological sample comprises plasma.

[0417] In some embodiments, the first plurality of biomolecule types, the second plurality of biomolecule types, or both include one or more polymeric amino acids. A polymeric amino acid can refer to any biomolecule comprising two or more amino acids. The amino acids can be the same or different. The amino acids can be covalently bonded. The covalent bonds can be peptide bonds. The polymeric amino acids can comprise peptides, proteins, or both.

[0418] In some embodiments, the biological sample includes plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, duct lavage, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage, sweat, sulcus fluid, semen, prostatic fluid, sputum, feces, bronchial lavage, fluid from swab, bronchial aspirate, fluidized 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 a cell culture medium. In some embodiments, the composition comprises no more than 60% of the biological sample by volume. In some embodiments, the composition comprises no more than 25% of the biological sample by volume. In some embodiments, the composition comprises no more than 15% of the biological sample by volume. In some embodiments, the composition comprises at least 1% of the biological sample by volume. In some embodiments, the composition comprises at least 4% of the biological sample by volume. In some embodiments, the composition comprises at least 10% by volume of the biological sample. In some embodiments, the composition comprises at least 20% by volume of the biological sample.

[0419] The systems and methods of the present disclosure are used to measure biological samples. In some cases, the biological sample may contain cells or be acellular. 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 fluidized solid, such as a tissue homogenate or a fluid extracted from the 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 an object. In some cases, the object may be a human or non-human. In some cases, the object may be a plant, a fungus, or an archaea. In some cases, the biological sample may contain a variety of proteins or proteomic data, which may be analyzed after protein adsorption or binding to the surface of various sensor element (e.g., particle) types in the set and subsequent digestion of the protein corona.

[0420] In some cases, the biological sample may include plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, ductal lavage, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage, sweat, sulcus fluid, semen, prostatic fluid, sputum, feces, bronchial lavage, fluid from swab, bronchial aspirate, fluidized solid, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof. In some cases, the biological sample may include multiple 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 biological material from a single source.

[0421] In some cases, biological samples can be diluted or pre-treated. In some cases, biological samples can be exhausted before or after contacting with a surface disclosed herein (for example, biological samples include serum). In some cases, biological samples can be physically (for example, homogenized or ultrasonically treated) or chemically treated before or after contacting with a surface disclosed herein. In some cases, biological samples can be diluted before or after contacting with a surface disclosed herein. In some cases, dilution medium can include a buffer or salt, or purified water (for example, distilled water). In some cases, biological samples can be provided in multiple partitions, wherein each partition can experience varying degrees of dilution. In some cases, biological samples can experience at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, 15 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, 200 times, 500 times or 1000 times of dilution. In some cases, the biological sample can be diluted with a buffer that changes the pH of the biological sample. In some cases, the pH can be changed to about 2, 3, 4, 5, 6, 7, 8, 9, 10. In some cases, the pH can be changed to 9-10. In some cases, the pH is changed by diluting with a pH 9.5 Tris buffer. In some cases, the biological sample is divided into portions, and the portions can be adjusted to different pHs before being processed using the methods disclosed herein.

[0422] In some cases, a biological sample can include a plurality of biomolecules. In some cases, a plurality of biomolecules can include polyamino acids. In some cases, polyamino acids include peptides, proteins, or combinations thereof. In some cases, a plurality of 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 class that defines a group of biomolecules (e.g., proteins, nucleic acids, carbohydrates) that have common characteristics.

[0423] Proteomic analysis

[0424] As used herein, "proteomic analysis", "protein analysis", etc. may refer to any system or method for analyzing proteins in a sample, including the systems and methods disclosed herein. The systems and methods disclosed herein are used for assays using one or more surfaces. In some cases, the surface may include the surface of a high surface area material, such as a nanoparticle, a particle, or a porous material. As used herein, "surface" may refer to a surface for assaying polyamino acids. When the composition, physical properties, or uses of the particles are described herein, it is understood that, in some cases, the surface of the particles may include the same composition, the same physical properties, or the same uses thereof. Similarly, when the surface composition, physical properties, or uses thereof are described herein, it is understood that the particles may include a surface to include the same composition, the same physical properties, or the same uses thereof.

[0425] The material of the particle and the surface can include metals, polymers, magnetic materials and lipids. In some cases, the magnetic particles can be iron oxide particles. Examples of metal materials include any one or any combination of the following: gold, silver, copper, nickel, cobalt, palladium, platinum, iridium, osmium, rhodium, ruthenium, rhenium, vanadium, chromium, manganese, niobium, molybdenum, tungsten, tantalum, iron, cadmium or any alloy thereof. In some cases, the particles disclosed herein can be magnetic particles, such as superparamagnetic iron oxide nanoparticles (SPION). In some cases, the magnetic particles can be ferromagnetic particles, ferrimagnetic particles, paramagnetic particles, superparamagnetic particles or any combination thereof (e.g., particles can include ferromagnetic materials and ferrimagnetic materials).

[0426] The present disclosure describes a set of particles or surfaces. In some cases, the set can contain more than one different surface type. The sets described herein can vary in the number of surface types in a single set and the diversity of surface types. For example, the surface in the set can vary according to size, polydispersity, shape and morphology, surface charge, surface chemistry and functionalization, and substrate material. In some cases, the set can be incubated with a sample of a polyamino acid, polyamino acid concentration, nucleic acid, nucleic acid concentration, or any combination thereof to be analyzed. In some cases, the polyamino acid in the sample is adsorbed onto different surfaces to form one or more biomolecule adsorption layers. The identity of the biomolecules in the one or more adsorption layers and their concentrations can depend on the physical properties of the different surfaces and the physical properties of the biomolecules. Therefore, each surface type in the set can have different adsorbed biomolecules due to the adsorption of different biomolecule sets, different concentrations of specific biomolecules, or combinations thereof. Each surface type in the set can have mutually exclusive adsorbed biomolecules, or can have overlapping adsorbed biomolecules.

[0427] In some cases, the panels disclosed herein can be used to identify the number of different biomolecules disclosed herein within a wide dynamic range of a given biological sample. For example, the panel can enrich a subset of biomolecules in a sample that can be identified within a wide dynamic range of biomolecules present in a sample (e.g., a plasma sample). In some cases, enrichment can be selective—for example, biomolecules in a subset can be enriched, but biomolecules outside the subset may not be enriched and / or depleted. In some cases, a subset can contain proteins with different post-translational modifications. For example, a first particle type in a particle panel can enrich for a protein or protein group with a first post-translational modification, a second particle type in a particle panel can enrich for the same protein or the same protein group with a second post-translational modification, and a third particle type in a particle panel can enrich for the same protein or the same protein group lacking a post-translational modification. In some cases, panels comprising any number of different particle types disclosed herein enrich and identify a single protein or protein group by binding to different domains, sequences, or epitopes of the protein or protein group. For example, a first particle type in a particle assembly can enrich for a protein or group of proteins by binding to a first domain of the protein or group of proteins, and a second particle type in the particle assembly 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, an assembly comprising any number of different 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, an assembly comprising any number of different particle types disclosed herein can enrich and identify biomolecules over a dynamic range of at most 5, 6, 7, 8, 9, 10, 15, or 20 orders of magnitude.

[0428] A panel may have more than one surface type. Increasing the number of surface types in a panel can be a way to increase the number of proteins that can be identified in a given sample.

[0429] The particle or surface may include a polymer. The polymer may constitute a core material (e.g., the core of the particle may include a particle), a layer (e.g., the particle may include a polymer layer between its core and its shell), a shell material (e.g., the surface of the particle may be coated with a polymer) or any combination thereof. Examples of polymers include any one or any combination of polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumarate, 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 (such as a copolymer of polyalkylene glycol (e.g., PEG) and polyester (e.g., PLGA)). The polymer may include crosslinking. The various polymers in the particle may be phase separated, or may include a degree of phase separation.

[0430] Examples of lipids that can be used to form particles or surfaces of the present disclosure include cationic, anionic, and neutrally charged lipids. For example, the particles and / or surfaces can be made of any one or any combination of the following: dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysinephosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, cholesterol, and any combination thereof.

[0431] Particle sets can include combinations of particles with silica and polymer surfaces. For example, a particle set can include particles coated with a silica outer layer and particles coated with a poly(dimethylaminopropylmethacrylamide) (PDMAPMA) outer layer. Particle sets consistent with the present disclosure can also include two or more particles selected from the following: 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. Particle sets consistent with the present disclosure may also include two or more particles selected from the group consisting of surfactant-free carboxylate-functionalized particles, 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 methacrylate-benzylamine)-coated particles, and poly(N-[3-(dimethylamino)propyl]methacrylamide-co-[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (P(DMAPMA-co-SBMA))-coated particles. Particles. Particle sets consistent with the present disclosure may include silica-coated particles, N-(3-trimethoxysilylpropyl)diethylenetriamine-coated particles, poly(N-(3-(dimethylamino)propyl)methacrylamide)(PDMAPMA)-coated particles, phosphate-sugar 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, particle sets may include silica-coated particles, amine-functionalized particles, amine-functionalized polymer-coated particles, and carboxylate-functionalized particles.

[0432] Particle sets consistent with the present disclosure can include silica functionalized particles, amine functionalized particles, silicon alkoxide functionalized particles, carboxylate functionalized particles and benzyl or phenyl functionalized particles. Particle sets consistent with the present disclosure can include silica functionalized particles, amine functionalized particles, silicon alkoxide functionalized particles, polystyrene functionalized particles and sugar functionalized particles. Particle sets 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 sets consistent with the present disclosure can include 5 kinds of particles, including silica functionalized particles, amine functionalized particles, silicon alkoxide functionalized particles.

[0433] Different surfaces or different particles of the present disclosure may differ in one or more physicochemical properties. One or more physicochemical properties are selected from: composition, size, surface charge, hydrophobicity, hydrophilicity, roughness, density surface functionalization, surface morphology, surface curvature, porosity, core material, shell material, shape and any combination thereof. Surface functionalization may include macromolecular functionalization, small molecule functionalization or any combination thereof. Small molecule functionalization may include aminopropyl functionalization, amine functionalization, boric acid functionalization, carboxylic acid functionalization, alkyl functionalization, N-succinimide ester functionalization, monosaccharide functionalization, phosphate sugar functionalization, sulfated sugar functionalization, ethylene glycol functionalization, streptavidin functionalization, methyl ether functionalization, trimethoxysilyl propyl functionalization, silica functionalization, triethoxypropylaminosilane functionalization, thiol functionalization, PCP functionalization, citrate functionalization, lipoic acid functionalization, ethyleneimine functionalization. The particle kit may comprise a plurality of particles having a plurality of small molecule functionalizations selected from the group consisting of silica functionalization, trimethoxysilylpropyl functionalization, dimethylaminopropyl functionalization, phosphate sugar functionalization, amine functionalization, and carboxyl functionalization.

[0434] Small molecule functionalization can include polar functional groups. Non-limiting examples of polar functional groups include carboxyl, hydroxyl, thiol, cyano, nitro, ammonium, imidazolium, sulfonium, pyridinium, pyrrolidinium, phosphonium or any combination thereof. In some embodiments, the functional group is an acidic functional group (e.g., sulfonic acid, carboxyl, etc.), a basic functional group, carbamoyl, hydroxyl, aldehyde, etc. In some cases, the polar functional group can include primary amine, secondary amine, tertiary amine, quaternary amine, cyclic secondary amine, primary amide, secondary amide, tertiary amide, imino, pyridyl, pyrimidinyl, pyrrolidinium, imidazolyl, guanidine, guanidinium or any combination thereof.

[0435] Small molecule functional groups can include ions or ionizable functional groups. Non-limiting examples of ions or ionizable functional groups include ammonium groups, imidazolium groups, sulfonium groups, pyridinium groups, pyrrolidinium groups, phosphonium groups. Small molecule functional groups can include polymerizable functional groups. Non-limiting examples of polymerizable functional groups include vinyl and (methyl) acrylic acid groups. In some embodiments, the functional group is pyrrolidinyl acrylate, acrylic acid, methacrylic acid, acrylamide, 2-(dimethylamino)ethyl methacrylate, hydroxyethyl methacrylate, etc.

[0436] Surface functionalization can include charge. For example, particles can be functionalized to carry a net neutral surface charge, a net positive surface charge, a net negative surface charge, or a zwitterionic surface. Surface charge can be a determining factor in the type of biomolecules collected on the particles. Therefore, optimizing the particle assembly can include selecting particles with different surface charges, which can not only increase the number of different proteins collected on the particle assembly, but also increase the possibility of identifying the biological state of the sample. The particle assembly can include positively charged particles and negatively charged particles. The particle assembly can include positively charged particles and neutral particles. The particle assembly can include positively charged particles and zwitterionic particles. The particle assembly can include neutral particles and negatively charged particles. The particle assembly can include neutral particles and zwitterionic particles. The particle assembly can include negative particles and zwitterionic particles. The particle assembly can include positively charged particles, negatively charged particles, and neutral particles. The particle assembly can include positively charged particles, negatively charged particles, and zwitterionic particles. The particle assembly can include positively charged particles, neutral particles, and zwitterionic particles. The particle set can include negatively charged particles, neutral particles, and zwitterionic particles. In some cases, the positively charged particles can have a zeta potential greater than 0, 5, 10, 15, 20, 25, 50, or 100 mV. In some cases, the negatively charged particles can have a zeta potential less than 0, -5, -10, -15, -20, -25, -50, or -100 mV.

[0437] The particle can include a single surface, such as a specific small molecule, or multiple surface functionalizations, such as multiple different small molecules. The surface functionalization can affect the composition of the biomolecule corona of the particle. Such surface functionalizations can include small molecule functionalizations or macromolecular functionalizations. The surface functionalization can be coupled to a particle material, such as a polymer, a metal, a metal oxide, an inorganic oxide (e.g., silica), or another surface functionalization.

[0438] Surface functionalization can include small molecule functionalization, macromolecular functionalization or two or more such functionalized combinations. In some cases, macromolecular functionalization can include biomacromolecules, such as proteins or polynucleotides (e.g., 100-mer DNA molecules). Macromolecular functionalization can include proteins, polynucleotides or polysaccharides, or can be comparable to the species of any of the above-mentioned types in size. 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 pKa. 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 pKa. In some cases, small molecule functionalization can include small organic molecules, such as alcohols (e.g., octanol), amines, alkanes, alkenes, alkynes, heterocycles (e.g., piperidinyl), heteroaromatic groups, thiols, carboxylates, 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 phosphate sugars, sugar acids, or sulfated sugars.

[0439] In some cases, macromolecular functionalization can include a specific form of attachment to the particle. In some cases, the macromolecule can be tethered to the particle via a joint. In some cases, the joint can keep the macromolecule in a position close to the particle, thereby limiting its movement and reorientation relative to the particle, or the macromolecule can be extended away from the particle. In some cases, the joint can be rigid (e.g., a polyolefin joint) or flexible (e.g., a nucleic acid joint). In some cases, the length of the joint is at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30nm. In some cases, the length of the joint is at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30nm. Therefore, the surface functionalization on the particle can protrude outside the main crown associated with the particle. In some cases, the surface functionalization can also be located under or within the biomolecule crown formed on the particle surface. In some cases, the macromolecule can be tethered to a specific position, such as the C-terminus of a protein, or can be tethered to many possible sites. For example, the peptide may be covalently attached to the particle via any of its surface exposed lysine residues.

[0440] In some cases, the particles can be contacted with a biological sample (e.g., a biological fluid) to form a biomolecule corona. In some cases, the biomolecule corona can include at least two biomolecules that do not share a common binding motif. The particles and the biomolecule corona can be separated from the biological sample, for example, by centrifugation, magnetic separation, filtration, or gravity separation. A variety of separation techniques can be used to separate the particle type and the biomolecule corona from the biological sample. Non-limiting examples of separation techniques include magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density- or gradient-based centrifugation, gravity separation, or any combination thereof. The separated particles and biomolecule corona can be subjected to protein corona analysis. Protein corona analysis can include identifying one or more proteins in the biomolecule 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 contacted with a biological sample in sequence and separated from the biological sample before contacting a subsequent particle type with the biological sample. In some cases, biomolecules adsorbed on particles can have a compressed (eg, smaller) dynamic range compared to a given original biological sample.

[0441] In some cases, the particles of the present disclosure can be used to serially interrogate a sample by incubating a first particle type with the sample to form a biomolecule corona on the first particle type, isolating the first particle type, incubating a second particle type with the sample to form a biomolecule corona on the second particle type, isolating the second particle type, and repeating the interrogation (by incubation with the sample) and isolation of any number of particle types. In some cases, the biomolecule corona on each particle type used to serially interrogate the sample can be analyzed by protein corona analysis. The supernatant can be analyzed for biomolecule content following serial interrogation with one or more particle types.

[0442] In some cases, the methods disclosed herein can identify a large number of unique biomolecules (e.g., proteins) in a biological sample (e.g., a biological fluid). In some cases, the surfaces disclosed herein can be incubated with the biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with the biological sample to adsorb up to 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with the biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecule groups. 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 10000 unique sets of biomolecules. In some cases, several different types of surfaces can be used alone 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.

[0443] In some cases, the methods of the present disclosure can identify a large number of unique protein variants in a biological sample. In some cases, the methods 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 10000 unique protein variants. In some cases, the methods may 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 10000 unique protein variants. In some cases, the surfaces disclosed herein can be 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 10000 unique protein variants. In some cases, the surfaces disclosed herein can be 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 10000 unique protein variants. In some cases, several different types of surfaces can be used alone or in combination to identify a large number of proteins in a particular biological sample. In other words, the surface can be multiplexed to bind and identify a large number of biomolecules in a biological sample.

[0444] The biomolecules collected on the particles can be subjected to further analysis. In some cases, the methods can include collecting a biomolecule corona or a subset of biomolecules from the biomolecule corona. In some cases, the collected biomolecule corona or a subset of biomolecules from the biomolecule corona can be subjected to further particle-based analysis (e.g., particle adsorption).

[0445] In some cases, the collected biomolecule corona or a collected subset of biomolecules from the biomolecule corona can be purified or fractionated (e.g., by chromatography). In some cases, the collected biomolecule corona or a collected subset of biomolecules from the biomolecule corona can be analyzed (e.g., by mass spectrometry).

[0446] In some cases, the panels disclosed herein can be used to identify a plurality of proteins, peptides, proteomes, or classes of proteins using a protein analysis workflow described herein (e.g., a protein corona analysis workflow). In some cases, the panels disclosed herein can be 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 can be 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 can be 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 can be 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, the panels disclosed herein can be 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, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 peptides.In some cases, the panels disclosed herein can be 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, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 peptides. In some cases, the peptides can be tryptic peptides. In some cases, the peptides can be semi-tryptic peptides. In some cases, protein analysis can include contacting a sample with different types of surfaces (e.g., particle sets), forming adsorbed biomolecule layers on different types of surfaces, and identifying biomolecules in the adsorbed biomolecule layers (e.g., by mass spectrometry). As disclosed herein, feature intensity can refer to the intensity of discrete peaks ("features") seen on a plot of mass-to-charge ratio versus intensity in a mass spectrometry run from a sample. In some cases, these features can correspond to variable ionization fragments of peptides and / or proteins. In some cases, using the data analysis methods of this article, feature intensities can be classified 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 a protein identified using a unique identification sequence. For example, if in a sample, a peptide sequence shared between two proteins (protein 1: XYZZX and protein 2: XYZYZ) is determined, the protein group can be an "XYZ protein group" with two members (protein 1 and protein 2). In some cases, if the peptide sequence is unique to a single protein (protein 1), the proteome can be a "ZZX" proteome with one member (protein 1). In some cases, each proteome can be supported by more than one peptide sequence. In some cases, proteins detected or identified in accordance with the present disclosure can refer to different proteins detected in a sample (e.g., different proteins relative to other proteins detected using mass spectrometry). In some cases, analysis of proteins present in different coronas corresponding to different surface types in a set will produce a large number of feature intensities. In some cases, this number will decrease as the feature intensities are processed into different peptides, the number will decrease further as different peptides are processed into different proteins, and the number will decrease further as the peptides are grouped into proteomes (two or more proteins that share different peptide sequences).

[0447] In some cases, the methods disclosed herein include separating one or more particle types from one sample or more than one sample (e.g., biological samples or samples of continuous interrogation). Magnets can be used to quickly isolate or separate particle types from samples. In addition, multiple samples isolated in space can be processed in parallel. In some cases, the methods disclosed herein are used to isolate or separate particle types from unbound proteins in samples. In some cases, particle types can be separated by a variety of methods, including but not limited to magnetic separation, centrifugation, filtration or gravity separation. In some cases, the particle set can be incubated with multiple spatially isolated samples, wherein each spatially isolated sample is located in a hole of a well plate (e.g., a 96-well plate). In some cases, the particles in each hole of the well plate can be separated from the unbound proteins present in the spatially isolated samples by placing the entire plate on a magnet. In some cases, this can simultaneously pull down the superparamagnetic particles in the particle set. In some cases, the supernatant in each sample can be removed to remove unbound proteins. In some cases, these steps (incubation, pull down) can be repeated to effectively clean the particles, thereby removing the residual background unbound proteins that may exist in the sample.

[0448] In some cases, the systems and methods disclosed herein can also illustrate the interactions between protein classes or protein classes. In some cases, protein classes can include a collection of proteins that share a common function (e.g., amine oxidases or proteins involved in angiogenesis); proteins that share a common physiological, cellular or subcellular localization (e.g., peroxisomal proteins or membrane proteins); proteins that share a common cofactor (e.g., heme or flavoprotein); proteins that correspond to a specific biological state (e.g., hypoxia-related proteins); proteins containing specific structural motifs (e.g., cup folds); functionally related proteins (e.g., part of the same metabolic pathway); or proteins with post-translational modifications (e.g., ubiquitinated or citrullinated proteins). In some cases, a protein class can include at least 2 proteins, 5 proteins, 10 proteins, 20 proteins, 40 proteins, 60 proteins, 80 proteins, 100 proteins, 150 proteins, 200 proteins or more.

[0449] In some cases, a variety of different analytical techniques can be used to identify, measure, and quantify proteomic data of biological samples. For example, proteomic data can be generated using SDS-PAGE or any gel-based separation technique. 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.

[0450] In some cases, determination can include protein particle collection, protein digestion and mass spectrometry (e.g., MS, LC-MS, LC-MS / MS). In some cases, digestion can include chemical digestion, such as by cyanogen bromide or 2-nitro-5-thiocyanatobenzoic acid (NTCB). In some cases, digestion can include enzymatic digestion, such as by trypsin or pepsin. In some cases, digestion can include enzymatic digestion by multiple proteases. In some cases, digestion can 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 metalloproteinase, glycoprotein endopeptidase, proline, aminopeptidase, isopentenyl protease, caspase, kex2 endoproteinase or any combination thereof. In some cases, digestion can cut peptides at random positions. In some cases, digestion can cut peptides at specific positions (e.g., at methionine) or sequences (e.g., glutamic acid-histidine-glutamic acid). In some cases, digestion can distinguish similar proteins. For example, a determination can be made that 8 different proteins are broken down into a single protein group with a first digestion method, and broken down into 8 independent proteins with different signals with a second digestion method. In some cases, digestion can generate an average peptide fragment length of 8 to 15 amino acids. In some cases, digestion can generate an average peptide fragment length of 12 to 18 amino acids. In some cases, digestion can generate an average peptide fragment length of 15 to 25 amino acids. In some cases, digestion can generate an average peptide fragment length of 20 to 30 amino acids. In some cases, digestion can generate an average peptide fragment length of 30 to 50 amino acids.

[0451] In some cases, the protein or peptide can be prepared for mass spectrometry. In some cases, the protein or peptide can be treated with an alkylating agent. For example, the protein or peptide can be treated with N-ethylmaleimide and iodoacetamide. In some cases, the protein or peptide can be treated with a reducing agent. For example, the protein or peptide is treated with dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP). In some cases, the protein or peptide is digested, alkylated and reduced before analysis. In some cases, the protein or peptide is digested, alkylated and reduced before using mass spectrometry analysis.

[0452] In some cases, determination can generate and analyze proteomics data quickly. In some cases, starting from input biological samples (e.g., oral or nasal smears, plasma or tissues), the methods disclosed herein can generate and analyze proteomics data in less than about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24 or 48 hours. In some cases, analysis may include identifying protein groups. In some cases, analysis may include identifying protein classes. In some cases, analysis may include quantifying the abundance of biomolecules, peptides, proteins, proteomes or protein classes. In some cases, analysis may include identifying the abundance ratio of two biomolecules, peptides, proteins, proteomes or protein classes. In some cases, analysis may include identifying biological states.

[0453] Examples of the particle types disclosed herein may be carboxylate (citrate) superparamagnetic iron oxide nanoparticles (SPIONs), phenol-formaldehyde coated SPIONs, silica coated SPIONs, polystyrene coated SPIONs, carboxylated poly(styrene-co-methacrylic acid) coated SPIONs, N-(3-trimethoxysilylpropyl)diethylenetriamine coated SPIONs, poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coated SPIONs, 1,2,4,5-pyromellitic acid coated SPIONs, poly(vinylbenzyltrimethylammonium chloride) (PVBTMAC) coated SPIONs, carboxylate, PAA coated SPIONs. ON, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coated SPION, carboxylate microparticles, polystyrene carboxyl functionalized particles, carboxylic acid coated particles, silica particles, carboxylic acid particles of about 150 nm in diameter, amino surface microparticles of about 0.4-0.6 μm in diameter, silica amino functionalized microparticles of about 0.1-0.39 μm in diameter, Jeffamine surface particles of about 0.1-0.39 μm in diameter, polystyrene microparticles of about 2.0-2.9 μm in diameter, silica particles, carboxylated particles with a pristine coating of about 50 nm in diameter, particles coated with a dextran-based coating of about 0.13 μm in diameter, or low acidity silica silanol coated particles. In some cases, the particles may lack functionalized specific binding moieties for specific binding on their surface. In some cases, the particles may lack functionalized proteins for specific binding on their surface. In some cases, the surface functionalized particles do not contain antibodies or T cell receptors, chimeric antigen receptors, receptor proteins, or variants or fragments thereof. In some cases, the ratio between surface area and mass can determine the properties of the particles. The particles disclosed herein can have a surface area of ​​3 to 30 cm 2 / mg, 5 to 50cm 2 / mg, 10 to 60cm 2 / mg, 15 to 70cm 2 / mg, 20 to 80cm 2 / mg, 30 to 100 cm 2 / mg, 35 to 120cm 2 / mg, 40 to 130cm 2 / mg, 45 to 150cm 2 / mg, 50 to 160cm 2 / mg, 60 to 180cm 2 / mg, 70 to 200cm 2 / mg, 80 to 220cm 2 / mg, 90 to 240cm 2 / mg, 100 to 270cm 2 / mg, 120 to 300cm 2 / mg, 200 to 500cm 2 / mg, 10 to 300cm 2 / mg, 1 to 3000cm 2 / mg, 20 to 150cm 2 / mg, 25 to 120cm 2 / mg or 40 to 85cm 2 / mg surface area to mass ratio. Small particles (e.g., 50 nm in diameter or less) can have significantly higher surface area to mass ratios, in part because diameter depends more on mass than on surface area. In some cases (e.g., for small particles), particles can have a surface area to mass ratio of 200 to 1000 cm 2 / mg, 500 to 2000cm 2 / mg, 1000 to 4000cm 2 / mg, 2000 to 8000cm 2 / mg or 4000 to 10000cm 2 / mg of surface area to mass ratio. In some cases (e.g., for large particles), the particles may have a surface area of ​​1 to 3 cm 2 / mg, 0.5 to 2cm 2 / mg, 0.25 to 1.5 cm 2 / mg or 0.1 to 1cm 2The particle can include a variety of physical properties. The physical properties of the particle can include composition, size, surface charge, hydrophobicity, hydrophilicity, amphiphilicity, surface functionality, surface morphology, surface curvature, porosity, core material, shell material, shape, zeta potential and any combination thereof. The particle can have a core-shell structure. In some cases, the core material can include metals, polymers, magnetic materials, paramagnetic materials, oxides and / or lipids. In some cases, the shell material can include metals, polymers, magnetic materials, oxides and / or lipids.

[0454] In some cases, the particles may include nanoparticles. In some cases, the particles may include microparticles. In some cases, the first particle, the second particle, or both particles in the particle set are nanoparticles. In some cases, the first particle, the second particle, or both particles in the particle set are microparticles. In some cases, the first particle may be a nanoparticle and the second particle may be a microparticle.

[0455] In some cases, the particles may comprise 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 may comprise a diameter of at most 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 may comprise 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 um. In some cases, the particles may comprise a diameter of at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 um.

[0456] In some cases, the first size of the first particle is at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 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 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 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 a first diameter and the second size is a second diameter. In some cases, the first size is a first average size and the second size is a second average size. In some cases, the first average size and the second average size are average sizes or median sizes.

[0457] In some cases, the particle set comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 parts of the first particle to about 1 part of the second particle. In some cases, the particle set comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 parts of the first particle to about 1 part of the second particle. In some cases, the particle set comprises about 15 parts of the first particle to about 6 parts of the 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 set comprises about 5 to about 20 parts by weight of the first particle to about 1 part by weight of the second particle. In some cases, the particle set comprises about 10 to about 15 parts by weight of the first particle to about 1 part by weight of the second particle.

[0458] Proteomics information

[0459] In some cases, proteomic information or data can refer to information about substances containing peptides and / or protein components. In some cases, proteomic information can include primary structure information, secondary structure information, tertiary structure information, or quaternary information about peptides or proteins. In some cases, proteomic information can include information about protein-ligand interactions, wherein the ligand can include any of the various biomolecules and substances that can be found in living organisms, such as nucleotides, nucleic acids, amino acids, peptides, proteins, monosaccharides, polysaccharides, lipids, phospholipids, hormones, or any combination thereof.

[0460] In some cases, proteomic information can include information about a single cell, tissue, organ, system of tissue and / or organ (such as cardiovascular, respiratory, digestive or nervous system) or entire multicellular organism. In some cases, proteomic information can include information about an individual (e.g., a human individual or a bacterial individual) or a population of individuals (e.g., a human or bacterial community diagnosed with cancer). Proteomic information can include information from various life forms, including life forms from archaea, bacteria, eukaryotes, protozoa, pseudobacteria (Chromista), plant kingdom, fungi or animal kingdom. In some cases, proteomic information can include information from viruses.

[0461] In some cases, proteomic information may include information related to exons and introns in the code of life. In some cases, proteomic information may include information about primary structural variation, secondary structural variation, tertiary structural variation, or quaternary structural variation of relevant peptides and / or proteins. In some cases, proteomic information may include information about exon expression variation, including alternative splicing variation, structural variation, or both. In some cases, proteomic information may include conformational information, post-translational modification information, chemical modification information (e.g., phosphorylation), cofactor (e.g., salt or other regulating chemicals) association information, or substrate association information of peptides and / or proteins.

[0462] In some cases, the proteomic information may include information related to various protein variants in the sample. In some cases, the proteomic information may include information related to peptide variants, protein variants, or both. In some cases, the proteomic information may include information related to splice variants, allelic variants, post-translational modification variants, or any combination thereof.

[0463] In some cases, a splice variant (also referred to in some cases as an "alternative splicing" variant, a "differential splicing" variant, or an "alternative RNA splicing" variant) can refer to a protein expressed by an alternative splicing process. In some cases, the alternative splicing process can express one or more splice variants from a set of exons via different combinations of exons. In some cases, a combination can include a different exon sequence compared to another combination. In some cases, a combination can include a different subset of exons compared to another combination. In some cases, a splice variant can include a reordered amino acid sequence of another splice variant.

[0464] In some cases, allelic variant can refer to the protein expressed from the gene comprising mutation compared with the reference gene. In some cases, the reference gene can be the gene of a cell, an individual or a population of individuals. In some cases, compared with the genetic reference of the reference gene, mutation can be a base substitution, a base deletion or a base insertion of the genetic sequence of the gene. In some cases, allelic variant can comprise an amino acid substitution in the amino acid sequence of another allelic variant.

[0465] In some cases, post-translational modification can refer to a protein that is modified after expression. Proteins can be modified by various enzymes. In some cases, the enzyme that can modify a protein can be a kinase, a protease, a ligase, a phosphatase, a transferase, a phosphotransferase, or any other enzyme for performing any of the modifications disclosed herein.

[0466] In some cases, the peptide variants or protein variants may include post-translational modifications. In some cases, the post-translational modifications include acylation, alkylation, prenylation, flavination, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfonation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, SUMOylation, pseudo-succinylation, myristoylation, palmitoylation, prenylation, farnesylation, geranylgeranylation, glycosylphosphatidylinositolization, glycosylphosphatidylinositol anchor formation, lipid acylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinyl Schiff base formation, diphtheria amide formation, ethanolamine phosphoglycerol functionalization, hydroxybutyric acid formation, β-lysine addition, acetylation, formylation , methylation, amidation, amide bond formation, butylation, γ-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate formation, phosphoramidate formation, adenylation, uridylylation, propionylation, pyroglutamate formation, glutathionylation, sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, glycation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, citrullination, deamidation, elimination, disulfide bond formation, proteolytic cleavage, isoaspartate formation, racemization, protein splicing, chaperone-assisted folding, or any combination thereof.

[0467] In some cases, proteomic information can be encoded as digital information. In some cases, proteomic information can include one or more elements representing proteomic information. In some cases, element can represent primary structure information, secondary structure information, tertiary structure information or four-level information about peptide or protein. In some cases, element can represent the protein-ligand interaction of peptide or protein. In some cases, element can represent the source (for example, specific cells, tissue, organ, organism, individual or individual colony) of peptide or protein. In some cases, element can represent the type of protein variant. In some cases, element can be any other data type provided by numeral, vector, array or this paper.

[0468] Nonspecific binding

[0469] The surface can bind biomolecules by variable selective adsorption (e.g., adsorption of a biomolecule or a group of biomolecules when the particle is in contact with a biological sample containing the biomolecule or the group of biomolecules, the adsorption having variable selectivity depending on factors including, for example, the physicochemical properties of the particle) or non-specific binding. Non-specific binding can refer to a class of binding interactions that exclude 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, wherein the sequence or three-dimensional structure provided by the template molecule favors the binding of a target molecule containing a complementary sequence or a complementary three-dimensional structure, and disfavors the binding of non-target molecules that do not contain a complementary sequence or a complementary three-dimensional structure.

[0470] Nonspecific binding can comprise one or combination of various chemical and physical interactions and effects.Nonspecific binding can comprise electromagnetic force, such as electrostatic interaction, London dispersion, van der Waals interaction or dipole-dipole interaction (for example, between permanent dipole and induced dipole).Nonspecific binding can be mediated by covalent bond, such as disulfide bond.Nonspecific binding can be mediated by hydrogen bond.Nonspecific binding can comprise solvophobic effect (for example, hydrophobic effect), in which one object is repelled by solvent environment and is forced to reach the boundary of solvent, such as the surface of another object.Nonspecific binding can comprise entropy effect, such as in consumption power, or heat energy is raised to above critical solution temperature (for example, lower critical solution temperature).Nonspecific binding can comprise kinetic effect, in which one binding molecule can have faster binding kinetics than another binding molecule.

[0471] 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). Multiple targets can have similar non-specific binding affinities that are within about one, two, or three orders of magnitude (e.g., as measured by non-specific binding free energy, equilibrium constant, competitive adsorption, etc.). This can be contrasted with specific binding, which can involve a higher binding affinity for a given target molecule compared to a non-target molecule.

[0472] Biomolecules can be adsorbed onto a surface at various densities via non-specific binding. In some cases, a biomolecule or protein can be adsorbed onto a surface 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed at a density of up to 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 In some cases, the biomolecule or protein can be adsorbed 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 / mm 2 Density adsorption.

[0473] 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 may 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.

[0474] In some cases, the concentration of the protein in the biological sample can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 orders of magnitude. In some cases, the concentration of the protein in the biological sample can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 orders of magnitude.

[0475] Composition Improvement Determination

[0476] In some cases, the methods of the present disclosure may include using a composition improvement assay. In some cases, a non-targeted assay may be a composition improvement assay. In some cases, a composition improvement assay may improve the acquisition of a subset of biomolecules in a biological sample. In some cases, a composition improvement assay may improve the detection of a subset of biomolecules in a biological sample. In some cases, a composition improvement assay may improve the identification of a subset of biomolecules in a biological sample. In some cases, a subset of biomolecules may be a low abundance biomolecule. In some cases, a subset of biomolecules may be a rare biomolecule. In some cases, a composition improvement assay may be used to compress the dynamic range of a biological sample. In some cases, the dynamic range may be compressed by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude.

[0477] In some cases, the composition improvement assay may include providing one or more surface areas comprising one or more surface types. In some cases, the composition improvement assay may include contacting a biological sample with one or more surface areas to produce an adsorbed biomolecule set on the one or more surface areas. In some cases, the composition improvement assay may include desorbing at least a portion of the adsorbed biomolecule set from the one or more surface areas to produce a polyamino acid set. In some cases, the composition improvement assay may include contacting a biological sample with one or more surface areas to capture the biomolecule set on the one or more surface areas. In some cases, the composition improvement assay may include releasing at least a portion of the biomolecule set from the one or more surface areas to produce a polyamino acid set. In some cases, the one or more surface areas are arranged on a single continuous surface. In some cases, the one or more surface areas are arranged on one or more discrete surfaces. In some cases, the one or more discrete 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, trifunctional, or N-functional particles.

[0478] In some cases, the composition improvement assay may include providing a plurality of surface areas comprising a plurality of surface types. In some cases, the composition improvement assay may include contacting a biological sample with a plurality of surface areas to produce an adsorbed biomolecule set on the plurality of surface areas. In some cases, the composition improvement assay may include desorbing at least a portion of the adsorbed biomolecule set from the plurality of surface areas to produce a polyamino acid set. In some cases, the composition improvement assay may include contacting a biological sample with a plurality of surface areas to capture the biomolecule set on the plurality of surface areas. In some cases, the composition improvement assay may include releasing at least a portion of the biomolecule set from the plurality of surface areas to produce a polyamino acid set. In some cases, the plurality of surface areas are arranged on a single continuous surface. In some cases, the plurality of surface areas are arranged on a plurality of discrete surfaces. In some cases, the plurality of discrete surfaces are 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, trifunctional, or N-functional particles.

[0479] Example

[0480] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the present disclosure; it should be understood that in view of their exemplary nature, other procedures, methods or techniques known to those skilled in the art may also be used instead.

[0481] Example 1: Proteograph using alternative pH conditions for canopy incubation of different nanoparticles TM Assays for protein profiling of biological fluids

[0482] The biological fluid sample can be incubated with different nanoparticles (NPs) in a solution containing water and / or biological pH to form a protein corona on the NPs. This embodiment provides the use of alternative fluid compositions with different pH containing different NPs to improve the binding between NPs and proteins in the biological fluid sample. In some cases, the use of alternative fluid compositions can improve the proteome coverage depth.

[0483] Plasma samples with various pH buffers were mixed in the same volume ratio, such as Figure 2A-2B The mixture was added to the dried NP particles and incubated. After the incubation was complete, 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 loosely bound proteins off the surface of the corona. Figure 2CThe NPs were washed with the wash compositions shown in . Afterwards, the proteins of the corona were subjected to digestion and clean-up steps. The proteins were analyzed using LC-MS / MS using a PharmaFluidics column in data-dependent acquisition (DDA) mode with maximum injection mass per condition. Various environments for corona formation were investigated with different NPs to achieve deeper proteome depth.

[0484] The data showed that by using some pH buffers, a 30% to 40% increase in proteome counts was observed for several NPs, such as Figure 3 For NP 2 with pH 9.5 Tris buffer, an increase of about 42% in the number of proteomes was observed, and for NP 5 under the same conditions, an increase of 48% in the number of proteomes was observed.

[0485] The data showed that peptide yields were increased by using some pH buffers, e.g. Figure 4 As shown in . Using citrate buffer at pH 3, peptide yields increased for all nanoparticles tested.

[0486] The data show that the miscleavage rate increases by using some pH buffers, e.g. Figure 5 As shown in . Using citrate buffer at pH 3, the miscleavage rate increased for all nanoparticles tested.

[0487] For NP 4, a low Jaccard index (JI) was observed between pH 7 (in water) and high pH (using TRIS buffer, pH = 9.5), even though the number of proteomes was similar between the two conditions, as shown in Figure 2. Figure 6A-6D as shown in . Fig. 7A Functional annotation of the proteome shown in Figures 7B-7D The range of physicochemical properties of the proteins shown in also varies significantly. The protein / NP ratio can be adjusted to further improve the proteomic depth coverage (e.g., by exploiting the Vroman effect).

[0488] Example 2: Using Multiplexed Nanoparticles to Proteograph TM Assays for protein profiling of biological fluids

[0489] Nanoparticles can be incubated separately from biological samples (e.g., in separate wells). In order to improve the throughput of analyzing biological samples, it can be advantageous to incubate a given biological product in fewer wells multiplexed with nanoparticles. The present embodiment provides a multiplexing method for mixing NPs with the same charge having different physicochemical properties to improve proteome coverage depth and determination throughput. The multiplexing method can incubate various NPs under different pH conditions.

[0490] Possible combinations of NPs with similar charges (e.g., all positive or all negative zeta potential as measured by DLS), as well as different ratios between similarly charged NPs were explored. Combinations of similarly charged NPs were multiplexed prior to incubating the biological sample with the NPs. The performance of the multiplexed NPs and single NPs were compared.

[0491] The results showed that by multiplexing multiple NPs into a single well, the number of identified proteomes increased for many sample types, e.g. Fig. 22 As shown in Fig.25 As shown in , by further incorporating high pH into the design, a 2-well particle set increased the number of identified proteomes by approximately 30% compared to a 5-particle set performed with 5 wells. This also reduced the mass spectrometry time per sample by 60%. The best performing 4- or 5-well particle sets had mixed buffer conditions including pH 6, water, and pH 9.5.

[0492] Fig. 22 Shown is a comparison of the number of protein groups identified between a multiplex assay compared to a singleplex assay according to some embodiments. Improved number of protein groups was observed with NP 1:NP 4 at a mass ratio of about 6 to 15.

[0493] Fig.23 A comparison of the number of protein groups identified between various multiplexed assays with different Vroman conditions according to some embodiments is shown. The combinations had between -5% and +8% of the number of protein groups identified when compared to the reference particle set of 5 particles. When NP 4 and NP 1 were combined, stable performance was observed for all samples (as shown in the dashed box).

[0494] Fig.24 A comparison of the number of protein groups identified between various multiplexed assays according to some embodiments is shown.Multiplexed NPs with standard pH conditions performed better than single NPs (dashed boxes compared to solid boxes).

[0495] Fig.25 A comparison of the number of protein groups identified between various multiplexed assays with different pH conditions according to some embodiments is shown. NP 5 and NP 2 were multiplexed at pH 9.5, while NP 4 and NP 1 were multiplexed at standard pH. For all sample types, an average improvement of +9% over the reference 5 particle set was observed.

[0496] Example 3: Proteograph using low sample volumes TM Protein profiling of biofluids

[0497] 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. TM These diluted plasma samples and undiluted control plasma samples were processed in triplicate and analyzed using LC-MS / MS. The total number of proteomes 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. Although all dilutions reduced the total number of proteomes detected in the process plasma samples, pH 9.5 Tris buffer provided significantly higher proteome counts compared to other dilutions.

[0498] Example 4: Using Proteograph TM Workflow Comparison of Sample Dilution for Protein Profiling of Biofluids

[0499] Using Proteograph TM v1.2 Workflow Different dilution volumes of pooled human plasma and mouse serum samples were analyzed in triplicate. The sample volumes tested were 250 μL, 125 μL, 50 μL, 25 μL, and 10 μL. Sample volumes below 250 μL were diluted with 1M pH 9.5 buffer to obtain a total volume of 250 μL. For example, 50 μL of pooled plasma samples were diluted with 200 μL of 1M pH 9.5 Tris buffer. Proteograph TM The v1.2 workflow processes the final volume in triplicate. Briefly, a 40 μL volume is aliquoted into 5 separate wells and each well is combined with 40 μL of a different nanoparticle suspended in water. The combined mixture is then incubated to form a biomolecular corona, followed by washing, digestion, and preparation for analysis by mass spectrometry. The processed peptide samples are analyzed using LC-MS / MS in DIA mode.

[0500] The number of proteomes and peptides identified in each sample is provided in Table 1 below:

[0501]

[0502] The results showed that the number of identified proteomes and peptides decreased with dilution, but even when a 1:24 dilution of a 10 μL sample volume was analyzed, the majority of proteomes and peptides were identified relative to the undiluted sample. The coefficient of variation for each dilution was below 25%.

[0503] About 1060 protein groups were identified in undiluted pooled human plasma samples (i.e., 250 μL plasma sample volumes), which are also annotated in the Human Plasma Proteome Project (HPPP). These protein groups were sorted using abundance data from the HPPP and grouped into quartiles, the first quartile containing the most abundant proteins, and the fourth quartile containing the least abundant proteins. Compared with undiluted 250 μL sample volumes, the number of proteins not identified in each quartile for each dilution is shown in Table 2 below:

[0504]

[0505] The results showed that dilution reduced the number of protein groups identified in each quartile, with a greater loss for less abundant protein groups. Even for a 1:24 dilution of the 10 μL plasma sample, the majority of protein groups were still identified in the fourth quartile compared to the undiluted sample.

[0506] The Jaccard index of each diluted plasma sample relative to the undiluted 250 μL plasma sample was determined, which is an indicator of the similarity of the protein groups identified between samples. The Jaccard index between samples was: 250 μL vs. 125 μL - 0.93; 250 μL vs. 50 μL - 0.87; 250 μL vs. 25 μL - 0.83; and 250 μL vs. 10 μL - 0.70. These results indicate that similar protein groups can be identified when diluting samples.

[0507] Example 5: System for Selective Enrichment and Detection of Biomolecules

[0508] This embodiment illustrates the use of an automated system (instrument): including pipelines for providing various consumable materials (e.g., nanoparticle formulations, solvents, reagents, etc.), using the automated system to perform assays, using a mass spectrometer to generate assay results, and then using data analysis software to analyze the results and display the results to a user.

[0509] The following describes an example method for an automated system that includes a computer-readable medium containing machine-executable code. (1) A user (i.e., an operator) prepares samples (e.g., by thawing frozen samples), reagents (e.g., diluting reagents), and particles (e.g., reconstituting lyophilized particles into a multiplexed particle suspension). The prepared samples, reagents, and particles are loaded into the automated system. The automated system then automatically performs the experimental steps from this point on, including: (2) initialization of the apparatus (chassis, MPE2, Hamilton heater oscillator (HHS), Inheco CPAC), (3) pipetting the sample to the assay plate within 5 minutes, (4) pipetting the particles to the assay plate within 15 minutes, (5) incubation at 37°C within 60 minutes, (6) washing the assay plate within 30 minutes, (7) adding lysis, reduction and alkylation buffers to the assay plate within 10 minutes, (8) incubation at 95°C on HHS within 10 minutes, (9) cooling the assay plate at room temperature within 20 minutes, (10) adding trypsin / LysC enzyme within 8 minutes, (11) washing with HHS within 180 minutes. Incubate at 37°C, (12) add stop solution which is performed within 3 minutes, (13) pull down particles which is performed within 5 minutes, (14) process samples using SPE plate on MPE2 which is performed within 8 minutes, (15) process samples using detergent A which is performed within 8 minutes using SPE plate on MPE2, (16) process samples using detergent B-1 which is performed within 8 minutes using SPE plate on MPE2, (17) process samples using detergent B-2 which is performed within 8 minutes using SPE plate on MPE2, and (18) elute samples using SPE plate on MPE2 which is performed within 5 minutes. (19) The user can then clean up the automated system after the experiment is complete. The total duration of the experiment is approximately 7 hours.

[0510] The series of experimental steps previously described may include additional steps, may exclude some steps, or may have variations in each step. These variations may be implemented so that the user can choose which variation to use. For example, there may be variations in step (1) where the user can dilute the sample (e.g., plasma samples up to 20 times their original volume), select different volumes for the assay (e.g., any range from 40 μL to 100 μL), thaw the sample to a specific temperature (e.g., room temperature or 4°C), single or multiplex nanoparticles (e.g., 2, 3, 4, 5, or any number of particles per partition), or perform an intervention step on the sample (e.g., hemolysis / lipid concentration). In some cases, the background of biomolecules other than proteins may alter the protein corona depending on the physicochemical properties of the particles. In some cases, the background of biomolecules may also form part of the biomolecule corona. In some cases, the intervention step may include titrating different concentrations of certain biomolecules (e.g., lipids) at different concentrations.

[0511] There can be variations in any incubation step, where the duration of the incubation can be varied (e.g., 5 minutes or overnight), the pH of the incubated solution can be varied (e.g., pH 3.8, 5.0, or 7.4), the ionic strength of the incubated solution can be varied (e.g., 0, 50, or 150 mM), and the rate at which the incubated solution is shaken can be varied (e.g., 0, 150, or 300 RPM).

[0512] There may be variations in any washing steps where some or all of the components in the solution may or may not be resuspended. Some or all of the components in the solution may be separated, for example, by applying a magnetic field to capture magnetic particles.

[0513] There can be variations in the cleavage, reduction or alkylation steps, wherein progressive denaturation can occur. The temperature of the solution can vary (e.g., 50°C or 95°C). There can be a step in which, for example, various durations (e.g., 3 hours or overnight) of digesting a protein or peptide by using trypsin of various concentrations (1X, 2X concentration of trypsin standard amounts). In some cases, the standard amount of trypsin can be trypsin of about 1 / 10 to about 1 / 100 mass compared to the mass of the protein. A protein or peptide can be digested in a progressive manner, for example, by using trypsin / LysC.

[0514] There can be variations in the elution step. The elution volume can be varied (e.g., 75, 150, or 300 μL), clean dry air (CDA) or nitrogen can be supplied at various pressures (anywhere from 0 to 50 psi), and different types of solid phase extraction (SPE) plates can be used (e.g., Thermal Fisher SPE plates, iST, C18, or other substrates).

[0515] In some cases, the automated system can be configured to run 8 to 16 samples at a time. Biomolecules in biological samples (i.e., biological fluids) can be measured with 5 different methods per sample. Measurements can be performed on a variety of biological fluids, including plasma, cell extracts, and lysates. The measurement can be performed automatically and completed in 7-8 hours, where the peptides are ready to be injected into liquid chromatography (LC) or MS for detection. Unbiased measurements allow the LC / MS time to be reduced, and these measurements can be independent of the LC / MS detector or method, for example: using the DIA SWATH (data-independent acquisition) method on Sciex 6600+, with no more than 30 minutes of gradient length per fraction (sample to sample), and / or no more than 1 hour of gradient length DDA (data-dependent acquisition) method on Thermo Orbitrap Lumos. DIA SWATH (data-independent acquisition) and DDA (data-independent acquisition) are modes of MS, the difference being the way peptides are analyzed and the way proteins are reconstructed based on MS raw data. Because intact proteins can be measured, the measurement can reveal protein-protein interactions in experimental data.

[0516] In some cases, the automated system may include a 96-well plate that can accommodate up to 16 samples with 5 nanoparticle inquiries. In some cases, the amount of the required sample volume may be less than or equal to 240 μL or 40 μL. In some cases, reagents may be stored while maintaining stability for more than 9 months at 4 ° C or for more than 6 months at room temperature. In some cases, the assay may be run within 7 hours. In some cases, the MS experiment run time may be within 120 minutes. In some cases, the MS experiment may be run with ScanningSWATH. In some cases, ScanningSWATH may refer to a fast MS acquisition mode for short gradients (as low as a few minutes). In some cases, ScanningSWATH may refer to a fast MS acquisition mode using a scanning quadrupole. In some cases, ScanningSWATH may use Sciex timsTOF fast IMS-IMS, which may involve ion mobility separation, and may involve pre-separation of ions based on the charge / dipole and shape properties of the ions. In some cases, the automated system can include analytical tools, including visualization (e.g., group analysis, PCA) tools or quality control tools, which can be integrated into a cloud-based computing system. In some cases, the protein detection method implemented on the automated system can show a 5-fold advantage (i.e., an advantage in the number of protein groups detected) over the shallow plasma method and a 3-fold advantage over the depleted plasma method. In some cases, the protein detection method implemented on the automated system can have a 5% accuracy improvement (lower CV) over a published data set (e.g., Geyer et al. Mol. Syst. Biol. 13, 942 (2017)).

[0517] List of implementation plans

[0518] The following list of embodiments of the present invention should be considered to disclose various features of the present invention, which may be considered specific to the particular embodiment in which they are discussed, or may be combined with various other features listed in other embodiments. Therefore, just because a feature is discussed in a particular embodiment does not necessarily limit the use of the feature to that embodiment.

[0519] Embodiment 1. A method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in a second fluid composition; and (c) performing downstream assays on the biomolecule types of the first plurality of biomolecule types and the second plurality of biomolecule types, wherein the first fluid composition comprises a first pH and the second fluid composition comprises a second pH, wherein the first pH and the second pH are different or the same.

[0520] Embodiment 2. A method according to embodiment 1, wherein the second infinite dilution limiting solvation enthalpy or solvation free energy of the second biomolecules in the second biomolecule subset is different when the second biomolecules are in the second fluid composition compared to when the second biomolecules are in the first fluid composition.

[0521] Embodiment 3. A method according to embodiment 1 or 2, wherein the first fluid composition comprises a first set of intensity physical properties that mediate the selective enrichment of the first plurality of biomolecule types, and the second fluid composition comprises a second set of intensity physical properties that mediate the selective enrichment of the second plurality of biomolecule types, wherein the first set of intensity physical properties and the second set of intensity physical properties are different.

[0522] Embodiment 4. A method according to any one of Embodiments 1-3, wherein the first fluid composition comprises a first ratio between a first sample volume and a first surface area of ​​a first surface, and the second fluid composition comprises a second ratio between a second sample volume and a second surface area of ​​a second surface, wherein the first ratio and the second ratio are different.

[0523] Embodiment 5. A method according to any one of embodiments 1-4, wherein the first fluid composition and the second fluid composition comprise different temperatures.

[0524] Embodiment 6. A method according to any one of Embodiments 1-5, wherein the first fluid composition comprises a first ionic strength and the second fluid composition comprises a second ionic strength, wherein the first ionic strength and the second ionic strength are different.

[0525] Embodiment 7. The method of any one of Embodiments 1-6, wherein the first fluid composition and the second fluid composition comprise different salts.

[0526] Embodiment 8. The method of any one of Embodiments 1-7, wherein the first fluid composition and the second fluid composition comprise different solvents.

[0527] Embodiment 9. The method of any one of Embodiments 1-8, wherein the first fluid composition, the second fluid composition, or both comprise a buffer comprising tris(hydroxymethyl)aminomethane.

[0528] Embodiment 10. The method of any one of Embodiments 1-9, wherein the first fluid composition, the second fluid composition, or both comprise a buffer comprising citrate.

[0529] Embodiment 11. The method of any one of Embodiments 1-10, wherein the first fluid composition, the second fluid composition, or both comprise a pH between 2 and 4.

[0530] Embodiment 12. The method of any one of Embodiments 1-11, wherein the first fluid composition, the second fluid composition, or both comprise a pH between 5 and 7.

[0531] Embodiment 13. The method of any one of Embodiments 1-12, wherein the first fluid composition, the second fluid composition, or both comprise a pH between 9 and 10.

[0532] Embodiment 14. A method according to any of Embodiments 1-13, wherein the first fluid composition, the second fluid composition, or both comprise a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0533] Embodiment 15. A method according to any of Embodiments 1-14, wherein the first fluid composition, the second fluid composition, or both comprise a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0534] Embodiment 16. The method of any one of Embodiments 1-15, further comprising diluting the sample to prepare the first composition, the second composition, or both.

[0535] Embodiment 17. A method according to embodiment 16, wherein the diluting comprises adding a buffer.

[0536] Embodiment 18. A method according to embodiment 17, wherein the buffer comprises a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0537] Embodiment 19. The method of embodiment 17 or 18, wherein the buffer comprises a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0538] Embodiment 20. A method according to any one of embodiments 17-19, wherein the sample and the buffer comprise different pH.

[0539] Embodiment 21. A method according to any of embodiments 16-20, wherein the sample contains at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biological molecules.

[0540] Embodiment 22. A method according to any one of embodiments 16-21, wherein the sample contains at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules per mL of the sample.

[0541] Embodiment 23. A method according to any one of embodiments 16-22, wherein the sample comprises biomolecules from at most about 1000, 100, 10 or 1 cell.

[0542] Embodiment 24. A method according to any one of embodiments 16-23, wherein the sample contains at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 microliters.

[0543] Embodiment 25. A method according to any one of embodiments 16-24, wherein the sample comprises a complex biological sample.

[0544] Embodiment 26. A method according to embodiment 25, wherein the complex biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, duct lavage, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration samples, tissue homogenates, lymph fluid, cell culture samples or any combination thereof.

[0545] Embodiment 27. A method according to embodiment 26, wherein the biological sample comprises plasma.

[0546] Embodiment 28. A method according to any one of Embodiments 1-27, wherein the selective enrichment of the first plurality of biomolecule types comprises contacting the first fluid composition with a first surface to adsorb the first plurality of biomolecule types onto the first surface.

[0547] Embodiment 29. A method according to any one of Embodiments 1-28, wherein the selective enrichment of the second plurality of biomolecule types comprises contacting the second fluid composition with a second surface to adsorb the second plurality of biomolecule types onto the second surface.

[0548] Embodiment 30. A method according to any one of Embodiments 1-29, wherein the first surface, the second surface, or both comprise particles.

[0549] Embodiment 31. A method according to Embodiment 30, wherein the particles are porous particles.

[0550] Embodiment 32. The method according to embodiment 30 or 31, wherein the particles are microparticles.

[0551] Embodiment 33. A method according to embodiment 30 or 31, wherein the particles are nanoparticles.

[0552] Embodiment 34. A method according to any one of Embodiments 30-33, wherein the particles comprise a paramagnetic material.

[0553] Embodiment 35. A method according to Embodiment 34, wherein the paramagnetic material is a superparamagnetic material.

[0554] Embodiment 36. A method according to Embodiment 34 or 35, wherein the paramagnetic material comprises iron oxide.

[0555] Embodiment 37. A method according to any one of Embodiments 1-36, wherein the selective enrichment of the first plurality of biomolecule types comprises contacting the first fluid composition with a first plurality of surface types to adsorb the first plurality of biomolecule types onto the first plurality of surface types.

[0556] Embodiment 38. A method according to any one of Embodiments 1-37, wherein the selective enrichment of the second plurality of biomolecule types comprises contacting the second fluid composition with a second plurality of surface types to adsorb the second plurality of biomolecule types onto the second plurality of surface types.

[0557] Embodiment 39. A method according to any one of Embodiments 1-38, wherein the first plurality of surface types, the second plurality of surface types, or both comprise the same charge sign.

[0558] Embodiment 40. A method according to any one of Embodiments 1-39, wherein the first plurality of surface types, the second plurality of surface types, or both comprise the same sign of zeta potential.

[0559] Embodiment 41. A method according to any one of Embodiments 1-40, wherein the first plurality of surface types, the second plurality of surface types, or both comprise acidic functional groups.

[0560] Embodiment 42. The method of any one of Embodiments 1-41, wherein the acidic functional group comprises a Bronsted-Lowry acid or a Lewis acid functional group.

[0561] Embodiment 43. A method according to any one of Embodiments 1-42, wherein the first plurality of surface types, the second plurality of surface types, or both comprise carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boric acid groups, boric ester groups, dihydrocarbyl boronic acid groups, dihydrocarbyl boronic ester groups, silica groups, silanol groups, polymers, or any combination thereof.

[0562] Embodiment 44. The method of any one of Embodiments 1-43, further comprising, prior to (c), washing the first plurality of biomolecule types with a first wash composition and washing the second plurality of biomolecule types with a second wash composition.

[0563] Embodiment 45. The method of Embodiment 44, wherein the first fluid composition and the first washing composition comprise at least one common strength physical property.

[0564] Embodiment 46. The method of Embodiment 44 or 45, wherein the first fluid composition and the first wash composition comprise at least one common solvent.

[0565] Embodiment 47. The method of any one of Embodiments 44-46, wherein the first fluid composition and the first wash composition comprise at least one different intensive physical property.

[0566] Embodiment 48. The method of any one of Embodiments 44-47, wherein the second fluid composition and the second wash composition comprise at least one common strength physical property.

[0567] Embodiment 49. The method of any one of Embodiments 44-48, wherein the second fluid composition and the second wash composition comprise at least one common solvent.

[0568] Embodiment 50. The method of any one of Embodiments 44-49, wherein the second fluid composition and the second wash composition comprise at least one different intensive physical property.

[0569] Embodiment 51. The method of any one of Embodiments 44-50, wherein the first washing composition and the second washing composition comprise at least one common strength physical property.

[0570] Embodiment 52. The method of any one of Embodiments 44-51, wherein the first cleaning composition and the second cleaning composition are the same.

[0571] Embodiment 53. The method of any one of Embodiments 44-52, wherein the first washing composition and the second washing composition comprise at least one different strength physical property.

[0572] Embodiment 54. The method of any one of Embodiments 44-53, wherein the first washing composition releases the first plurality of biomolecule types adsorbed on the first surface from the first surface.

[0573] Embodiment 55. The method of Embodiment 54, further comprising purifying the first plurality of biomolecule types to produce a first purified composition.

[0574] Embodiment 56. The method of Embodiment 55, wherein the purifying comprises drying the plurality of biomolecule types to remove the first wash composition.

[0575] Embodiment 57. The method of Embodiment 56, further comprising reconstituting the first purified composition with a first reconstitution composition to produce a first reconstituted composition.

[0576] Embodiment 58. The method of any one of Embodiments 44-57, wherein the second washing composition releases the second plurality of biomolecule types deposited on the second surface from the second surface.

[0577] Embodiment 59. The method of Embodiment 58, further comprising purifying the second plurality of biomolecule types to produce a second purified composition.

[0578] Embodiment 60. The method of Embodiment 59, wherein the purifying comprises drying the second plurality of biomolecule types to remove the second wash composition.

[0579] Embodiment 61. The method of Embodiment 60, further comprising reconstituting the second purified composition with a second reconstitution composition to produce a second reconstituted composition.

[0580] Embodiment 62. A method according to any one of embodiments 1-61, wherein the downstream determination comprises mass spectrometry.

[0581] Embodiment 63. A method according to embodiment 62, wherein the mass spectrometry comprises LC-MS / MS.

[0582] Embodiment 64. A method according to any one of Embodiments 1-63, wherein the downstream assay comprises protein sequencing.

[0583] Embodiment 65. A method according to any one of embodiments 1-64, wherein the downstream assay comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with an antibody pair capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the antibody pair comprises complementary single-stranded nucleic acid sequences attached thereto such that when the antibody pair binds to the at least one biomolecule type, the complementary nucleic acids hybridize to form a double-stranded nucleic acid, wherein the double-stranded nucleic acid is configured to form a binding complex with a polymerase and a plurality of nucleotides, nucleosides, nucleotide analogs and / or nucleoside analogs to perform an amplification reaction to produce a detectable signal.

[0584] Embodiment 66. A method according to any one of Embodiments 1-65, wherein the downstream assay comprises contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with one or more aptamers capable of binding to at least one of the first plurality of biomolecule types, the second plurality of biomolecule types, or both, wherein the one or more aptamers are coupled to a surface via a cleavable linker.

[0585] Embodiment 67. A method according to Embodiment 66, wherein the surface is a particle surface.

[0586] Embodiment 68. A method according to Embodiment 67, wherein the cleavable linker is photocleavable.

[0587] Embodiment 69. A method according to embodiment 68, further comprising contacting the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a macromolecular competitor, wherein the macromolecular competitor is configured to reduce the dissociation of a complex comprising the one or more aptamers and the first plurality of biomolecule types, the second plurality of biomolecule types, or both in a fluid composition.

[0588] Embodiment 70. A method according to embodiment 69, wherein the macromolecular competitor is further configured to bind to a biomolecule type different from the first plurality of biomolecule types, the second plurality of biomolecule types, or both.

[0589] Embodiment 71. A method according to embodiment 70, wherein the macromolecular competitor is a polyanionic macromolecule.

[0590] Embodiment 72. A method according to any one of embodiments 1-71, wherein the downstream determination comprises nucleic acid sequencing.

[0591] Embodiment 73. The method of any one of embodiments 1-72, wherein (a) and (b) are performed on one machine.

[0592] Embodiment 74. The method of any one of Embodiments 1-72, wherein (a) and (b) are performed on different machines.

[0593] Embodiment 75. The method of any one of Embodiments 1-74, wherein (a) and (b) are performed in parallel.

[0594] Embodiment 76. The method of any one of Embodiments 1-74, wherein (a) and (b) are performed serially.

[0595] Embodiment 77. A method according to any of Embodiments 1-76, wherein the first fluid composition and the second fluid composition are part of the same sample.

[0596] Embodiment 78. A method according to any one of Embodiments 1-76, wherein the first fluid composition and the second fluid composition are from different samples.

[0597] Embodiment 79. A method according to any one of Embodiments 1-78, wherein the first fluid composition and the second fluid composition are selectively enriched in parallel.

[0598] Embodiment 80. A method according to any one of Embodiments 1-78, wherein the first fluid composition and the second fluid composition are selectively enriched in series.

[0599] Embodiment 81. A method according to any of embodiments 1-80, wherein the selective enrichment is performed at a rate of enriching at least 100, 1000, 10,000, 100,000 or 1,000,000 biomolecules per hour.

[0600] Embodiment 82. A method according to any of embodiments 1-81, wherein the selective enrichment is performed at a rate of enriching at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biological molecules per hour.

[0601] Embodiment 83. A method according to any one of Embodiments 1-82, wherein the first fluid composition and the second fluid composition are contacted in parallel.

[0602] Embodiment 84. A method according to any one of Embodiments 1-82, wherein the first fluid composition and the second fluid composition are contacted serially.

[0603] Embodiment 85. The method of any one of Embodiments 1-84, wherein the contacting is performed at a rate of depositing at least 100, 1000, 10,000, 100,000, or 1,000,000 biomolecules per hour.

[0604] Embodiment 86. A method according to any of Embodiments 1-85, wherein the contacting is performed at a rate of depositing at least 1, 10, 100, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 μg of the biomolecule per hour.

[0605] Embodiment 87. The method of any one of Embodiments 1-86, wherein the washing is performed on the first fluid composition and the second fluid composition in parallel.

[0606] Embodiment 88. The method of any one of Embodiments 1-86, wherein the washing is performed serially on the first fluid composition and the second fluid composition.

[0607] Embodiment 89. The method of any one of Embodiments 1-88, wherein the washing is performed at a rate of at least 100, 1000, 10,000, 100,000, or 1,000,000 biomolecules per hour.

[0608] Embodiment 90. A method according to any one of embodiments 1-89, wherein the washing is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biomolecules per hour.

[0609] Embodiment 91. A method according to any one of Embodiments 1-90, wherein the purification is performed on the first fluid composition and the second fluid composition in parallel.

[0610] Embodiment 92. A method according to any one of Embodiments 1-90, wherein the purification is performed on the first fluid composition and the second fluid composition in series.

[0611] Embodiment 93. The method of any one of Embodiments 1-92, wherein the purifying is performed at a rate of at least 100, 1000, 10,000, 100,000, or 1,000,000 purified per hour.

[0612] Embodiment 94. The method of any one of Embodiments 1-92, wherein the purification is performed at a rate of purifying at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 μg of the biomolecule per hour.

[0613] Embodiment 95. A method according to any one of Embodiments 1-94, wherein the reconstruction of the first fluid composition and the second fluid composition is performed in parallel.

[0614] Embodiment 96. A method according to any one of Embodiments 1-94, wherein the reconstruction of the first fluid composition and the second fluid composition is performed serially.

[0615] Embodiment 97. A method according to any one of embodiments 1-96, wherein the reconstitution is performed at a rate of at least 100, 1000, 10000, 100000 or 1000000 reconstitutions per hour.

[0616] Embodiment 98. A method according to any of embodiments 1-97, wherein the reconstitution is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biomolecule per hour.

[0617] Embodiment 99. A method according to any one of Embodiments 1-98, wherein the downstream determination is performed on the first fluid composition and the second fluid composition in parallel.

[0618] Embodiment 100. A method according to any one of Embodiments 1-98, wherein the downstream determination is performed serially on the first fluid composition and the second fluid composition.

[0619] Embodiment 101. A method according to any one of embodiments 1-100, wherein the downstream determination is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0620] Embodiment 102. A method according to any of Embodiments 1-101, wherein the downstream assay is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of biomolecule per hour.

[0621] Embodiment 103. A method according to any of Embodiments 1-102, wherein the downstream assay is performed at a rate of identifying at least 100, 1000, 10,000, 100,000 or 1,000,000 biomolecules per hour.

[0622] Embodiment 104. A method according to any one of Embodiments 1-103, wherein the downstream assay is performed at a rate of identifying at least 100, 1000, 10,000, 100,000 or 1,000,000 protein groups per hour.

[0623] Embodiment 105. A method according to any of Embodiments 1-104, wherein the method is performed on the first fluid composition and the second fluid composition in parallel.

[0624] Embodiment 106. A method according to any of Embodiments 1-105, wherein the method is performed serially on the first fluid composition and the second fluid composition.

[0625] Embodiment 107. A method according to any of embodiments 1-106, wherein the method is performed at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 samples per hour.

[0626] Embodiment 108. A method according to any of Embodiments 1-107, wherein the method is performed at a rate of measuring at least 1, 10, 100, 1000, 10,000, 100,000, 1,000,000, or 10,000,000 μg of a biomolecule per hour.

[0627] Embodiment 109. A method according to any of embodiments 1-108, wherein the method is performed at a rate of identifying at least 100, 1000, 10,000, 100,000 or 1,000,000 biomolecules per hour.

[0628] Embodiment 110. The method of any one of Embodiments 1-109, wherein the method is performed at a rate of identifying at least 100, 1000, 10000, 100000, or 1000000 protein groups per hour.

[0629] Embodiment 111. A method according to any one of Embodiments 1-110, wherein when the first fluid composition and the second fluid composition are HeLa cell extracts, the downstream assay identifies at least about 100, 1000, 10000, 100000 or 1000000 biomolecules.

[0630] Embodiment 112. A method according to any one of embodiments 1-111, wherein when the first fluid composition and the second fluid composition are subjected to the downstream assay prior to the selective enrichment in (a) and (b), the first plurality of biomolecule types and the second plurality of biomolecule types together comprise at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 dynamic ranges.

[0631] Embodiment 113. A method according to any of Embodiments 1-112, wherein the first plurality of biomolecule types is depleted of at least a first biomolecule and enriched for a second biomolecule, wherein the first biomolecule is more abundant than the second biomolecule in the first fluid composition.

[0632] Embodiment 114. A method according to any of Embodiments 1-113, wherein the second plurality of biomolecule types is depleted of at least a first biomolecule and enriched for a second biomolecule, wherein the first biomolecule is more abundant than the second biomolecule in the second fluid composition.

[0633] Embodiment 115. A method according to any one of Embodiments 1-114, wherein the downstream assay has a first likelihood of identifying a low-abundance biomolecule in the first plurality of biomolecule types or the second plurality of biomolecule types that is greater than a second likelihood of the downstream assay identifying a low-abundance biomolecule in the first fluid composition or the second fluid composition, wherein the low-abundance biomolecule comprises less than about 1, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1000, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 ... -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 %.

[0634] Embodiment 116. A method according to embodiment 115, wherein the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 times.

[0635] Embodiment 117. A method according to any of embodiments 1-116, wherein the downstream identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1%.

[0636] Embodiment 118. A method according to any of embodiments 1-117, wherein the downstream identifies one or more biomolecules of the first plurality of biomolecule types, the second plurality of biomolecule types, or both with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1%.

[0637] Embodiment 119. A method according to embodiment 117 or 118, wherein 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, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 or 100000 peptides.

[0638] Embodiment 120. A method according to any of embodiments 117-119, wherein 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.

[0639] Embodiment 121. A method according to any 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.

[0640] Embodiment 122. A method according to any 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.

[0641] Embodiment 123. A method according to any 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.

[0642] Embodiment 124. The method of 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 wash composition.

[0643] Embodiment 125. The method of 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 wash composition.

[0644] Embodiment 126. The method of 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 reconstituted composition.

[0645] Embodiment 127. The method of any one of Embodiments 61-126, further comprising identifying a biological state associated with the second composition based at least in part on one or more physical properties of the second reconstituted composition.

[0646] Embodiment 128. A method according to any one of Embodiments 1-127, wherein the first surface is disposed in a first lyophilized composition, the first lyophilized composition comprising (i) at least one nanoparticle, and (ii) one or more modulators.

[0647] Embodiment 129. A method according to any one of Embodiments 1-128, wherein the second surface is disposed in a second lyophilized composition, the second lyophilized composition comprising (i) at least one nanoparticle, and (ii) one or more modulators.

[0648] Embodiment 130. The method of embodiment 128 or 129, wherein the one or more modifiers comprise a pH modifier, an ionic strength modifier, a viscosity modifier, or any combination thereof.

[0649] Embodiment 131. The method of any one of Embodiments 1-130, wherein the first pH and the second pH are different.

[0650] Embodiment 132. The method of any one of Embodiments 1-130, wherein the first pH and the second pH are the same.

[0651] Embodiment 133. A method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in a second fluid composition; and (c) performing downstream assays on biomolecule types within the first plurality of biomolecule types and the second plurality of biomolecule types, wherein the first fluid composition and the second fluid composition are different such that a first infinite dilution limiting solvation enthalpy or solvation free energy of the first biomolecule within the first plurality of biomolecule types is different when the first biomolecule is in the first fluid composition than when the biomolecule is in the second fluid composition.

[0652] Embodiment 134. A kit comprising: (a) a first reagent comprising a first pH; (b) a second reagent comprising a second pH; (c) a first surface configured to selectively enrich a first plurality of biomolecule types in a first fluid composition comprising the first surface and the first reagent; and (d) a second surface configured to selectively enrich a second plurality of biomolecule types in a second fluid composition comprising the second surface and the second reagent, wherein the first fluid composition comprises the first reagent and the second fluid composition comprises the second reagent, wherein the first pH and the second pH are different or the same.

[0653] Embodiment 135. A composition comprising: a suspension comprising: i. a first particle comprising (i) a first paramagnetic portion and (ii) a first surface chemical; ii. a second particle comprising (i) a second paramagnetic portion and (ii) a second surface chemical, wherein the second surface chemical is different from the first surface chemical; and iii. a plurality of biological molecules adsorbed on the first particle and the second particle.

[0654] Embodiment 136. A composition according to Embodiment 135, wherein the first particles and the second particles comprise the same charge sign.

[0655] Embodiment 137. A composition according to Embodiment 135 or 136, wherein the first particles and the second particles comprise the same sign of zeta potential.

[0656] Embodiment 138. A composition according to embodiment 136 or 137, wherein the same sign is a negative sign.

[0657] Embodiment 139. A composition according to any of Embodiments 135-138, wherein the first surface chemical, the second surface chemical, or both comprise acidic functional groups.

[0658] Embodiment 140. The composition of Embodiment 139, wherein the acidic functional groups comprise Bronsted-Lowry acid or Lewis acid functional groups.

[0659] Embodiment 141. A composition according to any of Embodiments 135-140, wherein the first surface chemical, the second surface chemical, or both comprise carboxylate groups, acrylate groups, methacrylate groups, acetal groups, hemiacetal groups, hemiketal groups, sulfonic acid groups, sulfinic acid groups, thiocarboxylic acid groups, phosphonic acid groups, phosphate groups, phosphodiester groups, boric acid groups, boric ester groups, dihydrocarbyl boronic acid groups, dihydrocarbyl boronic ester groups, silica groups, silanol groups, polymers, or any combination thereof.

[0660] Embodiment 142. The composition of any one of Embodiments 135-141, wherein the suspension is stable for at least about 1, 5, 10, 15, 30, or 60 minutes.

[0661] Embodiment 143. A composition according to any of Embodiments 135-142, wherein the time constant for destabilization of the suspension is at least about 1, 5, 10, 15, 30 or 60 minutes.

[0662] Embodiment 144. A composition according to any of Embodiments 135-143, wherein the average aggregation number of the first particles and the second particles in the suspension is at most about 1000, 100 or 10.

[0663] Embodiment 145. A composition according to any of Embodiments 135-144, wherein the suspension comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts of the first particles to about 1 part of the second particles.

[0664] Embodiment 146. A composition according to any of Embodiments 135-145, wherein the suspension comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts of the first particles to about 1 part of the second particles.

[0665] Embodiment 147. The composition of any one of Embodiments 135-146, wherein the suspension comprises about 15 parts of the first particles to about 6 parts of the second particles.

[0666] Embodiment 148. The composition of embodiment 147, wherein the parts are parts by weight, parts by volume, or parts by surface area.

[0667] Embodiment 149. The composition of any one of Embodiments 135-148, wherein the suspension comprises at least about 3, 4, 5, 6, 7, 8, 9, or 10 different particles.

[0668] Embodiment 150. The composition of any one of Embodiments 135-149, wherein the first particle, the second particle, or both are nanoparticles.

[0669] Embodiment 151. The composition of any of Embodiments 135-150, wherein the first particle, the second particle, or both are microparticles.

[0670] Embodiment 152. The composition of any one of Embodiments 135-151, wherein the first particle, the second particle, or both are porous.

[0671] Embodiment 153. A composition according to any of Embodiments 135-152, wherein the first size of the first particle is at least about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the second size of the second particle.

[0672] Embodiment 154. A composition according to embodiment 153, wherein the first size of the first particle is at most about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the second size of the second particle.

[0673] Embodiment 155. A composition according to any of Embodiments 153-154, wherein the first dimension is a first diameter and the second dimension is a second diameter.

[0674] Embodiment 156. A composition according to any of Embodiments 153-155, wherein the first size is a first average size and the second size is a second average size.

[0675] Embodiment 157. A composition according to embodiment 156, wherein the first average size and the second average size are average sizes or median sizes.

[0676] Embodiment 158. The composition of any one of Embodiments 135-152, 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.

[0677] Embodiment 159. A composition according to any of Embodiments 135-158, wherein the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01 or 0.001 nanograms of biomolecules.

[0678] Embodiment 160. The composition of any one of Embodiments 135-159, wherein the plurality of biomolecules comprises at most about 1000, 100, 10, 1, 0.1, 0.01, or 0.001 nanograms of biomolecules / mL.

[0679] Embodiment 161. A composition according to any of Embodiments 135-160, wherein the plurality of biomolecules comprises biomolecules from at most about 1000, 100, 10, or 1 cell.

[0680] Embodiment 162. A method comprising: assaying a plurality of biomolecules in a composition according to any one of Embodiments 135-161 to identify one or more biomolecules in the plurality of biomolecules.

[0681] Embodiment 163. A method according to embodiment 162, wherein the determination comprises performing mass spectrometry.

[0682] Embodiment 164. A method according to embodiment 163, wherein the mass spectrometry comprises LC-MS / MS.

[0683] Embodiment 165. A method according to Embodiment 162, wherein the determination comprises performing protein sequencing.

[0684] Embodiment 166. A method according to any one of embodiments 162-165, wherein the determination comprises contacting the multiple biomolecules with an antibody pair capable of binding to at least one biomolecule among the multiple biomolecules, wherein the antibody pair comprises a complementary single-stranded nucleic acid sequence attached thereto, such that when the antibody pair binds to the at least one biomolecule, the complementary nucleic acids hybridize to form a double-stranded nucleic acid, wherein the double-stranded nucleic acid is configured to form a binding complex with a polymerase and multiple nucleotides, nucleosides, nucleotide analogs and / or nucleoside analogs to perform an amplification reaction to produce a detectable signal.

[0685] Embodiment 167. A method according to any one of Embodiments 162-166, wherein the assay comprises contacting the plurality of biomolecules with one or more aptamers capable of binding to at least one of the plurality of biomolecules, wherein the one or more aptamers are coupled to a surface via a cleavable linker.

[0686] Embodiment 168. A method according to Embodiment 167, wherein the surface is a particle surface.

[0687] Embodiment 169. A method according to Embodiment 167 or 168, wherein the cleavable linker is photocleavable.

[0688] Embodiment 170. A method according to any one of Embodiments 162-169, further comprising contacting the plurality of biomolecules with a macromolecular competitor configured to reduce the dissociation of a complex comprising the one or more aptamers and the biomarker in a fluid composition.

[0689] Embodiment 171. A method according to embodiment 170, wherein the macromolecular competitor is further configured to bind to a biological molecule different from the biomarker.

[0690] Embodiment 172. A method according to embodiment 171, wherein the macromolecular competitor is a polyanionic macromolecule.

[0691] Embodiment 173. A method according to any one of Embodiments 167-172, wherein the determination comprises performing nucleic acid sequencing.

[0692] Embodiment 174. A method according to any one of embodiments 162-173, wherein the sample comprises the plurality of biomolecules.

[0693] Embodiment 175. A method according to embodiment 174, wherein the sample is a complex biological sample.

[0694] Embodiment 176. A method according to embodiment 175, wherein the complex biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, duct lavage, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration samples, tissue homogenates, lymph fluid, cell culture samples or any combination thereof.

[0695] Embodiment 177. A method according to Embodiment 176, wherein the biological sample comprises plasma.

[0696] Embodiment 178. A method according to any one of Embodiments 162-177, wherein the plurality of biomolecules comprises one or more polyamino acids.

[0697] Embodiment 179. A method according to any one of Embodiments 162-178, wherein the determination is performed at a rate of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 100 samples per hour.

[0698] Embodiment 180. A method according to any one of Embodiments 162-179, wherein the measuring is performed at a rate of at least 1, 10, 100, 1000, 10000, 100000, 1000000 or 10000000 μg of the biomolecule per hour.

[0699] Embodiment 181. A method according to any of Embodiments 162-180, wherein the assay is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000 or 1000000 biomolecules per hour.

[0700] Embodiment 182. A method according to any one of Embodiments 162-181, wherein the determining is performed at a rate of identifying at least 1, 10, 100, 1000, 10000, 100000 or 1000000 protein groups per hour.

[0701] Embodiment 183. The method of any one of Embodiments 162-182, wherein when the suspension comprises a HeLa cell extract, the assay identifies at least about 100, 1000, 10,000, or 100,000 biomolecules.

[0702] Embodiment 184. A method according to any one of Embodiments 162-183, wherein when the multiple biological molecules in the biological sample are measured in the absence of the first particle and the second particle, the multiple biological molecules include at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 dynamic ranges.

[0703] Embodiment 185. A method according to any one of Embodiments 162-184, wherein the determining has a first likelihood of identifying a low-abundance biomolecule in the plurality of biomolecules that is greater than a second likelihood of the determining identifying the low-abundance biomolecule in the biological sample in the absence of the first particle and the second particle, wherein the low-abundance biomolecule comprises less than about 1, 10, or 20% of the first set of biomolecules or the second set of biomolecules by mass. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 %.

[0704] Embodiment 186. A method according to any one of embodiments 162-185, wherein the first likelihood is at least 2, 5, 10, 10 greater than the second likelihood. 2 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 times.

[0705] Embodiment 187. A method according to any of Embodiments 162-186, wherein the assay identifies one or more of the plurality of biomolecules with a coefficient of variation of at most about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1%.

[0706] Embodiment 188. A method according to any of Embodiments 162-187, wherein the assay identifies one or more of the plurality of biomolecules with a coefficient of variation of at least about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1%.

[0707] Embodiment 189. A system for analyzing multiple biological samples, comprising: (a) multiple partitions, comprising a first partition and a second partition; (b) multiple reagent reservoirs, comprising a first reagent comprising a first pH and a second reagent comprising a second pH, wherein the first pH and the second pH are different or the same; (c) multiple substrates, comprising a first substrate comprising a first surface chemical and a second substrate comprising a second surface chemical; (d) one or more transfer devices, which are operably connected to the multiple partitions, the multiple reagent reservoirs and the multiple substrates; and (e) a computer, which comprises at least one processor and instructions executable by the at least one processor to perform the following steps: i. using the one or more transfer devices to generate a first fluid composition in the first partition, the first fluid composition comprising the first substrate, the first reagent and a first plurality of biomolecules, wherein the first plurality of biomolecules are adsorbed on the first substrate; ii. using the one or more transfer devices to generate a second fluid composition in the second partition, the second fluid composition comprising the second substrate, the second reagent and a second plurality of biomolecules, wherein the second plurality of biomolecules are adsorbed on the second substrate; and iii. using the one or more transfer devices to prepare the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry.

[0708] Embodiment 190. A system for analyzing multiple biological samples, comprising: (a) partitions; (b) a reagent reservoir containing reagents; (c) multiple substrates, comprising a first substrate containing a first surface chemical and a second substrate containing a second surface chemical, wherein the first surface chemical and the second surface chemical are different; (d) one or more transfer devices, which are operably connected to the partitions, the reagent reservoir and the multiple substrates; and (e) a computer, which comprises at least one processor and instructions executable by the at least one processor to perform the following steps: i. using the one or more transfer devices to generate a fluid composition in the partition, the fluid composition comprising the multiple substrates, the reagents and a plurality of biological molecules, wherein the multiple biological molecules are adsorbed on the substrates; and ii. using the one or more transfer devices to prepare the multiple biological molecules for mass spectrometry.

[0709] Embodiment 191. A method comprising: (a) forming a first suspension comprising a first plurality of particles using a first portion of a biological sample, wherein the first plurality of particles comprises first particles and second particles, wherein the first particles have a first functionalized surface chemistry that is different from a second functionalized surface chemistry of the second particles, and wherein the first particles and the second particles both have a negative charge; (b) forming a second suspension comprising a second plurality of particles using a second portion of the biological sample, wherein the second plurality of particles comprises third particles and fourth particles, wherein the third particles have a third functionalized surface chemistry that is different from a fourth functionalized surface chemistry of the fourth particles, and wherein the third particles and the fourth particles both have a positive charge; (c) enriching biological molecules that have been adsorbed to the first plurality of particles and the second plurality of particles; and (d) performing downstream assays on the enriched biological molecules.

[0710] Embodiment 192. A method according to Embodiment 191, wherein the first suspension and the second suspension each have a pH between 9 and 10.

[0711] Embodiment 193. A method according to Embodiment 192, wherein the first suspension and the second suspension each contain Tris buffer.

[0712] Embodiment 194. A method according to Embodiment 191, wherein the first suspension has a pH between 9 and 10.

[0713] Embodiment 195. A method according to Embodiment 194, wherein the first suspension comprises Tris buffer.

[0714] Embodiment 196. A method according to Embodiment 191, 194 or 195, wherein the second suspension has a pH between 6 and 8.

[0715] Embodiment 197. A method according to any of Embodiments 191-196, wherein the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0716] Embodiment 198. A method according to any of Embodiments 191-197, wherein the third particle is a magnetic particle and comprises an outer layer having an amine surface functionalization, and the fourth particle is a magnetic particle and comprises an outer layer having an amine surface functionalization.

[0717] Embodiment 199. The method of any one of Embodiments 191-198, wherein the outer layer of the third particle comprises a polymer.

[0718] Embodiment 200. A method according to any of Embodiments 191-199, wherein 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.

[0719] Embodiment 201. A method according to any of Embodiments 191-200, wherein 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.

[0720] Embodiment 202. A method according to any one of Embodiments 191-201, 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.

[0721] Embodiment 203. A method according to any one of Embodiments 191-202, 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.

[0722] Embodiment 204. A method according to any one of Embodiments 191-203, wherein each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle.

[0723] Embodiment 205. A kit comprising: (a) a first composition comprising: a first particle and a second particle, wherein the first particle has a first functionalized surface chemistry different from the second functionalized surface chemistry of the second particle, and wherein the first particle and the second particle both have a negative charge, wherein 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 chemistry different from the fourth functionalized surface chemistry of the fourth particle, and wherein the third particle and the fourth particle both have a positive charge, wherein the second composition is a solid.

[0724] Embodiment 206. A kit according to Embodiment 205, wherein the kit further comprises a buffer having a pH between 9 and 10.

[0725] Embodiment 207. A kit according to Embodiment 205, wherein the kit further comprises a Tris buffer having a pH between 9 and 10.

[0726] Embodiment 208. The kit of Embodiment 205, wherein both the first composition and the second composition are lyophilized.

[0727] Embodiment 209. A kit according to any one of Embodiments 205-208, wherein the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

[0728] Embodiment 210. A kit according to any one of Embodiments 205-209, wherein the third particle is a magnetic particle and comprises an outer layer having an amine surface functionalization, and the fourth particle is a magnetic particle and comprises an outer layer having an amine surface functionalization.

[0729] Embodiment 211. The kit of any one of Embodiments 205-210, wherein the outer layer of the third particle comprises a polymer.

[0730] Embodiment 212. A kit according to any one of Embodiments 205-211, wherein 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.

[0731] Embodiment 213. A kit according to any one of Embodiments 205-212, wherein 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.

[0732] Embodiment 214. A kit according to any one of Embodiments 205-213, 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.

[0733] Embodiment 215. A kit according to any one of Embodiments 205-214, 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.

[0734] Embodiment 216. The kit of any one of Embodiments 205-215, wherein each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle.

[0735] Embodiment 217. A suspension comprising: (a) a first particle comprising an outer layer having a negatively charged surface functionalization; (b) a biological sample; and (c) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein the biological molecule is adsorbed to the outer layer of the first particle.

[0736] Embodiment 218. A suspension comprising: (a) first particles comprising a first outer layer having a negatively charged surface functionalization; (b) second particles comprising a second outer layer having a negatively charged surface functionalization, 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 the biological molecules are adsorbed to the first outer layer of the first particles and the second outer layer of the particles.

[0737] Embodiment 219. A suspension according to Embodiment 218, wherein the first outer layer comprises a silanol and the second outer layer comprises a carboxylate.

[0738] Embodiment 220. A suspension comprising: (a) first particles comprising a first outer layer having a positively charged surface functionalization; (b) second particles comprising a second outer layer having a positively charged surface functionalization, 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 the biological molecules are adsorbed to the first outer layer of the first particles and the second outer layer of the particles.

[0739] Embodiment 221. A suspension according to Embodiment 220, wherein the first outer layer and the second outer layer comprise amine groups.

[0740] Embodiment 222. A suspension according to any one of Embodiments 217-221, wherein the biomolecules comprise at least 100 different peptides or proteins.

[0741] Embodiment 223. The suspension of any one of Embodiments 217-222, wherein the first particles and optionally the second particles are magnetic.

[0742] Embodiment 224. The suspension of any one of Embodiments 217-223, wherein the buffer is Tris buffer.

[0743] Embodiment 225. A method comprising: (a) 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, wherein biomolecules from the biological sample are adsorbed onto the one or more surfaces; (b) enriching the biomolecules that have been adsorbed to the surfaces; (c) measuring the enriched biomolecules to identify one or more of the enriched biomolecules from the biological sample.

[0744] Embodiment 226. A method according to Embodiment 225, wherein the buffer is configured to maintain the pH of the composition between 9 and 10.

[0745] Embodiment 227. A method according to Embodiment 225, wherein the buffer is configured to maintain the pH of the composition at about 9.5.

[0746] Embodiment 228. A method according to any one of Embodiments 225-227, wherein the buffer is Tris buffer.

[0747] Embodiment 229. A method according to any one of embodiments 225-207, wherein the biological sample comprises plasma, serum, urine, cerebrospinal fluid, synovial fluid, tears, saliva, whole blood, milk, nipple aspirate, duct lavage, vaginal secretions, nasal fluid, ear fluid, gastric juice, pancreatic juice, trabecular fluid, lung lavage fluid, sweat, sulcular fluid, semen, prostatic fluid, sputum, feces, bronchial lavage fluid, fluid from a swab, bronchial aspirate, fluidized solids, fine needle aspiration sample, tissue homogenate, lymph fluid, cell culture sample, or any combination thereof.

[0748] Embodiment 230. A method according to any one of Embodiments 225-229, wherein the biological sample is plasma or serum.

[0749] Embodiment 231. A method according to any one of Embodiments 225-225, wherein the biological sample is a cell culture medium.

[0750] Embodiment 232. A method according to any one of Embodiments 225-231, wherein the composition comprises no more than 60% by volume of the biological sample.

[0751] Embodiment 233. A method according to any one of Embodiments 225-232, wherein the composition comprises no more than 25% by volume of the biological sample.

[0752] Embodiment 234. A method according to any one of Embodiments 225-233, wherein the composition comprises no more than 15% by volume of the biological sample.

[0753] Embodiment 235. A method according to any one of Embodiments 225-234, wherein the composition comprises at least 1% by volume of the biological sample.

[0754] Embodiment 236. A method according to any one of Embodiments 225-235, wherein the composition comprises at least 4% by volume of the biological sample.

[0755] Embodiment 237. A method according to any one of Embodiments 225-236, wherein the composition comprises at least 10% by volume of the biological sample.

[0756] Embodiment 238. A method according to any one of Embodiments 225-237, wherein the composition comprises at least 20% by volume of the biological sample.

[0757] Embodiment 239. A method according to any one of Embodiments 225-238, wherein the assay comprises SDS-PAGE, gel-based separation techniques, immunoassay, ELISA, high performance liquid chromatography, mass spectrometry, Edman degradation, or immunoaffinity techniques.

[0758] Embodiment 240. A method according to any one of Embodiments 225-238, wherein the determining comprises mass spectrometry.

[0759] Embodiment 241. A method according to any one of Embodiments 225-238, wherein the determination comprises LC-MS / MS.

[0760] Embodiment 242. A method according to any one of Embodiments 225-241, wherein the determination includes quantifying the biological molecule.

[0761] Embodiment 243. A method according to any one of Embodiments 225-242, wherein the determining comprises identifying one or more proteins in the biological sample.

[0762] Embodiment 244. A method according to embodiment 243, wherein at least 100 different proteins are identified in the biological sample.

[0763] Embodiment 245. A method according to embodiment 243, wherein at least 250 different proteins are identified in the biological sample.

[0764] Embodiment 246. A method according to embodiment 243, wherein at least 500 different proteins are identified in the biological sample.

[0765] Embodiment 247. A method according to any one of Embodiments 225-246, wherein the one or more surfaces are contained on particles dispersed in the composition during the incubation.

[0766] Embodiment 248. A method according to Embodiment 247, wherein the particles are nanoparticles.

[0767] Embodiment 249. A method according to Embodiment 248, wherein the nanoparticles have an average diameter of less than 500 nanometers.

[0768] Embodiment 250. A method according to Embodiment 247, wherein the particles are microparticles.

[0769] Embodiment 251. A method according to any one of Embodiments 247-250, wherein the particles are magnetic.

[0770] Embodiment 252. A method according to any one of Embodiments 247-251, wherein the particle comprises an outer layer of poly(dimethylaminopropylmethacrylamide) (PDMAPMA).

[0771] Embodiment 253. A method according to any one of Embodiments 247-251, wherein the particle comprises an outer layer of silica.

[0772] Embodiment 254. The method of any one of Embodiments 247-251, wherein the particle comprises a carboxylate-functionalized outer layer.

[0773] Embodiment 255. A method according to any one of Embodiments 247-251, wherein the particle comprises an amine-functionalized outer layer.

[0774] Embodiment 256. A method according to any one of Embodiments 247-251, wherein the particle comprises a polymer outer layer.

[0775] Embodiment 257. A method according to any one of Embodiments 247-251, wherein the particle comprises an outer layer of polyacrylamide.

[0776] Embodiment 258. The method of any one of Embodiments 247-251, wherein the particles comprise an acrylate polymer.

[0777] Embodiment 259. A method according to any one of Embodiments 225-258, further comprising: (a) incubating a second composition in contact with one or more second surfaces, wherein the second composition comprises the biological sample, wherein biological molecules from the biological sample are adsorbed onto the one or more second surfaces; (b) enriching the biological molecules that have been adsorbed to the one or more second surfaces; (c) measuring the enriched biological molecules to identify one or more of the enriched biological molecules from the one or more second surfaces in the biological sample.

[0778] Embodiment 260. The method of Embodiment 259, wherein the second composition has a pH between 5 and 9.

[0779] Embodiment 261. A method according to Embodiment 259, wherein the second composition has a pH between 6 and 8.

[0780] Embodiment 262. The method of Embodiment 259, wherein the second composition has a pH between 8 and 11.

[0781] Embodiment 263. A method according to Embodiment 259, wherein the second composition has a pH between 9 and 10.

[0782] Embodiment 264. The method of Embodiment 259, wherein the second composition does not comprise a buffer.

[0783] Embodiment 265. A method according to Embodiment 259, wherein the second composition comprises a buffer different from the buffer configured to maintain the pH of the composition between 8 and 11.

[0784] Embodiment 266. A method according to Embodiment 259, wherein the second composition comprises the same buffer as the composition.

[0785] Embodiment 267. A method according to Embodiment 259, wherein both the first composition and the second composition comprise a Tris buffer having a pH between 9 and 10.

[0786] Embodiment 268. The method of any one of Embodiments 259-267, wherein said incubating of said composition and said incubating of said second composition occur simultaneously.

[0787] Embodiment 269. A method according to any one of Embodiments 259-268, wherein the assay identifies the enriched biomolecules with a coefficient of variation of at most 30%.

[0788] Embodiment 270. A method according to any one of Embodiments 259 to 268, wherein the assay identifies the enriched biomolecules with a coefficient of variation of at most 25%.

[0789] Embodiment 271. A method according to any one of Embodiments 259-249, wherein the method further comprises digesting the protein adsorbed to the first surface before determining the biomolecule.

[0790] Embodiment 272. A method according to any one of Embodiments 259-271, wherein the method further comprises treating the protein within the biomolecule corona with a reducing agent prior to measuring the biomolecule.

[0791] Embodiment 273. A method according to any one of Embodiments 259-272, wherein the method further comprises treating a protein within the biomolecule corona with an alkylating agent prior to assaying the biomolecule.

[0792] Embodiment 274. A suspension comprising: (a) first particles having a first functionalized surface chemist...

Claims

1. A method comprising: (a) forming a first suspension comprising a first plurality of particles with a first portion of a biological sample, wherein the first plurality of particles comprises first particles and second particles, wherein the first particles have a first functionalized surface chemistry that is different from a second functionalized surface chemistry of the second particles, and wherein the first particles and the second particles both have a negative charge; (b) forming a second suspension comprising a second plurality of particles with a second portion of the biological sample, wherein the second plurality of particles comprises third particles and fourth particles, wherein the third particles have a third functionalized surface chemistry that is different from a fourth functionalized surface chemistry of the fourth particles, and wherein the third particles and the fourth particles both have a positive charge; (c) enriching biomolecules that have adsorbed to the first plurality of particles and the second plurality of particles; and (d) performing downstream assays on the enriched biomolecules. 2 . The method according to claim 1 , wherein the first suspension and the second suspension each have a pH between 9 and 10.

3. The method of claim 2, wherein the first composition, the first suspension, and the second suspension each comprise a Tris buffer. The method of claim 1 , wherein the first composition has a pH between 9 and 10. The method of claim 4 , wherein the first composition comprises Tris buffer.

6. The method of claim 5, wherein the second composition has a pH between 6 and 8.

7. The method of claim 1, wherein the first particle is a magnetic particle and comprises an outer layer having a silanol surface functionalization, and the second particle is a magnetic particle and comprises an outer layer having a carboxylate surface functionalization.

8. The method of claim 1, wherein the third particle is a magnetic particle and comprises an outer layer having an amine surface functionalization, and the fourth particle is a magnetic particle and comprises an outer layer having an amine surface functionalization.

9. The method of claim 1, wherein the outer layer of the third particle comprises a polymer.

10. The method of claim 1, wherein 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.

11. The method of claim 1, wherein 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.

12. The method of claim 1, wherein a ratio of an average diameter of the first particles to an average diameter of the second particles is from about 3:2 to about 2:

3.

13. The method of claim 1, wherein a ratio of an average diameter of the third particles to an average diameter of the fourth particles is from about 3:2 to about 2:

3.

14. The method of claim 1, wherein each of the first particle, the second particle, the third particle, and the fourth particle is a superparamagnetic iron oxide nanoparticle.

15. A kit comprising: (a) a first composition comprising: a first particle and a second particle, wherein the first particle has a first functionalized surface chemistry that is different from a second functionalized surface chemistry of the second particle, and wherein the first particle and the second particle both have a negative charge, wherein 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 chemistry that is different from a fourth functionalized surface chemistry of the fourth particle, and wherein both the third particle and the fourth particle have a positive charge, wherein the second composition is a solid.

16. The kit of claim 15, wherein the first composition and the second composition are both lyophilized.

17. A suspension comprising: (a) a first particle comprising an outer layer having a negatively charged surface functionalization; (b) biological samples; and (c) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein the biomolecule is adsorbed to the outer layer of the first particle.

18. A suspension comprising: (a) a first particle comprising a first outer layer having a negatively charged surface functionalization; (b) a second particle comprising a second outer layer having a negatively charged surface functionalization, wherein the second outer layer is different from the first outer layer; (c) biological samples; and (d) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein the biomolecule is adsorbed to the first outer layer of the first particle and the second outer layer of the particle.

19. A suspension comprising: (a) a first particle comprising a first outer layer having a positively charged surface functionalization; (b) a second particle comprising a second outer layer having a positively charged surface functionalization, wherein the second outer layer is different from the first outer layer; (c) biological samples; and (d) a buffer configured to maintain the pH of the suspension between 9 and 10, wherein the biomolecule is adsorbed to the first outer layer of the first particle and the second outer layer of the particle.

20. A method comprising: (a) 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, wherein biomolecules from the biological sample are adsorbed onto the one or more surfaces; (b) enriching the biomolecules that have been adsorbed to the surface; (c) assaying the enriched biomolecules to identify one or more of the enriched biomolecules from the biological sample.

21. A suspension comprising: (a) a first particle having a first functionalized surface chemistry; (b) a second particle having a second functionalized surface chemistry, wherein the first functionalized surface chemistry is different from the second functionalized surface chemistry of the second particle, and wherein the first particle and the second particle both have a negative charge; (c) biological samples; and (d) a buffer configured to maintain a pH of about 8 to about 11.

22. A suspension comprising: (a) a first particle having a first functionalized surface chemistry; (b) a second particle having a second functionalized surface chemistry, 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 positive charge; and (c) Biological samples.

23. A method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in the second fluid composition; and (c) performing a downstream assay on a biomolecule type from the first plurality of biomolecule types and the second plurality of biomolecule types, Wherein the first fluid composition comprises a first pH and the second fluid composition comprises a second pH, wherein the first pH and the second pH are different or the same.

24. A method comprising: (a) selectively enriching a first plurality of biomolecule types in a first fluid composition; (b) selectively enriching a second plurality of biomolecule types in the second fluid composition; and (c) performing a downstream assay on a biomolecule type from the first plurality of biomolecule types and the second plurality of biomolecule types, wherein the first fluid composition and the second fluid composition are different such that a first infinite dilution limiting solvation enthalpy or solvation free energy of the first biomolecule in the first plurality of biomolecule types is different when the first biomolecule is in the first fluid composition than when the biomolecule is in the second fluid composition.

25. A kit comprising: (a) comprising a first reagent at a first pH; (b) comprising a second reagent at a second pH; (c) a first surface configured to selectively enrich a first plurality of biomolecule types in a first fluid composition comprising the first surface and the first reagent; and (d) a second surface configured to selectively enrich a second plurality of biomolecule types in a second fluid composition comprising the second surface and the second reagent, Wherein the first fluid composition comprises the first reagent and the second fluid composition comprises the second reagent, wherein the first pH and the second pH are different or the same.

26. A composition comprising: A suspension comprising: i. a first particle comprising (i) a first paramagnetic portion and (ii) a first surface chemical; ii. a second particle comprising (i) a second paramagnetic portion and (ii) a second surface chemical, wherein the second surface chemical is different from the first surface chemical; and iii. A plurality of biomolecules adsorbed on the first particle and the second particle.

27. A system for analyzing a plurality of biological samples, comprising: (a) a plurality of partitions, comprising a first partition and a second partition; (b) a plurality of reagent reservoirs comprising a first reagent comprising a first pH and a second reagent comprising a second pH, wherein the first pH and the second pH are different or the same; (c) a plurality of substrates comprising a first substrate comprising a first surface chemistry and a second substrate comprising a second surface chemistry; (d) one or more transfer devices operably connected to the plurality of partitions, the plurality of reagent reservoirs, and the plurality of substrates; and (e) a computer comprising at least one processor and instructions executable by the at least one processor to perform steps comprising: i. using the one or more transfer devices to generate a first fluid composition in the first partition, the first fluid composition comprising the first substrate, the first reagent, and a first plurality of biomolecules, wherein the first plurality of biomolecules are adsorbed on the first substrate; ii. using the one or more transfer devices to generate a second fluid composition in the second partition, the second fluid composition comprising the second substrate, the second reagent, and a second plurality of biomolecules, wherein the second plurality of biomolecules are adsorbed on the second substrate; and iii. preparing the first plurality of biomolecules and the second plurality of biomolecules for mass spectrometry using the one or more transfer devices.

28. A system for analyzing a plurality of biological samples, comprising: (a) Zoning; (b) a reagent reservoir containing a reagent; (c) a plurality of substrates comprising a first substrate comprising a first surface chemistry and a second substrate comprising a second surface chemistry, wherein the first surface chemistry and the second surface chemistry are different; (d) one or more transfer devices operably connected to the partitions, the reagent reservoirs, and the plurality of substrates; and (e) a computer comprising at least one processor and instructions executable by the at least one processor to perform steps comprising: i. Using the one or more transfer devices to create a fluid combination in the partition The fluid composition comprises the plurality of substrates, the reagents, and a plurality of biomolecules, wherein the plurality of biomolecules are adsorbed on the substrates; and ii. preparing the plurality of biomolecules for mass spectrometry using the one or more transfer devices.

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