Systems and methods for assaying the secretome

Abstract

In some aspects, the present disclosure provides a method for identifying one or more biomolecules. In some embodiments, the method comprises generating a set of biomolecules by incubating cells in a supplemented medium under conditions sufficient for the cells to generate the set of biomolecules. In some embodiments, the method comprises contacting at least a portion of the supplemented medium with one or more surfaces to adsorb the set of biomolecules. In some embodiments, the method comprises removing the one or more surfaces and the set of biomolecules from at least a portion of the supplemented medium to create a separated sample. In some embodiments, the method comprises releasing the set of biomolecules from the one or more surfaces in the separated sample. In some embodiments, the method comprises detecting at least a subset of the set of biomolecules, thereby identifying one or more biomolecules.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,191, filed June 14, 2022; U.S. Provisional Patent Application No. 63 / 348,674, filed June 3, 2022; U.S. Provisional Patent Application No. 63 / 323,976, filed March 25, 2022; and U.S. Provisional Patent Application No. 63 / 299,859, filed January 14, 2022; each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] background The conditioned medium of different cell cultures can be used for various in vitro biological applications.In some cases, the in vitro biological application can include characterizing the biomolecules secreted from cell cultures in conditioned medium under different conditions.In some cases, characterizing the biomolecules secreted in in vitro biological applications can help to gain deeper insight into the biological functions observed. Summary of the Invention [Means for solving the problem]

[0003] overview In some aspects, the disclosure provides a method for identifying one or more biomolecules, the method comprising: (a) providing a supplemented medium; (b) generating a set of biomolecules by incubating cells in the supplemented medium under conditions sufficient for the cells to generate the set of biomolecules; (c) contacting at least a portion of the supplemented medium with one or more surfaces, whereby the set of biomolecules is adsorbed; (d) removing the one or more surfaces and the set of biomolecules from at least a portion of the supplemented medium, whereby a separated sample is created; (e) releasing the set of biomolecules from the one or more surfaces in the separated sample; and (f) detecting at least a subset of the set of biomolecules, thereby identifying one or more biomolecules.

[0004] In some embodiments, after the contacting step of (b), the set of biomolecules, when adsorbed onto one or more surfaces, comprises a reduced dynamic range compared to the original dynamic range of the set of biomolecules in the supplemented medium.

[0005] In some embodiments, in (a), the step of incubating the cells in the supplemented medium is carried out for less than about 5 seconds, less than 5 minutes, less than 5 hours, or less than 5 days.

[0006] In some embodiments, the cells are infected or mutated.

[0007] In some embodiments, the cell is a viable cell, including a cancer cell, an epithelial cell, a bone cell, a muscle cell, an adipocyte, a tissue cell, a senescent cell, a pluripotent cell, a stem cell, or a neural cell.

[0008] In some embodiments, the cells are cancer cells that are biopsied cells from a patient.

[0009] In some embodiments, the particle comprises a surface.

[0010] In some embodiments, the particles are nanoparticles.

[0011] In some embodiments, the contacting step of (b) further comprises contacting the biological sample with a second surface to adsorb the second plurality of biomolecules onto the second surface.

[0012] In some embodiments, the detecting step comprises mass spectrometry.

[0013] In some embodiments, the conditions sufficient for the cells to produce the set of biomolecules in the supplemented medium include a predetermined temperature, a predetermined pressure, a predetermined flow regime, a predetermined solvent environment, or a combination thereof.

[0014] In some embodiments, the conditions are held constant.

[0015] In some embodiments, the supplemented medium comprises in part or in whole serum, plasma, cerebrospinal fluid (CSF), synovial fluid (SF), urine, tears, gingival crevicular fluid, semen, whole blood, milk, nipple aspirate, needle aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, pulmonary lavage, prostatic fluid, sputum, excreta, bronchial washings, fluid from swabs, bronchial aspirates, sweat, saliva, or any combination thereof.

[0016] In some embodiments, the supplemented medium comprises cell culture supernatant, co-culture secretome, exosomes, tissue or cell lysate, or any combination thereof.

[0017] In some embodiments, the supplemented medium comprises a synthetically supplemented medium.

[0018] In some embodiments, the set of biomolecules comprises one or more biomarkers, molecular signatures, secreted proteins, absorbed proteins, secretomes, exosomes, or any combination thereof.

[0019] In some embodiments, the method further comprises repeating (a)-(e) for a second supplemented medium comprising a second set of biomolecules, the second supplemented medium being generated by incubating the cells in the supplemented medium for different lengths of time.

[0020] In some embodiments, the providing step is performed by an automated fluidic system.

[0021] In some embodiments, the automated fluidic system comprises a microfluidic system.

[0022] In some embodiments, the automated fluid system provides replenishment medium at different times.

[0023] In some embodiments, the generating step comprises active secretion of at least a portion of the set of biomolecules.

[0024] In some embodiments, the generating step comprises passive release of at least a portion of the set of biomolecules.

[0025] In some embodiments, the generating step comprises release of exosomes or liposomes by the cells.

[0026] In some embodiments, the generating step comprises apoptosis of the cells.

[0027] In some embodiments, the generating step comprises necroptosis of the cells.

[0028] In some embodiments, a biomolecule of the set of biomolecules is a complex.

[0029] In some embodiments, a biomolecule of the set of biomolecules is a protein.

[0030] In some embodiments, a biomolecule of the set of biomolecules is a polypeptide.

[0031] In some embodiments, a biomolecule of the set of biomolecules is a nucleic acid.

[0032] In some embodiments, the cell is part of a plurality of cells.

[0033] In some embodiments, the plurality of cells are of the same type of cell.

[0034] In some embodiments, the cells are from a tissue sample, an organoid, an immortalized cell line, or any combination thereof.

[0035] In some embodiments, the cells are stem cells.

[0036] In some embodiments, conditions sufficient for cells to exchange the set of biomolecules with supplemented medium include one or more of the presence or absence of organic compounds, the presence or absence of inorganic compounds, the presence or absence of autocrine signaling molecules, the presence or absence of paracrine signaling molecules, the presence or absence of antigens, the presence or absence of one or more co-cultured cells, the presence or absence of radiation, the presence or absence of one or more toxins, the presence or absence of protein aggregates, the presence or absence of one or more proteins, the presence or absence of active viral particles, the presence or absence of inactivated viral particles, the presence or absence of applied heating or cooling, the presence or absence of applied mechanical stress, the presence or absence of an electrical stimulus, the presence or absence of a transposon, the presence or absence of an exosome, the presence or absence of a liposome, the presence or absence of a coated nucleic acid, the presence or absence of a shock, or any combination thereof.

[0037] In some embodiments, the conditions are varied over time.

[0038] In some embodiments, the contacting step of (b) is performed such that a portion of the supplemented medium contacts one or more surface regions of the one or more surfaces, wherein the one or more surface regions do not comprise a specific targeting moiety.

[0039] In some embodiments, the method further comprises determining, at least in part, based on the identification, one or more protein-protein interactions, biomarkers, molecular signatures, biomolecules absorbed by the cell, biomolecules secreted by the cell, biomolecules dissociated from the cell surface, biomolecules cleaved or released from the surface, macromolecular complexes budding, released, cleaved from the surface, biomolecules passively released by the cell, conventional and non-conventional released proteins, apoptotic release of biomolecules, necrotic released biomolecules, post-translational modifications, cell-cell interactions, cell-cell signaling, or any combination thereof.

[0040] In some embodiments, the biomolecule secreted by the one or more cells is a conventional secretion, a non-conventional secretion, a type 1 non-conventional secretion, or a type 2 non-conventional secretion.

[0041] In some embodiments, the set of biomolecules comprises a biomolecular assembly.

[0042] In some embodiments, the biomolecular assembly comprises a quaternary protein, a vesicle, or an exosome.

[0043] In some embodiments, the cell is a eukaryote or a prokaryote.

[0044] In some embodiments, the method further comprises a plurality of cells comprising the cells.

[0045] In some embodiments, the plurality of cells is contained within a tissue, an organoid, an organism, or multiple organisms.

[0046] In some embodiments, the plurality of cells includes at least a first cell of a first type and a second cell of a second type, such that the first cell exchanges one or more biomolecules of the set of biomolecules with the second cell.

[0047] In some embodiments, the first cell and the second cell are co-cultured.

[0048] In some embodiments, the first cells are included in a feeder culture for the second cells.

[0049] In some embodiments, the cells are derived from an immortalized cell line.

[0050] In some embodiments, the cell is a HeLa cell.

[0051] In some embodiments, the cells are stem cells.

[0052] In some embodiments, the cells are contained in a primary cell culture.

[0053] In some embodiments, the plurality of cells is disposed in a plurality of separate volumes, each volume comprising a different supplemented medium or incubation condition.

[0054] In some embodiments, the releasing step comprises the use of a protease.

[0055] In some embodiments, the one or more surfaces include at least two surfaces that comprise distinct physicochemical properties, such that the at least two surfaces adsorb different patterns of biomolecule abundance from a set of biomolecules.

[0056] In some embodiments, the method further comprises determining that the one or more biomolecules are produced by the cells and were not originally present in the supplemented medium.

[0057] In some embodiments, the supplemented medium comprises fetal bovine serum.

[0058] In some aspects, the disclosure provides a method for monitoring cellular activity, the method comprising: (a) incubating cells such that the cells produce a biological sample containing a plurality of biomolecules; (b) contacting the biological sample with a surface, where the plurality of biomolecules are adsorbed onto the surface; (c) separating the surface from the biological sample; (d) releasing at least a portion of the plurality of biomolecules on the surface; (e) detecting at least a portion of the plurality of biomolecules, thereby identifying the plurality of biomolecules; and (f) repeating (a)-(d) after a predetermined amount of time, thereby monitoring cellular activity.

[0059] In some embodiments, the cells generate the biological sample by any one of producing, releasing, adsorbing, digesting, or modifying a biomolecule of the plurality of biomolecules.

[0060] In some embodiments, the method further comprises altering incubation conditions for the cells based at least in part on the activity of the cells.

[0061] In some embodiments, the incubating step comprises exposing the cells to a supplemented medium, where the supplemented medium obscures detection of at least a portion of the plurality of biomolecules.

[0062] In some embodiments, the method further comprises analyzing the activity of the cells.

[0063] In some embodiments, at least a portion of the plurality of biomolecules comprises a dynamic range of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0064] In some embodiments, at least a portion of the plurality of biomolecules comprises at least about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 biomolecules.

[0065] In some embodiments, (b) comprises contacting the biological sample with a plurality of surfaces.

[0066] In some embodiments, the releasing step in (d) is carried out using a protease to enzymatically cleave the surface.

[0067] In some embodiments, the surface is disposed on a magnetic substrate, and the separating step in (c) is performed using a magnetic field to separate the magnetic substrate from the biological sample.

[0068] In some aspects, the disclosure provides a method for identifying low abundance biomolecules in a biological sample, the method comprising: (a) incubating cells in a predetermined environment such that the cells produce a biological sample; (b) contacting the biological sample with a surface, where a plurality of low abundance biomolecules in the biological sample are adsorbed onto the surface; (c) separating the surface from the biological sample; (d) releasing at least a portion of the plurality of low abundance biomolecules on the surface; and (e) detecting at least a portion of the plurality of low abundance biomolecules, thereby identifying the plurality of low abundance biomolecules.

[0069] In some embodiments, the method further comprises, prior to (c), adding a biomolecule to the biological sample, wherein the biomolecule reduces detectability of the plurality of low abundance biomolecules in the biological sample.

[0070] In some embodiments, the signal of the step of detecting low abundance biomolecules is about 7 orders of magnitude higher than the signal from direct digestion of the cells in the medium.

[0071] In some embodiments, the incubating step comprises exposing the cells to a supplemented medium, where the supplemented medium obscures detection of at least a portion of the plurality of low abundance biomolecules.

[0072] In some embodiments, the plurality of low abundance biomolecules comprises at least about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 biomolecules.

[0073] In some embodiments, (b) comprises contacting the biological sample with a plurality of surfaces.

[0074] In some embodiments, the releasing step in (d) is carried out using a protease to enzymatically cleave the surface.

[0075] In some embodiments, the surface is disposed on a magnetic substrate, and the separating step in (c) is performed using a magnetic field to separate the magnetic substrate from the biological sample.

[0076] In some aspects, the present disclosure provides an apparatus for assaying a biological sample, the apparatus comprising: a substrate comprising a surface; a cell culture chamber comprising cells; a loading unit operably coupled to the substrate and the cell culture chamber; and a computer readable medium comprising machine executable code that, when executed by a processor, implements a method comprising: (a) providing a controlled environment for cells in the cell culture chamber for a predetermined duration such that the cells produce a biological sample in the cell culture chamber; (b) transferring at least a portion of the biological sample from the cell culture chamber to the substrate using the loading unit, thereby contacting at least a portion with the surface to adsorb biomolecules in at least a portion of the biological sample onto the surface; and (c) assaying at least a portion of the biomolecules to detect biomolecules in the biological sample.

[0077] In some embodiments, the device comprises multiple cell culture chambers.

[0078] In some embodiments, a first cell culture chamber of the plurality of cell culture chambers is provided with a first controlled environment and a second cell culture chamber of the plurality of cell culture chambers is provided with a second controlled environment.

[0079] In some embodiments, the transferring step is performed using one or more fluid connections and one or more pumps included in the loading unit.

[0080] In some embodiments, the transferring step is performed using one or more pipettes and one or more pumps included in a loading unit.

[0081] In some aspects, the disclosure provides a method for identifying one or more biomolecules, the method comprising: (a) incubating cells under conditions sufficient for the cells to produce exosomes, the exosomes comprising a plurality of biomolecules; (b) contacting the exosomes with one or more surfaces, capturing at least a portion of the exosomes; (c) removing the one or more surfaces and at least a portion of the exosomes from the cells, creating a separated sample; (d) releasing at least a portion of the exosomes from the one or more surfaces in the separated sample; and (e) detecting at least a subset of the plurality of biomolecules in at least a portion of the exosomes, thereby identifying the one or more biomolecules.

[0082] In some aspects, the present disclosure provides a method for monitoring cellular activity comprising: (a) incubating cells such that the cells produce a biological sample comprising exosomes, the exosomes comprising a plurality of biomolecules; (b) contacting the biological sample with a surface, wherein at least a portion of the exosomes are captured on the surface; (c) releasing at least a portion of the exosomes from the surface; (d) detecting at least a portion of the plurality of biomolecules in at least a portion of the exosomes, thereby identifying the plurality of biomolecules; and (e) repeating (a)-(d) after a predetermined amount of time, thereby monitoring cellular activity.

[0083] In some aspects, the disclosure provides a method for identifying low abundance biomolecules in a biological sample, the method comprising: (a) incubating cells in an environment such that the cells produce a biological sample comprising exosomes, the exosomes comprising a plurality of low abundance biomolecules; (b) contacting the biological sample with a surface, wherein at least a portion of the exosomes in the biological sample are captured on the surface; (c) releasing at least a portion of the exosomes from the surface; and (d) detecting at least a portion of the plurality of low abundance biomolecules in at least a portion of the exosomes, thereby identifying the plurality of low abundance biomolecules.

[0084] In some aspects, the present disclosure provides an apparatus for assaying a biological sample, the apparatus comprising: a substrate comprising a surface; a cell culture chamber comprising cells; a loading unit operably coupled to the substrate and the cell culture chamber; and a computer readable medium comprising machine executable code that, when executed by a processor, implements a method comprising: (a) providing a controlled environment to cells in the cell culture chamber for a predetermined duration such that the cells produce a biological sample in the cell culture chamber comprising exosomes, the exosomes comprising one or more biomolecules; (b) transferring at least a portion of the biological sample from the cell culture chamber to the substrate using the loading unit, thereby contacting at least a portion with the surface to adsorb exosomes in at least a portion of the biological sample onto the surface; and (c) assaying the exosomes to detect one or more biomolecules in the biological sample.

[0085] In some aspects, the present disclosure provides a method of identifying a biomolecule comprising: (a) processing one or more exosomes to release a plurality of biomolecules in the one or more exosomes to an environment external to the one or more exosomes, wherein a subset of the biomolecules in the plurality of biomolecules comprises a first distribution of relative abundance in the one or more exosomes; (b) performing a composition refinement assay on the plurality of biomolecules to increase from the first distribution to a second distribution of relative abundance for the subset of biomolecules; and (c) assaying the plurality of biomolecules to identify the subset of biomolecules.

[0086] In some embodiments, the processing step is performed, at least in part, by adding a lysis buffer to the one or more exosomes.

[0087] In some embodiments, the processing step is performed, at least in part, by providing ultrasound energy to one or more exosomes.

[0088] In some embodiments, the processing step is performed, at least in part, by freezing and thawing the one or more exosomes.

[0089] In some embodiments, the processing step is performed, at least in part, by heating the one or more exosomes.

[0090] In some embodiments, the processing step is performed, at least in part, by shearing one or more exosomes.

[0091] In some embodiments, the processing step is carried out, at least in part, by disrupting one or more exosomes.

[0092] In some embodiments, the subset of biomolecules includes at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 unique biomolecules.

[0093] In some embodiments, the composition improvement assay includes contacting a plurality of biomolecules with one or more surfaces to adsorb a subset of the biomolecules to the one or more surfaces, such that when the subset of biomolecules is adsorbed to the one or more surfaces, the subset of biomolecules comprises a second distribution.

[0094] In some embodiments, the contacting increases the visibility of the subset of biomolecules in the assaying step of (c) when the subset of biomolecules are adsorbed to one or more surfaces compared to the visibility of the subset of biomolecules when the subset of biomolecules is in one or more exosomes, and the subset of biomolecules are low abundance biomolecules that comprise less than about 1 mass percent of the biomolecules in the one or more exosomes.

[0095] In some aspects, the present disclosure provides a method of identifying a biomolecule, the method comprising: (a) contacting a biological sample containing one or more exosomes with a plurality of particles to non-specifically bind a subset of the one or more exosomes onto the plurality of particles, the plurality of particles comprising distinct physicochemical properties, and the one or more exosomes comprising a plurality of biomolecules; (b) processing the one or more exosomes to release the plurality of biomolecules into an environment external to the one or more exosomes; and (c) assaying the plurality of biomolecules to identify at least a subset of the biomolecules in the plurality of biomolecules.

[0096] The processing step is carried out, at least in part, by adding a lysis buffer to the one or more exosomes.

[0097] In some embodiments, the processing step is performed, at least in part, by providing ultrasound energy to one or more exosomes.

[0098] In some embodiments, the processing step is performed, at least in part, by freezing and thawing the one or more exosomes.

[0099] In some embodiments, the processing step is performed, at least in part, by heating the one or more exosomes.

[0100] In some embodiments, the processing step is performed, at least in part, by shearing one or more exosomes.

[0101] In some embodiments, the processing step is carried out, at least in part, by disrupting one or more exosomes.

[0102] In some embodiments, the subset of biomolecules includes at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 unique biomolecules.

[0103] In some embodiments, the plurality of particles increases the visibility of the subset of biomolecules in the assaying step of (c) when the subset of biomolecules are adsorbed to the plurality of particles compared to the visibility of the subset of biomolecules when the subset of biomolecules is within one or more exosomes, and the subset of biomolecules are low abundance biomolecules that comprise less than about 1 mass percent of the plurality of biomolecules in the one or more exosomes.

[0104] In some aspects, the disclosure provides a method for characterizing a biological preparation comprising: contacting the biological preparation with one or more surfaces, where a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces, where the plurality of biomolecules includes product biomolecules and impurities; and assaying the plurality of biomolecules, where a difference between a composition of the plurality of biomolecules and a reference composition is determined, where the difference indicates the purity or activity of the biological preparation.

[0105] In some embodiments, the biological preparation comprises a cell culture.

[0106] In some embodiments, the plurality of biological molecules comprises a secretome or exosomes from a cell culture.

[0107] In some embodiments, the plurality of biological molecules comprises a portion of one or more cells of a cell culture.

[0108] In some embodiments, the cell culture comprises a host organism that produces a product biomolecule.

[0109] In some embodiments, the cell culture comprises a host organism that produces the impurity.

[0110] In some embodiments, the cell culture includes a contaminating organism that produces an impurity.

[0111] In some embodiments, the impurities include host cell proteins.

[0112] In some embodiments, the host cell protein comprises a protease, lipase, or isomerase.

[0113] In some embodiments, the host cell proteins include passively released proteins or actively released proteins.

[0114] In some embodiments, the host cell protein comprises a portion of a host organism.

[0115] In some embodiments, the moiety comprises a cell membrane, an organelle, or both.

[0116] In some embodiments, the cell culture comprises multiple host organism species or strains.

[0117] In some embodiments, the cell culture comprises multiple species or strains.

[0118] In some embodiments, the biological preparation comprises a plurality of impurities.

[0119] In some embodiments, the biological preparation comprises a plurality of product biomolecules.

[0120] In some embodiments, the host organism comprises a prokaryotic host organism or a eukaryotic host organism.

[0121] In some embodiments, prokaryotic host organisms include Escherichia coli, Streptomyces sp., acetic acid bacteria, lactic acid bacteria, thermophilic Bacillus sp., Clostridium thermocellus, Agrobacterium tumefaciens, Thermus aquaticus, Bacillus coagulans, Pseudomonas stutzeri, Acetobacter sp., Micrococcus sp., Haemophilus influenzae, or Leuconostoc mesenteroides.

[0122] In some embodiments, the eukaryotic host organism comprises yeast, a fungus, a HeLa cell, a stem cell, a cancer cell, a genetically modified cell, or algae.

[0123] In some embodiments, the genetically modified cell comprises an exogenous nucleic acid sequence encoding a product biomolecule.

[0124] In some embodiments, the impurity is a proteoform of a biomolecule of the plurality of biomolecules.

[0125] In some embodiments, the impurities are proteoforms of the product biomolecule.

[0126] In some embodiments, a proteoform comprises a splicing variant, an allelic variant, or a post-translationally modified variant.

[0127] In some embodiments, the post-translational modification variant is acylation, alkylation, prenylation, flavinylation, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, sumoylation, neddylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hypusine formation, beta-lysine addition, acetylation, formylation, methylation, Post-translational modifications include amidation, amide bond formation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester formation, phosphoramidate formation, adenylation, uridylylation, propionylation, pyroglutamate formation, glutathionylation, sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, glycosylation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, citrullination, deamidation, eliminylation, disulfide bond formation, proteolytic cleavage, isoaspartate formation, racemization, protein splicing, chaperone-mediated folding, or any combination thereof.

[0128] In some embodiments, the difference between the first log(water-octanol partition coefficient) for the impurity and the second log(water-octanol partition coefficient) for the product biomolecule is less than about 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0129] In some embodiments, the difference between the first log(water-octanol partition coefficient) for the impurity and the second log(water-octanol partition coefficient) for the product biomolecule is greater than about 2, 1.5, 1, 0.9, 0.8, 0.7, about 0.6, about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0130] In some embodiments, the impurities and the product biomolecules are hydrophobic.

[0131] In some embodiments, the impurities and the product biomolecules are hydrophilic.

[0132] In some embodiments, the difference between the first pKa of the impurity and the second pKa of the product biomolecule is at most 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0133] In some embodiments, the difference between the first pKa of the impurity and the second pKa of the product biomolecule is at least 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0134] In some embodiments, the impurity and the product biomolecule are stereoisomers of one another.

[0135] In some embodiments, the impurity and the product biomolecule comprise enantiomers of one another.

[0136] In some embodiments, the difference between the first mass to charge ratio of the impurity and the second mass to charge ratio of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons / e-.

[0137] In some embodiments, the difference between the first mass to charge ratio of the impurity and the second mass to charge ratio of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons / e-.

[0138] In some embodiments, the impurity comprises a first histidine tag and the product biomolecule comprises a second histidine tag.

[0139] In some embodiments, the first histidine tag and the second histidine tag comprise the same number of amino acids.

[0140] In some embodiments, the difference between the first molecular weight of the impurity and the second molecular weight of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons.

[0141] In some embodiments, the difference between the first molecular weight of the impurity and the second molecular weight of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons.

[0142] In some embodiments, the method further comprises purifying the impure biological preparation prior to (a) to produce a biological preparation, wherein the impure biological preparation and the biological preparation comprises an impurity.

[0143] In some embodiments, the purifying step increases the purity or activity of the biological preparation as compared to an impure biological preparation.

[0144] In some embodiments, the impurities are present in lower abundance than the product biomolecule.

[0145] In some embodiments, the impurities are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.

[0146] In some embodiments, an impurity is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.

[0147] In some embodiments, the plurality of biomolecules comprises a reduced dynamic range on the one or more surfaces compared to the dynamic range of the plurality of biomolecules in a biological preparation.

[0148] In some embodiments, the reduced dynamic range is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less dynamic range.

[0149] In some embodiments, the reduced dynamic range is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less dynamic range.

[0150] In some embodiments, the biological preparation comprises a drug or a metabolite thereof.

[0151] In some embodiments, the impurities include a drug.

[0152] In some embodiments, the impurity comprises a metabolic product thereof.

[0153] In some embodiments, the product biomolecule comprises a drug.

[0154] In some embodiments, the host cell protein comprises a drug.

[0155] In some embodiments, the drug comprises an antibody, a biotherapeutic, or a chemotherapeutic biomolecule.

[0156] In some embodiments, the drug comprises methamphetamine, isotretinoin, an antibiotic, an antiplatelet agent, dutasteride, an antithrombotic agent, insulin, hepatitis B immunoglobulin, growth hormone, tamsulosin, finasteride, acitretin, etretinate, or any combination thereof.

[0157] In some embodiments, the impurities are capable of reducing the activity of the product biomolecule, and the impurities are produced by the host organism or by contaminating organisms that are foreign to the host organism.

[0158] In some embodiments, the biological preparation comprises a vaccine.

[0159] In some embodiments, the product biomolecule comprises a pathogenic bacterial antigen, a viral antigen, or a derivative thereof.

[0160] In some embodiments, the product biomolecule comprises an immunity protein or a derivative thereof.

[0161] In some embodiments, the product biomolecule comprises an enzyme.

[0162] In some embodiments, the enzyme is configured to degrade synthetic polymers, degrade oil, or catalyze ethanol production.

[0163] In some embodiments, the biological preparation comprises blood, plasma, platelets, clotting factors, or any combination thereof.

[0164] In some embodiments, the impurity comprises a biomolecule produced by a pathogen.

[0165] In some embodiments, the pathogen comprises Hepatitis B virus, Hepatitis C virus, COVID-19, or HIV.

[0166] In some embodiments, the biological preparation comprises a human consumable product or a livestock consumable product.

[0167] In some embodiments, the human or livestock consumable product comprises chicken, beef, pork, vegetables, fungi, fermented products, or meat substitutes.

[0168] In some embodiments, the impurities include bacterial biomolecules.

[0169] In some embodiments, the method further comprises estimating a shelf life of the biological preparation based on the difference.

[0170] In some embodiments, the method further comprises determining the suitability of the biological preparation for human or livestock consumption based on the difference.

[0171] In some embodiments, the product for human consumption comprises a fermented product.

[0172] In some embodiments, the fermentation product includes ethanol, acetic acid, or lactic acid.

[0173] In some embodiments, the fermented product comprises beer or wine.

[0174] In some embodiments, the fermented product comprises vinegar.

[0175] In some embodiments, the fermented product comprises yogurt.

[0176] In some embodiments, impurities may increase the risk of a complication when the biological preparation is administered to a subject.

[0177] In some embodiments, the impurities are expected to reduce the activity / stability of the product biomolecule to a human subject when administered to or ingested by a human subject.

[0178] In some embodiments, the impurity may be harmful to a human subject when administered to or ingested by a human subject.

[0179] In some embodiments, a biological preparation includes a host organism or a contaminant organism that may be harmful to a human subject when administered to or ingested by a human subject.

[0180] In some embodiments, the impairing comprises an immune response, blood clotting, an allergic reaction, food poisoning, or any combination thereof.

[0181] In some embodiments, the difference comprises the difference between the level of activity of the biological preparation and a reference activity level for the reference composition.

[0182] In some embodiments, the level of activity is assayed by contacting a plurality of biomolecules on one or more surfaces in an activity assay.

[0183] In some embodiments, the activity assay involves in vitro cell culture.

[0184] In some embodiments, the activity assay comprises a substrate.

[0185] In some embodiments, the activity assay comprises an immunoaffinity assay.

[0186] In some embodiments, the activity assay comprises an avidity assay.

[0187] In some embodiments, the difference comprises a difference between the safety level of the biological preparation and a reference safety level for the reference composition.

[0188] In some embodiments, the level of safety is assayed by contacting a plurality of biomolecules on one or more surfaces in a safety assay.

[0189] In some embodiments, the safety level comprises an immunogenicity level and the reference safety level comprises a reference immunogenicity level.

[0190] In some embodiments, the level of immunogenicity is assayed using one or more human blood samples or derivatives thereof by contacting a plurality of biomolecules on one or more surfaces.

[0191] In some embodiments, the one or more human blood samples or derivatives thereof comprise one or more white blood cells.

[0192] In some embodiments, the difference comprises a detectable level of an impurity.

[0193] In some embodiments, the difference comprises a level of a product biomolecule below a reference level of the product biomolecule.

[0194] In some embodiments, the difference comprises the difference between an impurity level and a reference impurity level.

[0195] In some embodiments, the method further comprises purifying the biological preparation based on the difference.

[0196] In some embodiments, the purifying step is performed if the difference is greater than 1, 2, 3, 4, 5, or 6 times the standard deviation of the measured impurity level.

[0197] In some embodiments, the purifying step is performed if the difference is greater than 1, 2, 3, 4, 5, or 6 times the standard error of the measurement of the impurity level, where the standard error is based on at least N assays.

[0198] In some embodiments, the reference composition is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.9, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, 99.99 The product biomolecule comprises a product biomolecule with a purity of 9.98, 99.99, 99.99, 99.991, 99.992, 99.992, 99.993, 99.994, 99.995, 99.996, 99.997, 99.998, 99.999, 99.999, 99.9991, 99.9992, 99.9993, 99.9994, 99.9995, 99.9996, 99.9997, 99.9998, or 99.9999 percent.

[0199] In some embodiments, the reference composition contains impurities at a purity 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.9 percent.

[0200] In some embodiments, the reference composition contains 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, or 900 ppm of an impurity.

[0201] In some embodiments, the reference composition contains 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, or 900 ppb of an impurity.

[0202] In some embodiments, the percent purity is based on the product signal intensity determined from the assaying step.

[0203] In some embodiments, the difference is determined using a machine learning algorithm.

[0204] In some embodiments, the machine learning algorithm is configured to receive one or more features representative of a composition of a plurality of biomolecules and output a variance based on the composition.

[0205] In some embodiments, the machine learning algorithm is trained to learn a reference impurity level based on a first plurality of samples that contain a purity or activity above a predetermined threshold, a second plurality of samples that contain a purity or activity below a predetermined threshold, or both.

[0206] In some embodiments, the method further comprises using a machine learning algorithm to classify the biological preparation as comprising a purity or activity above a predetermined threshold or comprising a purity or activity below a predetermined threshold based on the composition of the plurality of biomolecules.

[0207] In some embodiments, the method further comprises monitoring the biological preparation by assaying a plurality of biomolecules multiple times to determine a plurality of differences in the multiple times.

[0208] In some embodiments, the method further comprises determining a drift in genetic structure of the host organism based on the plurality of differences.

[0209] In some embodiments, the method further comprises determining a drift in the population of cell cultures based on the plurality of differences.

[0210] In some embodiments, the method further comprises determining a contaminant in a workflow for producing the biological preparation based on the plurality of differences.

[0211] In some aspects, the disclosure provides a method for detecting an impurity in a biological preparation comprising the steps of contacting the biological preparation with one or more surfaces, wherein a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces; and assaying the plurality of biomolecules, wherein a biomolecular fingerprint of the biological preparation is detected, wherein the biomolecular fingerprint comprises a signature of the impurity in the plurality of biomolecules.

[0212] In some aspects, the disclosure provides an apparatus for characterizing a biological preparation, the apparatus including: a first chamber configured to hold a biological preparation, the biological preparation comprising a plurality of biomolecules, the plurality of biomolecules including product biomolecules and impurities; a second chamber comprising one or more surfaces; a loader operably coupled to the first chamber and the second chamber, the loader configured to transfer the biological preparation between the first chamber and the second chamber; and a computer-readable medium for measuring purity or activity of a biological preparation comprising machine-executable code that, when executed by a processor, implements a method comprising using the loader to contact the biological preparation from the first chamber with one or more surfaces in the second chamber, wherein the plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces.

[0213] In some embodiments, the method further comprises assaying the plurality of biomolecules to determine a difference between the composition of the plurality of biomolecules and a reference composition, the difference being indicative of the purity or activity of the biological preparation.

[0214] In some embodiments, the apparatus further comprises a first separator operably coupled to the second chamber, the first separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the biological preparation.

[0215] In some embodiments, the first separator comprises a magnet.

[0216] In some embodiments, the apparatus further includes a second separator operably coupled to the second chamber, the second separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the one or more surfaces.

[0217] In some embodiments, the apparatus further comprises one or more purifiers for purifying the biological preparation.

[0218] In some embodiments, the method further comprises purifying the biological preparation using one or more purifiers based on product quality.

[0219] In some embodiments, the one or more purifiers comprise a plurality of purifiers.

[0220] In some embodiments, the multiple purifiers are provided in an arrangement such that a biological preparation purified in one of the multiple purifiers is fed to another of the multiple purifiers.

[0221] In some embodiments, the processing conditions for the multiple refiners are based on the product quality.

[0222] In some embodiments, the method further comprises purifying the biological preparation using one or more purifiers based on the difference to improve the purity or activity of the biological preparation.

[0223] In some embodiments, the device further comprises a third chamber operably coupled to the loader, the chamber containing an in vitro cell culture or a human blood sample or derivatives thereof.

[0224] In some embodiments, the method further comprises authenticating or rejecting the biological preparation based on the differences.

[0225] In some embodiments, the device further comprises a mass spectrometer for performing an assay to determine the difference.

[0226] In some embodiments, the first chamber comprises an incubator.

[0227] In some embodiments, the first chamber includes a heater or cooler operably connected to an incubator.

[0228] In some embodiments, the method includes controlling the temperature of the first chamber using a heater or a cooler.

[0229] In some embodiments, the first chamber is pressurized.

[0230] In some embodiments, the device comprises a plurality of chambers including a first chamber, each chamber of the plurality of chambers operably connected to a loader and a second chamber, and each chamber of the plurality of chambers contains a portion of a biological preparation.

[0231] In some embodiments, each chamber of the plurality of chambers comprises a different species or strain of host organism configured to produce at least a portion of the biological preparation.

[0232] In some embodiments, the device further comprises one or more particles comprising one or more surfaces and one or more supports.

[0233] In some embodiments, the one or more supports comprise a paramagnetic material.

[0234] In some embodiments, the paramagnetic material comprises a superparamagnetic material.

[0235] In some embodiments, the one or more surfaces comprise multiple surface types.

[0236] In some embodiments, the one or more particles include a plurality of particles comprising a plurality of surface types.

[0237] In some embodiments, the one or more particles comprise one or more microparticles.

[0238] In some embodiments, the one or more particles comprise one or more nanoparticles.

[0239] In some embodiments, the one or more particles comprise one or more porous particles.

[0240] In some embodiments, the one or more surfaces are configured to reduce the dynamic range of the multiple biomolecules in a biological preparation when the multiple biomolecules are adsorbed to the one or more surfaces.

[0241] In some embodiments, the impurities are present in lower abundance than the product biomolecule.

[0242] In some embodiments, the impurities are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.

[0243] In some embodiments, an impurity is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.

[0244] In some embodiments, the dynamic range of the plurality of biomolecules is reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude.

[0245] In some embodiments, the reduced dynamic range is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less than the dynamic range.

[0246] In some aspects, the present disclosure provides an apparatus for detecting impurities in a biological preparation, the apparatus comprising: a plurality of chambers operably connected to each other, the plurality of chambers comprising one or more surfaces; one or more fluid transfer devices operably coupled to the plurality of chambers; and a computer readable medium for detecting impurities in a biological preparation comprising machine executable code that, when executed by a processor, implements a method comprising contacting a biological preparation with one or more surfaces using the one or more fluid transfer devices, wherein a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces.

[0247] In some embodiments, the method further comprises assaying the plurality of biomolecules to detect a biomolecular fingerprint of the biological preparation, wherein the biomolecular fingerprint comprises signatures of contaminating biomolecules in the plurality of biomolecules.

[0248] In some embodiments, the apparatus further comprises a first separator operably coupled to the plurality of chambers, the first separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the biological preparation.

[0249] In some embodiments, the first separator comprises a magnet.

[0250] In some embodiments, the apparatus further includes a second separator operably coupled to the second chamber, the second separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the one or more surfaces.

[0251] In some embodiments, the apparatus further comprises one or more purifiers for purifying the biological preparation.

[0252] In some embodiments, the method further comprises purifying the biological preparation using one or more purifiers based on the biomolecular fingerprint.

[0253] In some embodiments, the one or more purifiers comprise a plurality of purifiers.

[0254] In some embodiments, the multiple purifiers are provided in an arrangement such that a biological preparation purified in one of the multiple purifiers is fed to another of the multiple purifiers.

[0255] In some embodiments, the processing conditions of the multiple purifiers are based on biomolecular fingerprints.

[0256] In some embodiments, the method further comprises purifying the biological preparation using one or more purifiers to reduce the signature of contaminating biomolecules based on the biomolecular fingerprint.

[0257] In some embodiments, the device further comprises a third chamber operably coupled to the loader, the chamber containing an in vitro cell culture or a human blood sample or derivatives thereof.

[0258] In some embodiments, the method further comprises authenticating or rejecting the biological preparation based on the biomolecular fingerprint.

[0259] In some embodiments, the apparatus further comprises a mass spectrometer for performing an assay for determining the biomolecular fingerprint.

[0260] In some embodiments, the multiple chambers comprise an incubator.

[0261] In some embodiments, the multiple chambers include a heater or cooler operably connected to the incubator.

[0262] In some embodiments, the method includes controlling the temperature of the multiple chambers using a heater or a cooler.

[0263] In some embodiments, multiple chambers are pressurized.

[0264] In some embodiments, each chamber of the plurality of chambers comprises a different species or strain of host organism configured to produce at least a portion of the biological preparation.

[0265] In some embodiments, the device further comprises one or more particles comprising one or more surfaces and one or more supports.

[0266] In some embodiments, the one or more supports comprise a paramagnetic material.

[0267] In some embodiments, the paramagnetic material comprises a superparamagnetic material.

[0268] In some embodiments, one or more surfaces comprise multiple surface types.

[0269] In some embodiments, the one or more particles include a plurality of particles comprising multiple surface types.

[0270] In some embodiments, the one or more particles comprise one or more microparticles.

[0271] In some embodiments, the one or more particles comprise one or more nanoparticles.

[0272] In some embodiments, the one or more particles comprise one or more porous particles.

[0273] In some embodiments, the one or more surfaces are configured to reduce the dynamic range of the multiple biomolecules in a biological preparation when the multiple biomolecules are adsorbed to the one or more surfaces.

[0274] In some embodiments, a contaminating biomolecule is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant in terms of count, mass, or mass spectrometry signal intensity than another biomolecule in the plurality of biomolecules.

[0275] In some embodiments, a contaminating biomolecule is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant in terms of counts, mass, or mass spectrometry signal intensity than another biomolecule in the plurality of biomolecules.

[0276] In some embodiments, the dynamic range of the plurality of biomolecules is reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude.

[0277] In some aspects, the disclosure provides a computer-implemented method for generating a quality metric for a biological preparation, the method comprising: (a) receiving a plurality of mass spectrometry datasets for a plurality of polyamino acids in the biological preparation, the plurality of polyamino acids comprising at least one product biomolecule and a plurality of impurities; (b) generating a plurality of polyamino acid identifications and a plurality of polyamino acid abundances for the plurality of polyamino acids based on the plurality of mass spectrometry datasets; and (c) processing the plurality of polyamino acid identifications to output a quality metric for the biological preparation.

[0278] In some embodiments, the abundance of the plurality of polyamino acids indicates the relative abundance between the plurality of polyamino acids.

[0279] In some embodiments, the plurality of polyamino acid identifications comprises at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, or 50000 polyamino acid identifications.

[0280] In some embodiments, the plurality of polyamino acids comprises a dynamic range of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 in a biological preparation.

[0281] In some embodiments, the plurality of polyamino acid abundances comprises a dynamic range of less than about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0282] In some embodiments, the plurality of impurities comprises a proteoform of a polyamino acid of the plurality of polyamino acids.

[0283] In some embodiments, a proteoform comprises a splicing variant, an allelic variant, or a post-translationally modified variant.

[0284] In some embodiments, the post-translational modification variant is acylation, alkylation, prenylation, flavinylation, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, sumoylation, neddylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hypusine formation, beta-lysine addition, acetylation, formylation, methylation, acetyl ... Post-translational modifications include amidation, amide bond formation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester formation, phosphoramidate formation, adenylation, uridylylation, propionylation, pyroglutamimate formation, glutathionylation, sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, glycosylation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, citrullination, deamidation, eliminylation, disulfide bond formation, proteolytic cleavage, isoaspartate formation, racemization, protein splicing, chaperone-mediated folding, or any combination thereof.

[0285] In some embodiments, the processing step comprises using a machine learning algorithm to score the biological preparation based on the plurality of polyamino acid identifications and the plurality of polyamino acid abundances to generate a quality metric.

[0286] In some embodiments, the machine learning algorithm is trained using a first set of samples above a predetermined quality metric threshold, a second set of samples below the determined quality metric threshold, or both.

[0287] In some embodiments, the processing step is based on a difference between the plurality of polyamino acid identifications and the plurality of polyamino acid abundances and the reference plurality of polyamino acid identifications and the reference plurality of polyamino acid abundances.

[0288] In some embodiments, the quality metric comprises a purity metric, an activity metric, a safety metric, or any combination thereof.

[0289] In some aspects, the disclosure provides a computer-implemented system including a digital processing device comprising at least one processor, an operating system configured to execute executable instructions, a memory, and a computer program comprising instructions executable by the digital processing device to receive a plurality of mass spectrometry datasets from a plurality of biological preparations; process the plurality of mass spectrometry datasets in real time to generate a plurality of quality metrics for the plurality of biological preparations; and provide process control instructions to a manufacturing process to produce the plurality of biological preparations.

[0290] In some embodiments, the process control instructions include authenticating or rejecting one or more biological preparations in the plurality of biological preparations.

[0291] In some embodiments, the process control instructions include effecting a change in one or more processing conditions of the manufacturing process.

[0292] In some embodiments, the processing step is performed using one or more cloud computing nodes.

[0293] In some embodiments, the processing step is carried out in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30, 60, 120, 180, 240, 300, or 360 minutes. In some embodiments, the plurality of biological preparations comprises a plurality of impurities. [Brief description of the drawings]

[0294] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0295] [Figure 1-1] 1A-1E illustrate a method for assaying the secretome using a composition refinement assay, according to some embodiments. [Figure 1-2] 1A-1E illustrate a method for assaying the secretome using a composition refinement assay, according to some embodiments. [Figure 1-3] 1A-1E illustrate a method for assaying the secretome using a composition refinement assay, according to some embodiments.

[0296] [Diagram 2] 2A-2D illustrate a method for assaying the secretome using a composition refinement assay, according to some embodiments.

[0297] [Figure 3-1] 3A-3E illustrate an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments. [Figure 3-2] 3A-3E illustrate an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments. [Figure 3-3] 3A-3E illustrate an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments. [Diagram 3-4] 3A-3E illustrate an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments. [Figure 3-5]3A-3E illustrate an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments.

[0298] [Figure 4] FIG. 4 illustrates multiple compartments, according to some embodiments.

[0299] [Diagram 5] FIG. 5 illustrates multiple compartments, according to some embodiments.

[0300] [Figure 6] FIG. 6 illustrates a transfer unit, according to some embodiments.

[0301] [Figure 7] FIG. 7 illustrates a transfer unit, according to some embodiments.

[0302] [Figure 8-1] FIG. 8 illustrates multiple transfer units, according to some embodiments. [Figure 8-2] FIG. 8 illustrates multiple transfer units, according to some embodiments.

[0303] [Figure 9] FIG. 9 illustrates a method for assaying the secretome using a composition refinement assay to determine a biological state, according to some embodiments.

[0304] [Figure 10] FIG. 10 illustrates an apparatus according to some embodiments.

[0305] [Figure 11] FIG. 11 illustrates multiple transfer units, according to some embodiments.

[0306] [Figure 12-1] FIG. 12 illustrates an apparatus and its components, according to some embodiments. [Figure 12-2] FIG. 12 illustrates an apparatus and its components, according to some embodiments. [Figure 12-3] FIG. 12 illustrates an apparatus and its components, according to some embodiments.

[0307] [Figure 13] FIG. 13 illustrates an apparatus and its components, according to some embodiments.

[0308] [Figure 14] FIG. 14 illustrates components of a cell culture medium, according to some embodiments.

[0309] [Figure 15] FIG. 15 shows a method for assaying the secretome using a composition refinement assay, according to some embodiments.

[0310] [Figure 16] 16A-16B show the peptide masses and protein group numbers, respectively, identified from the secretome, according to some embodiments.

[0311] [Figure 17] 17A-17B show the number of peptide and protein groups, respectively, identified by mass spectrometry experiments, according to some embodiments.

[0312] [Figure 18] FIG. 18 shows the number of protein groups identified exclusively in the improved secretome composition or direct digestion, according to some embodiments, as well as the number of protein groups identified in both experiments.

[0313] [Figure 19] 19A-19B show spectral counts versus count ranks of proteins identified in improved secretome composition and direct digestion, respectively, according to some embodiments.

[0314] [Figure 20] FIG. 20 shows the number of protein groups that were exclusively or mutually identified using some particles, according to some embodiments, compared to direct digestion.

[0315] [Figure 21] 21A-21C show violin plots of mass distribution, hydropathic index distribution, and isoelectric point distribution of proteins identified in direct digestion and improved secretome composition, respectively, according to some embodiments.

[0316] [Figure 22-1] 22A-22F show the improved secretome composition and the number of proteins identified in direct digests for various protein keywords according to some embodiments. The protein keywords considered were phosphorylated protein, tumor suppressor, glycoprotein, Alzheimer's disease, cytokine, and kinase. [Figure 22-2] 22A-22F show the improved secretome composition and the number of proteins identified in direct digests for various protein keywords according to some embodiments. The protein keywords considered were phosphorylated protein, tumor suppressor, glycoprotein, Alzheimer's disease, cytokine, and kinase. [Figure 22-3] 22A-22F show the improved secretome composition and the number of proteins identified in direct digests for various protein keywords according to some embodiments. The protein keywords considered were phosphorylated protein, tumor suppressor, glycoprotein, Alzheimer's disease, cytokine, and kinase.

[0317] [Figure 23] 23A-23C show different proteomics workflows, the number of protein groups identified in the workflows, and the coefficient of variation of peptides in the workflows, according to some embodiments.

[0318] [Figure 24] 24A-24B show a comparison of the number of protein groups and peptides identified using a label-free DDA workflow with the number of protein groups and peptides identified using a particle-based composition refinement workflow, according to some embodiments.

[0319] [Diagram 25] FIG. 25 illustrates a computer system configured to implement the methods or systems of the present disclosure, according to some embodiments.

[0320] [Figure 26] 26A-26I show non-limiting examples of surfaces according to some embodiments of the present disclosure. FIG. 26A illustrates a non-limiting example of a surface functionalized in one or more regions to capture biomolecules. FIG. 26B illustrates a non-limiting example of a surface including one or more wells or depressions to capture biomolecules. For example, the functionalized surface may be disposed in a 96-well plate or a 384-well plate. FIG. 26C illustrates a non-limiting example of a surface disposed on one or more particles. In some embodiments, one or more particles may be disposed in one or more wells or depressions. FIG. 26D illustrates a non-limiting example of a surface disposed on a channel or a plurality of particles packed in a porous material disposed in a channel. FIG. 26E illustrates a non-limiting example of a surface disposed on an interior surface of a channel. FIG. 26F-26I illustrate non-limiting examples of surfaces according to some embodiments of the present disclosure. The surface may include one, two, three, four, or any number of distinct surface regions. In some cases, the surface may be disposed on a particle. In some cases, the particle may be a porous particle.

[0321] [Figure 27] FIG. 27 illustrates generally a system and method for quality control of a biological sample, according to some embodiments.

[0322] [Figure 28] FIG. 28 shows an example of a plate layout for a single run of a Proteograph™ assay, according to some embodiments.

[0323] [Figure 29] FIG. 29 shows the number of protein groups identified by performing mass spectrometry on direct digest samples versus samples enriched using one or more surfaces, according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0324] Detailed Description Conditioned media from different cell cultures can be used for various in vitro biological applications. In some cases, the in vitro biological applications may include characterizing biomolecules secreted from cell cultures under different conditions into the conditioned media. In some cases, characterizing the secreted biomolecules in in vitro biological applications may help to gain deeper insight into the observed biological functions. However, in various in vitro systems, exogenous biomolecules may be provided to the cell cultures. For example, abundant proteins in fetal bovine serum (FBS) or a set of proteins sourced from different organisms may be included in the conditioned media for cell culture. The inclusion of exogenous biomolecules, such as those in FBS, may add complexity to the conditioned media. The exogenous biomolecules may affect the dynamic range of protein concentrations in the conditioned media, the exogenous biomolecules may suppress signals originating from the secreted biomolecules (e.g., from the cell culture), or the increased volume of the conditioned media may contribute to the dilution of the molecules of interest by the conditioned media.

[0325] The collection of biomolecules secreted by a cell can be referred to as a secretome. A secretome can include a variety of biomolecules, including members of biomolecular groups that can be classified based on composition (e.g., proteins, lipids, carbohydrates, and combinations thereof, e.g., glycoproteins, lipoproteins, glycolipids), as well as members of biomolecular groups that can be classified based on function (e.g., antibodies, cytokines, chemokines, hormones, growth factors, etc.).

[0326] The added complexity, wider dynamic range, or higher dilution in in vitro systems can create challenges similar in some ways to those in plasma proteomics. Plasma is estimated to contain unique proteins spanning concentrations that span at least 12-20 orders of magnitude (the "magnitude of the dynamic range"). High abundance biomolecules in plasma (e.g., albumin) can obscure detection of rare biomolecules of low abundance. Similarly, the secretome can contain biomolecules spanning a large dynamic range (e.g., more than 10, 11, or 12 orders of magnitude), whereby the presence of high abundance biomolecules can greatly obscure detection of biomolecules that fall into the low abundance range. A concomitant challenge is that the total available amount of biomolecules that fall into the low abundance range is too small for detection by some techniques.

[0327] In some aspects, the present disclosure provides systems and methods that can overcome some of these and other challenges by improving the composition of secretome collected from biological samples.The composition of secretome can be improved such that (1) high abundance biomolecules in supplemented media that obscure the detection of significant biomolecules in secretome are depleted, (2) low abundance biomolecules in secretome are enriched, and / or (3) the dynamic range of secretome in biological samples is compressed by changing the relative abundance ratio of biomolecules in secretome.The secretome with improved composition can then be analyzed by various assays, including those further provided in the present disclosure.The systems and methods of the present disclosure can enable discovery and derivation of insights based on detection and / or identification of biomolecules in secretome over a dynamic range that spans a larger number of orders of magnitude compared to some other assays.

[0328] The composition of the secretome can be improved using various techniques. In some cases, the techniques may include contacting a biological sample containing the secretome with one or more engineered surfaces, where the one or more engineered surfaces are configured to adsorb or bind biomolecules, thereby (1) depleting high abundance biomolecules in the supplemented medium that obscure the detection of significant biomolecules in the secretome, (2) enriching low abundance biomolecules in the secretome, and / or (3) compressing the dynamic range of the secretome in the biological sample by changing the relative abundance ratio of biomolecules in the secretome. The adsorbed or bound biomolecules can be released from the one or more engineered surfaces, thereby outputting an improved composition for use in a subsequent assay to detect the biomolecules.

[0329] In some aspects, the present disclosure describes systems and methods for applying a deep and scalable proteome profiling platform to directly analyze FBS-based or non-FBS-based cell culture media with an automated Proteograph™ Product Suite. In some cases, the systems and methods may utilize at least one, two, three, four, or five nanoparticles (NPs), which can be engineered to have different and / or distinct physicochemical properties to provide extensive coverage of large complex proteomes.

[0330] In some aspects, the disclosure describes a method for identifying one or more biomolecules. In some cases, the method includes providing a supplemented medium. In some cases, the method includes incubating cells in the supplemented medium under conditions sufficient for the cells to generate a set of biomolecules, thereby generating a set of biomolecules. In some cases, the method includes contacting at least a portion of the supplemented medium with one or more surfaces to adsorb the set of biomolecules. In some cases, the method includes removing the one or more surfaces and the set of biomolecules from at least a portion of the supplemented medium to generate a separated sample. In some cases, the method includes releasing the set of biomolecules from the one or more surfaces in the separated sample. In some cases, the method includes detecting at least a subset of the set of biomolecules, thereby identifying one or more biomolecules.

[0331] In some aspects, the disclosure describes an apparatus for assaying a biological sample. In some cases, the apparatus includes a substrate including one or more surfaces. In some cases, the apparatus includes one or more cell culture chambers including viable cells. In some cases, the apparatus includes one or more loading units operably coupled to the substrate and the cell culture chambers. In some cases, the apparatus includes a computer readable medium including machine executable code, which when executed by a processor, implements a method of the disclosure. In some cases, the method may include providing one or more controlled environments to cells in the one or more cell culture chambers for a predetermined duration. In some cases, a biological sample is produced from the cells in the cell culture chambers. In some cases, the method may include transferring at least a portion of the biological sample from the cell culture chambers to the substrate using one or more loading units. In some cases, the transferring step may cause at least a portion of the biological sample to contact one or more surfaces such that biomolecules in at least a portion of the biological sample may adsorb onto the one or more surfaces. In some cases, the method may include assaying at least a portion of the biomolecules to detect the biomolecules in the biological sample.

[0332] In some aspects, the disclosure provides a method for characterizing a biological preparation. In some embodiments, the method includes contacting the biological preparation with one or more surfaces to adsorb a plurality of biomolecules in the biological preparation onto the one or more surfaces. In some embodiments, the plurality of biomolecules includes product biomolecules and impurities. In some embodiments, the method includes assaying the plurality of biomolecules to determine a difference between a composition of the plurality of biomolecules and a reference composition. In some embodiments, the difference indicates the purity or activity of the biological preparation.

[0333] In some aspects, the present disclosure provides a method for detecting impurities in a biological preparation. In some embodiments, the method comprises contacting the biological preparation with one or more surfaces to adsorb a plurality of biomolecules in the biological preparation onto the one or more surfaces. In some embodiments, the method comprises assaying the plurality of biomolecules to detect a biomolecular fingerprint of the biological preparation. In some embodiments, the biomolecular fingerprint comprises a signature of an impurity in the plurality of biomolecules.

[0334] In some aspects, the present disclosure provides an apparatus for characterizing a biological preparation. In some embodiments, the apparatus includes a first chamber configured to hold the biological preparation. In some embodiments, the biological preparation includes a plurality of biomolecules. In some embodiments, the plurality of biomolecules includes product biomolecules and impurities. In some embodiments, the apparatus includes a second chamber including one or more surfaces. In some embodiments, the apparatus includes a loader operably coupled to the first chamber and the second chamber. In some embodiments, the loader is configured to transfer the biological preparation between the first chamber and the second chamber. In some embodiments, the apparatus includes a computer readable medium for measuring purity or activity of a biological preparation, the computer readable medium including machine executable code, the machine executable code, when executed by a processor, implementing a method for analyzing the biological preparation. In some embodiments, the method includes using the loader to contact the biological preparation from the first chamber with one or more surfaces in the second chamber to adsorb the plurality of biomolecules in the biological preparation onto the one or more surfaces.

[0335] In some aspects, the present disclosure provides an apparatus for detecting impurities in a biological preparation. In some embodiments, the apparatus includes a plurality of chambers operably connected to each other. In some embodiments, the plurality of chambers includes one or more surfaces. In some embodiments, the apparatus includes one or more fluid transfer devices operably coupled to the plurality of chambers. In some embodiments, the apparatus includes a computer readable medium for detecting impurities in a biological preparation, the computer readable medium including machine executable code, the machine executable code, when executed by a processor, implementing a method for analyzing a biological preparation. In some embodiments, the method includes contacting the biological preparation with one or more surfaces using one or more fluid transfer devices to adsorb a plurality of biomolecules in the biological preparation onto the one or more surfaces.

[0336] In some aspects, the present disclosure provides a computer-implemented method for generating a quality metric for a biological preparation. In some embodiments, the computer-implemented method includes receiving a plurality of mass spectrometry data sets for a plurality of polyamino acids in the biological preparation. In some embodiments, the plurality of polyamino acids includes at least one product biomolecule and a plurality of impurities. In some embodiments, the computer-implemented method includes generating a plurality of polyamino acid identifications and a plurality of polyamino acid abundances for the plurality of polyamino acids based on the plurality of mass spectrometry data sets. In some embodiments, the computer-implemented method includes processing the plurality of polyamino acid identifications to output a quality metric for the biological preparation.

[0337] In some aspects, the present disclosure provides a computer-implemented system. In some embodiments, the computer-implemented system includes a digital processing device including at least one processor, an operating system configured to execute executable instructions, a memory, and a computer program including instructions executable by the digital processing device to analyze a plurality of mass spectrometry data sets. In some embodiments, the computer program includes executable instructions for receiving a plurality of mass spectrometry data sets from a plurality of biological preparations. In some embodiments, the computer program includes executable instructions for processing the plurality of mass spectrometry data sets in real time to generate a plurality of quality metrics for the plurality of biological preparations. In some embodiments, the computer program includes executable instructions for providing a manufacturing process with process control instructions for making a plurality of biological preparations. Systems and methods for incubating cells - Patents.com

[0338] In some aspects, the disclosure provides a method for incubating cells. FIGS. 1A-1E illustrate a method for assaying a secretome using a compositional refinement assay, according to some embodiments. In some cases, the method includes providing a supplemented medium 101 to cells 102. In some cases, incubating the cells 102 in the supplemented medium 101 produces a set of biomolecules from the cells. In some cases, the cells are incubated under conditions sufficient for the cells 102 to produce the set of biomolecules 103. In some cases, the set of biomolecules is contacted with one or more surfaces 104 to adsorb the set of biomolecules 103.

[0339] In some cases, as shown in Figure ID, one or more surfaces 104 and the set of biomolecules 103 can be separated from a fluid composition 110 comprising the one or more surfaces 104 and the set of biomolecules 103 in an operation 105 to generate a separated sample 106. In some cases, the set of biomolecules 103 in the separated sample 106 can be released from the one or more surfaces 104. In some cases, at least a subset of the set of biomolecules 103 can be detected using a detector (109), thereby identifying one or more biomolecules.

[0340] 1E, the set of biomolecules 103 can be separated from one or more surfaces 104 and a fluid composition 110 comprising the set of biomolecules 103 by operation 105 to generate a separated sample 108. In some cases, at least a subset of the set of biomolecules 103 can be detected using a detector (109), thereby identifying one or more biomolecules.

[0341] In some cases, the incubating step is performed under conditions sufficient for the cells to produce the set of biomolecules. The conditions may include keeping constant or modulating various environmental factors and / or supplemental medium composition. The conditions may vary the type or amount of biomolecules released by the cells. In some cases, the conditions sufficient for the cells to exchange the set of biomolecules with supplemental medium include a predetermined temperature, a predetermined pressure, a predetermined flow regime, a predetermined solvent environment, or a combination thereof. In some cases, the conditions sufficient for the cells to exchange the set of biomolecules with the supplemented medium include one or more of the following: the presence or absence of an organic compound, the presence or absence of a therapeutic compound, the presence or absence of an inorganic compound, the presence or absence of an autocrine signaling molecule, the presence or absence of a paracrine signaling molecule, the presence or absence of an antigen, the presence or absence of one or more co-cultured cells, the presence or absence of radiation, the presence or absence of one or more toxins, the presence or absence of protein aggregates, the presence or absence of one or more proteins, the presence or absence of active virus particles, the presence or absence of inactivated virus particles, the presence or absence of applied heating or cooling, the presence or absence of applied mechanical stress, the presence or absence of electrical stimulation, the presence or absence of a transposon, the presence or absence of an exosome, the presence or absence of a liposome, the presence or absence of a coated nucleic acid, the presence or absence of a shock, or any combination thereof. In some cases, the supplemented medium comprises a synthetic supplemented medium. In some cases, the step of incubating the cells in the supplemented medium is performed while keeping the conditions constant. In some cases, incubating cells in supplemented medium is carried out under a first condition for a first amount of time, and then under a second condition for a second amount of time.In some cases, incubating cells in supplemented medium is carried out under continuously varying conditions.In some cases, incubating cells in supplemented medium is carried out for less than about 5 seconds, less than 5 minutes, less than 5 hours, or less than 5 days.In some cases, incubating the cells in supplemented medium is performed for at least about 5 seconds, 5 minutes, 5 hours, or 5 days. In some cases, incubating the cells in supplemented medium is performed at a temperature of at least about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65, or 70 degrees Celsius. In some cases, incubating the cells in supplemented medium is performed at a temperature of up to about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65, or 70 degrees Celsius. In some cases, incubating the cells in supplemented medium is performed at an absolute pressure of at least about 0.01, 0.1, 1, 10, 100, 1000, 10000, or 100000 bar. In some cases, incubating the cells in supplemented medium is performed at an absolute pressure of up to about 0.01, 0.1, 1, 10, 100, 1000, 10000, or 100000 bar. In some cases, incubating the cells in supplemented medium is performed by flowing supplemented medium over the cells at a Reynolds number of at least about 0.01, 0.1, 1, 10, 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, or 9000. In some cases, incubating the cells in the supplemented medium is performed by flowing the supplemented medium over the cells at a Reynolds number of at most about 0.01, 0.1, 1, 10, 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, or 9000. In some cases, incubating the cells in the supplemented medium is performed by flowing the supplemented medium in a laminar flow. In some cases, incubating the cells in the supplemented medium is performed by flowing the supplemented medium in a transient flow. In some cases, incubating the cells in the supplemented medium is performed by flowing the supplemented medium in a turbulent flow. In some cases, the method includes incubating the cells with a second supplemented medium. In some cases, the second supplemented medium includes a second set of biomolecules.In some cases, the second supplemented medium is generated by incubating cells in the supplemented medium for different lengths of time.

[0342] In some cases, the supplemented medium comprises in part or in whole serum, plasma, cerebrospinal fluid (CSF), synovial fluid (SF), urine, tears, gingival crevicular fluid, semen, whole blood, milk, nipple aspirate, needle aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic fluid, trabecular fluid, lung lavage, prostatic fluid, sputum, discharge, bronchial lavage, fluid from swabs, bronchial aspirant, sweat, saliva, or any combination thereof. In some cases, the supplemented medium comprises cell culture supernatant, co-culture secretome, tissue or cell lysate, or any combination thereof. In some cases, the supplemented medium comprises a vector. In some cases, the vector comprises one or more engineered nucleic acids encoding a protein product.

[0343] In some cases, the method includes generating a set of biomolecules by incubating cells in a supplemented medium. The cells may generate various types of biomolecules. In some cases, the set of biomolecules includes one or more biomarkers, molecular signatures, secreted proteins, absorbed proteins, exosomes, liposomes, lipids, hormones, glycoproteins, lipoproteins, glycolipids, peptides, cholesterol, carbohydrates, or any combination thereof. In some cases, the set of biomolecules includes one or more recombinant or engineered biomolecules. In some cases, the exchange of the set of biomolecules includes active secretion of at least a portion of the set of biomolecules. In some cases, the exchange of the set of biomolecules includes passive release of at least a portion of the set of biomolecules. In some cases, the exchange of the set of biomolecules includes the use of exosomes or liposomes by the cells. In some cases, the exchange of the set of biomolecules includes apoptosis of the cells. In some cases, the apoptosis of the cells releases the set of biomolecules into solution. In some cases, the released apoptotic biomolecules are exchanged with another cell. In some cases, the exchange of the set of biomolecules includes necroptosis of the cell. In some cases, the released necroptotic biomolecules are exchanged with another cell. In some cases, the biomolecules of the set of biomolecules are complexes. In some cases, the biomolecules of the set of biomolecules are proteins. In some cases, the biomolecules of the set of biomolecules are polypeptides. In some cases, the biomolecules of the set of biomolecules are nucleic acids. In some cases, the biomolecules in the set of biomolecules are carbohydrates, lipids, proteins, glycolipids, glycoproteins, lipoproteins, or any macromolecular assembly thereof. In some cases, the biomolecules secreted by one or more cells are conventional secretions, non-conventional secretions, type 1 non-conventional secretions, or type 2 non-conventional secretions. In some cases, the set of biomolecules includes a biomolecular assembly. In some cases, the biomolecular assembly includes a quaternary protein, a vesicle, or an exosome.

[0344] In some cases, the method includes contacting at least a portion of the supplemented medium with one or more surfaces to adsorb the set of biomolecules. In some cases, the contacting step is performed such that a portion of the supplemented medium contacts one or more surface regions of the one or more surfaces. In some cases, the one or more surface regions do not include a specific targeting moiety. In some cases, the surface may be included in one or more particles. In some cases, the surface region may be included in one or more particles. In some cases, the surface may be included in multiple particles. In some cases, the surface region may be included in multiple particles.

[0345] In some cases, the contacting step may include aliquoting at least a portion of the supplemented medium and then providing at least a portion of the supplemented medium in a chamber that includes one or more surfaces. In some cases, the aliquoting may be performed using one or more transfer units. In some cases, the one or more transfer units may include a pipette head, as shown in FIG. 6 or FIG. 8. In some cases, the one or more transfer units may include a fluid connection, as shown in FIG. 7.

[0346] In some cases, the contacting step may include providing one or more surfaces to a chamber containing a supplemented medium. For example, the one or more surfaces may be provided on one or more particles, which are added to the compartment containing the supplemented medium and / or cells. In some cases, the one or more particles may be provided in a fluid composition. In some cases, the fluid composition may be added to the compartment. In some cases, the fluid composition may be transferred using one or more transfer units. In some cases, the one or more transfer units may include a pipette head, as shown in FIG. 6 or FIG. 8. In some cases, the one or more transfer units may include a fluid connection, as shown in FIG. 7. In some cases, the composition of the set of biomolecules is improved for detection of the biomolecules. In some cases, the set of biomolecules, when adsorbed on the one or more surfaces, includes a reduced dynamic range compared to the original dynamic range of the set of biomolecules in the supplemented medium. In some cases, after the contacting step, the set of biomolecules is depleted of high abundance biomolecules. In some cases, after the contacting step, the set of biomolecules is depleted of easily detectable biomolecules. In some cases, after the contacting step, the set of biomolecules is enriched for low abundance biomolecules. In some cases, after the contacting step, the set of biomolecules is enriched for difficult to detect biomolecules.

[0347] In some cases, the contacting step includes contacting the biological sample with a second surface to adsorb a second plurality of biomolecules onto the second surface. In some cases, after the second contact, the composition of the set of biomolecules is improved for detection of the biomolecules. In some cases, the set of biomolecules, when adsorbed onto one or more surfaces, includes a reduced dynamic range compared to the original dynamic range of the set of biomolecules in the supplemented medium. In some cases, after the contact, the set of biomolecules is depleted of high abundance biomolecules. In some cases, after the contact, the set of biomolecules is depleted of easily detectable biomolecules. In some cases, after the contact, the set of biomolecules is enriched for low abundance biomolecules. In some cases, after the contact, the set of biomolecules is enriched for difficult to detect biomolecules.

[0348] In some cases, the method includes releasing a set of biomolecules from one or more surfaces in the separated sample. In some cases, the releasing includes the use of an enzyme. In some cases, the releasing includes the use of a protease. In some cases, the releasing includes the use of trypsin. In some cases, the releasing includes the use of a lysin. In some cases, the releasing includes the use of a buffer. In some cases, the releasing includes the use of a solvent. In some cases, the releasing includes fragmenting the biomolecules.

[0349] 2A-2D illustrate a method for assaying a secretome using a composition refinement assay, according to some embodiments. In some cases, the plurality of cells includes at least a first cell 201 of a first type and a second cell 202 of a second type, such that the first cell exchanges one or more biomolecules of the set of biomolecules with the second cell 203. In some cases, the first cell and the second cell are co-cultured. In some cases, the first cell is included in a feeder culture for the second cell. In some cases, the plurality of cells are disposed in a plurality of separate volumes. In some cases, each volume of the plurality of separate volumes includes a different supplemented medium or incubation condition.

[0350] In some cases, the method further comprises determining whether one or more biomolecules are produced by the cells and were not originally present in the supplemented medium. In some cases, at least a portion of the biomolecules in the supplemented medium are depleted. In some cases, the supplemented medium includes fetal bovine serum. In some cases, the fetal bovine serum is depleted.

[0351] In some cases, the method includes monitoring cellular activity. In some cases, the method includes incubating the cells such that a biological sample comprising a plurality of biomolecules is generated from the cells. In some cases, the method includes contacting the biological sample with a surface to adsorb the plurality of biomolecules onto the surface. In some cases, the method includes releasing at least a portion of the plurality of biomolecules on the surface. In some cases, the method includes detecting at least a portion of the plurality of biomolecules, thereby identifying the plurality of biomolecules. In some cases, the method includes analyzing cellular activity.

[0352] In some cases, the method includes incubating the cells after a predetermined amount of time such that the cells generate a second biological sample comprising a second plurality of biomolecules. In some cases, the method includes contacting the second biological sample with a second surface after a predetermined amount of time to adsorb the second plurality of biomolecules onto the surface. In some cases, the method includes releasing at least a second portion of the second plurality of biomolecules on the second surface after a predetermined amount of time. In some cases, the method includes detecting at least a second portion of the second plurality of biomolecules after a predetermined amount of time, thereby identifying the second plurality of biomolecules. In some cases, the biological sample is generated from the cells by any one of production, release, adsorption, digestion, or modification of the biomolecules of the plurality of biomolecules. In some cases, the cells participate in the supplemented medium by any one of production, release, adsorption, digestion, or modification of the biomolecules of the plurality of biomolecules.

[0353] In some cases, the method includes generating multiple biological samples at multiple different times. In some cases, multiple different times can be used to monitor changes in cell activity, biological sample composition, or both. For example, cells can be incubated under the same or changing conditions for some period of time (e.g., minutes, hours, days, weeks, months, or years), and biomolecules released from the cells can be sampled periodically (e.g., within seconds, minutes, hours, days, weeks, or months). The periodically sampled biomolecules can be assayed to generate a time series that can reveal the dynamics of the cell's activity over time. Changes in the biomolecules released from the cell can indicate changes in the cell's activity.

[0354] In some cases, the method includes altering incubation conditions of the cells based at least in part on the activity of the cells. In some cases, the incubation conditions that are altered may include temperature, pressure, flow rate, supplemented media composition, or any combination thereof.

[0355] In some cases, the method includes identifying a low abundance biomolecule in the biological sample. In some cases, the method includes incubating the cells in a predetermined environment. In some cases, a biological sample is produced from the cells. In some cases, the method includes adding an exogenous biomolecule to the biological sample. In some cases, the exogenous biomolecule reduces the detectability of the plurality of low abundance biomolecules in the biological sample. In some cases, the exogenous biomolecule can be a biomolecule that is not endogenously expressed or synthesized by the cell. In some cases, the method includes contacting the biological sample with a surface to adsorb the plurality of low abundance biomolecules in the biological sample onto the surface. In some cases, the method includes releasing at least a portion of the plurality of low abundance biomolecules on the surface. In some cases, the method includes detecting at least a portion of the plurality of low abundance biomolecules. In some cases, the method includes identifying the plurality of low abundance biomolecules.

[0356] In some cases, the step of detecting the low abundance biomolecule is at least about 7 orders of magnitude more certain when the biological sample is contacted with the surface. In some cases, the step of detecting the low abundance biomolecule can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude more certain when the biological sample is contacted with the surface. In some cases, the step of detecting the low abundance biomolecule can be up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude more certain when the biological sample is contacted with the surface. In some cases, the low abundance biomolecule in the set of biomolecules can be at least about 7 orders of magnitude less abundant or concentrated than the high abundance biomolecule in the set of biomolecules. In some cases, a low abundance biomolecule in a set of biomolecules may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant or concentrated than a high abundance biomolecule in the set of biomolecules. In some cases, a low abundance biomolecule in a set of biomolecules may be at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 orders of magnitude less abundant or concentrated than a high abundance biomolecule in the set of biomolecules.

[0357] In some cases, the identification of a set of biomolecules can lead to insights into biological processes occurring in cells.In some cases, the method includes, based at least in part on the identification, determining one or more protein-protein interactions, biomarkers, molecular signatures, biomolecules absorbed by cells, biomolecules secreted by cells, biomolecules dissociated from the cell surface, biomolecules cleaved or released from the surface, budding macromolecular complexes, biomolecules passively released by cells, conventional and non-conventional released proteins, apoptotic release of biomolecules, necrotic released biomolecules, post-translation modifications, cell-cell interactions, cell-cell communication, or any combination thereof.In some cases, the molecular signatures can include protein patterns and / or proteoforms that indicate biological states.

[0358] In some aspects, the present disclosure provides an apparatus for assaying a biological sample. FIG. 3A illustrates an apparatus for assaying a biological sample using a composition improvement assay, according to some embodiments. In some cases, the apparatus includes a substrate 301 including a surface. In some cases, the substrate 301 may be disposed in a substrate chamber 302. In some cases, the apparatus includes a cell culture chamber 303 including cells 305. In some cases, the apparatus includes a loading unit 304. In some cases, the loading unit is operably coupled to the substrate chamber 302, the cell culture chamber 303, or both. In some cases, the loading unit 304 may include a pump, a pipette device, a sample holder, or any other device configured to transfer at least a portion of a biological sample from one location to another. In some cases, the cell culture chamber 303 and the substrate chamber 302 may be included in a sample volume. In some cases, the cell culture chamber 303 and the substrate chamber 302 may constitute separate volumes. In some cases, the separate volumes may be fluidly connected. In some cases, the apparatus includes a second loading unit 306. In some cases, the second loading unit is operably coupled to the substrate chamber 301, the cell culture chamber 302, or both. In some cases, the second loading unit 306 is configured to transfer at least a portion of the biological sample from one location to another location. In some cases, the second loading unit 306 is configured to transfer at least a portion of the biological sample to a location other than the location to which the first loading unit 304 is configured to transfer at least a portion of the biological sample.

[0359] FIG. 3B illustrates an apparatus for assaying biological samples using a compositional refinement assay, according to some embodiments. In some cases, the apparatus may include multiple different cell lines 307. In some cases, the multiple different cell lines 307 can be incubated under the same conditions. For example, the apparatus may include multiple compartments (e.g., 96-well plates) that include multiple different cell lines. The multiple different cell lines 307 can be incubated under the same conditions. In some cases, multiple biological samples can be obtained from multiple different cell lines 307. In some cases, the multiple biological samples can be assayed to identify biomolecules produced by each cell line of the multiple cell lines 307. In some cases, the multiple biological samples can be assayed using multiple surface types 308. In some cases, the multiple surface types can include multiple different surface chemistries.

[0360] FIG. 3C illustrates a device for assaying biological samples using a compositional improvement assay, according to some embodiments. In some cases, the device may include multiple different supplemented media 309. In some cases, multiple different supplemented media 309 may be used to incubate one or more cells in multiple compartments. In some cases, the one or more cells may include one or more cell lines. In some cases, different biomolecules may be excreted by cells exposed to different supplemented media. In some cases, multiple biological samples may be obtained from multiple compartments, each of which includes one or more cells incubated with one of the multiple different supplemented media 309. In some cases, the multiple compartments may contain the same cell line (e.g., HeLa cells), but different supplemented media 309. For example, the supplemented media 309 may contain different organic compounds, such as drugs. In some cases, multiple biological samples may be assayed to identify biomolecules produced by one or more cells under the influence of the various supplemented media 309. In some cases, multiple biological samples may be assayed using one or more surface types. In some cases, one or more of the surface types may include the same surface chemistry or may include a number of different surface chemistries.

[0361] FIG. 3D illustrates an apparatus for assaying a biological sample using a composition-improved assay, according to some embodiments. In some cases, the apparatus may include a plurality of compartments 310 containing a plurality of cells. In some cases, the plurality of cells can be incubated with a supplemented medium. In some cases, the plurality of compartments can be transferred from a first location to a second location using a transfer unit 311. In some cases, one or more particles can be added to the plurality of compartments to assay one or more biomolecules in the plurality of compartments.

[0362] 3E illustrates an apparatus for assaying biological samples using a composition-improved assay, according to some embodiments. In some cases, the apparatus may include a first plurality of compartments 310 containing a plurality of cells. In some cases, the plurality of cells may be incubated with a supplemented medium. In some cases, the plurality of samples from the first plurality of compartments may be transferred to a second plurality of compartments 313 using a transfer unit 312. In some cases, the second plurality of compartments 313 may include one or more surfaces for assaying one or more biomolecules in the plurality of samples.

[0363] In some cases, the loading unit is configured to dispense a reagent, wash, or supplemental medium into the chamber. In some cases, the first loading unit, the second loading unit, or both, are configured to dispense a reagent, wash, or supplemental medium into the cell culture chamber, the substrate chamber, or both.

[0364] In some cases, the device includes a computer readable medium including machine executable code, which when executed by a processor, implements any of the methods disclosed herein. In some cases, the method includes providing a controlled environment for a predetermined duration to cells in a cell culture chamber. In some cases, a biological sample is produced from the cells in the cell culture chamber. In some cases, the method includes transferring at least a portion of the biological sample from the cell culture chamber to a substrate using a loading unit. In some cases, the method includes contacting the at least a portion with a surface to adsorb biomolecules in at least a portion of the biological sample onto the surface. In some cases, the method includes transferring at least a portion of the biological sample to an analytical instrument. In some cases, the analytical instrument includes a mass spectrometer. In some cases, the analytical instrument includes a sequencer. In some cases, the analytical instrument includes a chromatography column. In some cases, the method includes assaying at least a portion of the biomolecules to detect biomolecules in the biological sample. In some cases, the method includes assaying at least a portion of the biomolecules to detect the biomolecules in the biological sample using an analytical instrument. In some cases, the device includes a plurality of cell culture chambers. In some cases, a first controlled environment is provided to a first cell culture chamber 303 in the plurality of cell culture chambers. In some cases, a second controlled environment is provided to a second cell culture chamber 305 in the plurality of cell culture chambers.

[0365] In some aspects, the present disclosure provides a method for identifying one or more biomolecules. In some cases, the method includes incubating a cell under conditions sufficient for the cell to produce an exosome. In some cases, the exosome includes a plurality of biomolecules. In some cases, the method includes contacting the exosome with one or more surfaces to capture at least a portion of the exosome. In some cases, the method includes removing the one or more surfaces and at least a portion of the exosome from the cell to create a separated sample. In some cases, the method includes releasing at least a portion of the exosome from the one or more surfaces in the separated sample. In some cases, the method includes detecting at least a subset of the plurality of biomolecules in at least a portion of the exosome, thereby identifying one or more biomolecules.

[0366] In some aspects, the disclosure provides a method for monitoring cellular activity. In some cases, the method includes incubating cells such that a biological sample comprising exosomes is generated from the cells. In some cases, the exosomes include a plurality of biomolecules. In some cases, the method includes contacting the biological sample with a surface to capture at least a portion of the exosomes on the surface. In some cases, the method includes releasing at least a portion of the exosomes from the surface. In some cases, the method includes detecting at least a portion of the plurality of biomolecules in at least a portion of the exosomes. In some cases, the method includes identifying the plurality of biomolecules. In some cases, the method includes repeating any one or combination of steps after a predetermined amount of time. In some cases, the method includes monitoring cellular activity. In some cases, the exosomes can be enriched from the biological sample prior to contacting with one or more surfaces. In some cases, the exosomes can be enriched using size exclusion chromatography. In some cases, the exosomes can be enriched using differential ultracentrifugation. In some cases, exosomes can be enriched using ultrafiltration, precipitation, or immunoaffinity methods.

[0367] In some aspects, the disclosure provides a method for identifying low abundance biomolecules in a biological sample. In some cases, the method includes incubating cells in a predetermined environment. In some cases, a biological sample comprising exosomes is produced from the cells. In some cases, the exosomes comprise a plurality of low abundance biomolecules. In some cases, the method includes contacting the biological sample with a surface to capture at least a portion of the exosomes in the biological sample on the surface. In some cases, the method includes releasing at least a portion of the exosomes from the surface. In some cases, the method includes detecting at least a portion of the plurality of low abundance biomolecules in at least a portion of the exosomes. In some cases, the method includes identifying the plurality of low abundance biomolecules. In some cases, the plurality of low abundance biomolecules is less than about 1 percent, by mass, of the biomolecules in the exosomes, about 1×10 -1 Less than 1% (approximately 1×10 -2 Less than 1% (approximately 1×10 -3 Less than 1% (approximately 1×10 -4 Less than 1% (approximately 1×10 -5 Less than 1% (approximately 1×10 -6 Less than 1% (approximately 1×10 -7 Less than a percent, or about 1 × 10 -8 In some cases, the plurality of low abundance biomolecules constitutes more than about 1 percent, by mass, of the biomolecules in the exosome, such as about 1 x 10 -1 Over 10 percent, approximately 1×10 -2 More than 1 x 10 percent -3 More than 1 x 10 percent -4 Over 10 percent, approximately 1×10 -5 More than 1 x 10 percent -6 Over 10 percent, approximately 1×10 -7 percent or about 1×10 -8 Constitutes more than a percent.

[0368] In some aspects, the present disclosure provides an apparatus for assaying a biological sample. In some cases, the apparatus includes a substrate including a surface. In some cases, the apparatus includes a cell culture chamber including cells. In some cases, the apparatus includes a loading unit operably coupled to the substrate and the cell culture chamber. In some cases, the apparatus includes a computer readable medium including machine executable code when executed by a processor. In some cases, the computer readable medium implements a method including providing a controlled environment to cells in the cell culture chamber for a predetermined duration. In some cases, a biological sample including exosomes is produced from the cells in the cell culture chamber. In some cases, the exosomes include one or more biomolecules. In some cases, the computer readable medium implements a method including transferring at least a portion of the biological sample from the cell culture chamber to the substrate using a loading unit. In some cases, the computer readable medium implements a method including contacting at least a portion with a surface to adsorb exosomes in at least a portion of the biological sample onto the surface. In some cases, the computer readable medium implements a method that includes assaying the exosomes to detect one or more biomolecules in a biological sample.

[0369] In some aspects, the present disclosure provides a method for identifying a biomolecule.In some cases, the method comprises processing one or more exosomes so that a plurality of biomolecules in one or more exosomes are released to the environment outside the one or more exosomes.In some cases, a subset of the biomolecules of the plurality of biomolecules comprises a first distribution of relative abundance in one or more exosomes.In some cases, the method comprises performing a composition improvement assay on the plurality of biomolecules to increase the relative abundance of the subset of biomolecules from the first distribution to a second distribution.In some cases, the method comprises assaying the plurality of biomolecules to identify a subset of biomolecules.

[0370] In some aspects, the present disclosure provides a method for identifying a biomolecule. In some cases, the method includes contacting a biological sample containing one or more exosomes with a plurality of particles. In some cases, the method includes non-specifically binding a subset of one or more exosomes onto the plurality of particles. In some cases, the plurality of particles include distinct physicochemical properties. In some cases, the one or more exosomes include a plurality of biomolecules. In some cases, the method includes processing one or more exosomes such that the plurality of biomolecules are released into an environment outside the one or more exosomes. In some cases, the method includes assaying the plurality of biomolecules to identify at least a subset of the biomolecules of the plurality of biomolecules. Supplementary medium

[0371] Supplementary medium may contain various chemical constituents that may affect the secretome composition of cells. In some cases, supplementary medium may contain one or more nutrients, one or more drugs, one or more therapeutic compounds, one or more biomolecules, one or more pathogens, or any combination thereof. In some cases, supplementary medium may provide a supply of nutrients for one or more cells to grow and / or grow. In some cases, supplementary medium may contain one or more substances that reduce the visibility or detectability in downstream assays of at least some of the biomolecules excreted by the cells. For example, some nutrients of supplementary medium may be proteins that are provided in high abundance (compared to the biomolecules of interest excreted or potentially excreted by one or more cells). High abundance proteins may obscure the detection of low abundance biomolecules produced by one or more cells.

[0372] In some embodiments, nutrients may include any substance used by cells in metabolism, in some embodiments, nutrients are or include carbohydrates, lipids, fiber, minerals, proteins, vitamins, microorganisms, water, sugars, fats, ions, amino acids, peptides, alcohols, ketones, carboxylic acids, amines, amides, ethers, esters, bases, acids, small molecule supplements, commensal gut bacteria, hormones, salts of nutrients, metabolic products of nutrients, synthetically modified forms of nutrients, micronutrients, isotopes of nutrients, or any combination thereof.

[0373] In some embodiments, the drug may be configured to perturb cells. Any drug or candidate drug may be examined by the disclosed systems and methods. In some embodiments, the drug may include a chemotherapeutic agent, a hormone targeting agent, an agent configured to target a metabolic response, or an agent configured to affect a regulator of a metabolic response. In some embodiments, the drug is an analgesic, an anesthetic, an antibacterial, an antibiotic, an anticonvulsant, an anti-dementia agent, an antidepressant, an antidote, an antitoxin, an antiemetic, an antifungal, an anti-inflammatory, a corticosteroid, a nonsteroidal anti-inflammatory, an anti-migraine, an antimyasthenic, an antimycobacterial, an antineoplastic, an antiparasitic, an antiparkinsonian, an antipsychotic, an antiviral, an antiretroviral, an anti-hepatitis C agent, a tranquilizer, an anxiolytic, a bipolar, a blood glucose regulator, insulin, a blood product, an anticoagulant, a cardiovascular agent, a beta blocker, an ACE inhibitor, a central nervous system agent, an amphetamine, a dental agent, an oral agent, a dermatological agent, an enzyme replacement agent, a gastrointestinal agent, an H2 blocker, a proton pump inhibitor, a genitourinary agent. The therapeutic agent may include a therapeutic agent for the reproductive tract, a urogenital agent for the urinary tract, a hormone agent, an adrenal hormone agent, a pituitary hormone agent, a prostaglandin hormone agent, a sex hormone, an estrogen, a testosterone, an anabolic steroid, a thyroid agent, a hormone suppressant, an adrenal hormone suppressant, a parathyroid hormone suppressant, a pituitary hormone suppressant, a sex hormone suppressant, a thyroid hormone suppressant, an immunological agent, a vaccine, an antirheumatic agent, an inflammatory bowel disease agent, a metabolic bone disease agent, an ophthalmic agent, an otic agent, a respiratory agent, an antihistamine, a bronchodilator, a sedative, a hypnotic, a skeletal muscle relaxant, a therapeutic nutrient, a mineral, an electrolyte, a chemotherapy agent, a radiotherapy agent, or any combination thereof.

[0374] The supplemented medium may contain water and / or various co-solvents. In some cases, the supplemented medium may contain water, methanol, ethanol, propanol, butanol, acetone, polyethylene glycol (PEG), dimethylsulfoxide (DMSO), beta-cyclodextrin, buffer, or any combination thereof.

[0375] In some cases, the supplemented medium may include a natural medium. In some cases, the supplemented medium may include an artificial medium. In some cases, the artificial medium may include organic nutrients, inorganic nutrients, vitamins, salts, O2, CO2, serum proteins, carbohydrates, cofactors, or any combination thereof. In some cases, the artificial medium may be configured to improve short-term cell survival, improve long-term cell survival, promote cell growth or proliferation, or any combination thereof.

[0376] In some cases, the supplemented medium can include serum-containing medium. In some cases, the serum-containing medium can include fetal bovine serum. In some cases, the serum-containing medium can include carriers or chelating agents for solubilizing labile or water-insoluble nutrients, hormones and growth factors, protease inhibitors, or any combination thereof. In some cases, the serum-containing medium can bind toxins and / or neutralize toxins.

[0377] In some cases, the supplemented medium may include serum-free medium. In some cases, the serum-free medium may include Knockout Serum Replacement, Knockout DMEM, and mTESR medium, purified growth factors, lipoproteins, or any combination thereof.

[0378] In some cases, a supplemented medium may comprise a synthetic medium, which in some cases may comprise inorganic constituents, organic constituents, protein additives (e.g., growth factors), or any combination thereof.

[0379] In some cases, the supplemented medium may include a natural buffer system. In some cases, the natural buffer system may include CO2, which can balance the CO3 / HCO3 content in the supplemented medium.

[0380] In some cases, supplemented media may contain inorganic salts, amino acids, carbohydrates, proteins, peptides, fatty acids, lipids, vitamins, trace elements, antibiotics, albumin, growth factors, growth inhibitory hormones, transferrin, fibronectin, protease inhibitors, or any combination thereof.

[0381] In some cases, the supplemented medium is selected from the group consisting of HEPES, phenol red, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640, Eagle's Minimum Essential Medium (EMEM), Ham's nutrient mixture, any one of Ham's F-10, Ham's F-12, Coon's modified Ham's F-12, DMEM / F12, Iscove's Modified Dulbecco's Medium (IMDM), neurobasal medium, McCoy's 5A medium, Dynamis medium, Essential 8 (E8) medium, StemFlex culture medium, Airway Epithelial Cell basal medium, alpha modified minimum essential medium (α-MEM), StemMacs The medium may include iPS-Brew medium, TeSR-E8, mTeSR1, mTeSR Plus, Glasgow Minimum Essential Medium (GMEM), Opti-MEM I, SmGM-2, Fibroblast Growth Medium / FGM, StemPro-34 serum-free growth medium, mTESR1 medium, ECGM-2 medium, EGM2-Bullet kit medium, Williams'Medium E, medium M254, CnT07 medium, TNM-FH medium, Mammary Epithelial Cell Growth Basal Medium (MEBM), Complete Skeletal Muscle Medium, NeuroCult NS-A Basal Medium, Ham's F-10

[50] , Fibroblast Growth Kit-Low Serum, or any combination thereof.

[0382] In some cases, the supplemented medium comprises a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some cases, the supplemented medium comprises a pH of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some cases, the supplemented medium comprises an oxygen concentration of at least about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises an oxygen concentration of up to about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises a carbon dioxide concentration of at least about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises a carbon dioxide concentration of up to about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises an ammonia concentration of at least about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises an ammonia concentration of up to about 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 mg / L. In some cases, the supplemented medium comprises an ionic strength of at least about 0.001, 0.01, 0.1, 1, 2, 3, 4, or 5 M. In some cases, the supplemented medium comprises an ionic strength of at most about 0.001, 0.01, 0.1, 1, 2, 3, 4, or 5 M. Biological samples

[0383] A biological sample may include a single sample or multiple samples from a species, an individual organism, or a portion of an individual organism. In some embodiments, a biological sample may be obtained from an individual organism. In some embodiments, a biological sample may include multiple samples from a population of organisms. In some embodiments, a biological sample may include a gene. In some embodiments, a biological sample may include a tissue. In some embodiments, a biological sample may include an organ. In some embodiments, a biological sample may be obtained by performing a biopsy. In some embodiments, a biological sample may be obtained by performing a tissue biopsy. In some embodiments, a biological sample may include a tumor biopsy. In some embodiments, a biological sample may include a liquid biopsy. In some cases, a biological sample may be processed (e.g., lysed, mixed, centrifuged, fractionated, etc.). In some cases, a biological sample may include a medium that includes a biomolecule secreted by one or more cells. In some cases, a biological sample may be acellular or substantially acellular. In some cases, a biological sample may include multiple biomolecules. In some cases, a multiple biomolecule may include a polyamino acid. In some cases, the polyamino acid comprises a peptide, a protein, or a combination thereof. In some cases, the plurality of biomolecules may comprise nucleic acids, carbohydrates, polyamino acids, or any combination thereof. The biological sample may comprise members of any class of biomolecules, where "class" may refer to any named category that defines a group of biomolecules that have a common characteristic (e.g., protein, nucleic acid, carbohydrate).

[0384] In some embodiments, the biological sample may include cells. In some embodiments, a cell may refer to the basic unit of a living organism that includes at least a cell membrane and genetic material. In some embodiments, the biological sample may include cells of a single-celled organism. In some embodiments, the biological sample may include cells of a multicellular organism. In some embodiments, the biological sample may include bacterial cells. In some embodiments, the biological sample may include fungal cells. In some embodiments, the biological sample may include virally infected cells. In some embodiments, the biological sample may include mammalian cells. In some embodiments, the biological sample may include human cells. In some embodiments, the biological sample may include specialized cells in a multicellular organism. In some embodiments, the biological sample may include stem cells. In some embodiments, the biological sample may include healthy cells. In some embodiments, the biological sample may include cancerous cells. In some embodiments, the biological sample may include malignant cells. In some embodiments, the biological sample may include nucleic acids and various forms thereof. In some embodiments, the biological sample may include proteins and various forms thereof. In some cases, the cell is part of a plurality of cells. In some cases, the plurality of cells are cells of the same type. In some cases, the cell is a cell of a tissue sample, a cell of an organoid, a cell of an immortalized cell line, or any combination thereof. In some cases, the cell is a stem cell. In some cases, the cell is infected or mutated. In some cases, the cell is a viable cell, including a cancer cell, an epithelial cell, a bone cell, a muscle cell, a fat cell, a tissue cell, or a nerve cell. In some cases, the cancer cell is a cell obtained from a patient biopsy. In some cases, the cell is a eukaryotic or prokaryotic organism. In some cases, the biological sample may include yeast. In some cases. In some cases, the plurality of cells is contained within a tissue, an organoid, an organism, or a plurality of organisms. In some cases, the cell is from an immortalized cell line. In some cases, the cell is a HeLa cell. In some cases, the cell is a stem cell. In some cases, the cell is contained within a primary cell culture. In some cases, the cell includes a genetically modified cell.A genetically modified cell can contain nucleic acid that includes engineered or recombinant nucleic acid.

[0385] The subject may include any living organism. In some embodiments, the subject may be a cell. In some embodiments, the subject may include a bacterium, a mammalian cell, a human cell, a fungal cell, a bacterial colony, a mammalian tissue, a mammalian organ, a mammal, a human tissue, a human organ, a fungus, or any combination thereof. In some embodiments, the subject may include a cancer cell, a healthy cell, or both. In some embodiments, the cell may include a genetically modified cell.

[0386] In some embodiments, the biological sample may comprise secretome. In some cases, secretome may refer to the factors secreted by cells, tissues, or organisms into the extracellular space under some defined time and / or conditions. In some cases, factors may include soluble factors such as proteins, peptides, lipids, extracellular vesicles, or any combination thereof.

[0387] In some embodiments, the biological sample may contain exosomes. In some cases, the secretome may contain exosomes. In some cases, the biological sample contains enriched exosomes. In some cases, the biological sample contains preferentially enriched exosomes. For example, ultracentrifugation can be used to enrich exosomes from plasma or secretomes. In some cases, exosomes may contain at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 unique biomolecules. In some cases, exosomes may contain a narrower dynamic range of biomolecules than a human plasma sample. In some cases, exosomes may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dynamic ranges. In some cases, exosomes may comprise up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dynamic ranges. In some cases, biomolecules in exosomes may comprise higher visibility or detectability than the same biomolecules not in exosomes. In some cases, at least about 1, 10, 100, or 1000 biomolecules in exosomes may comprise higher visibility or detectability than the same biomolecules not in the same exosomes. In some cases, exosomes can be processed. In some cases, the exosomes can be at least partially processed by adding a lysis buffer to one or more exosomes.In some cases, the exosomes can be at least partially processed by providing ultrasonic energy to one or more exosomes.In some cases, the exosomes can be at least partially processed by freezing and thawing one or more exosomes.In some cases, the exosomes can be at least partially processed by heating one or more exosomes. In some cases, the exosomes can be at least partially processed by shearing one or more exosomes. In some cases, the exosomes can be at least partially processed by disrupting one or more exosomes.

[0388] In some cases, cells can secrete biomolecules through transmembrane proteins. In some cases, cells can secrete biomolecules through porosomes. In some cases, cells can secrete biomolecules through vesicles. In some cases, cells can secrete biomolecules through exosomes. In some cases, cells can secrete biomolecules through type 1 secretion machinery, type 2 secretion machinery, type 3 secretion machinery, type 4 secretion machinery, type 5 secretion machinery, type 6 secretion machinery, or any combination thereof. In some cases, cells can secrete conventional secretions, non-conventional secretions, type 1 non-conventional secretions, or type 2 non-conventional secretions.

[0389] In some cases, the secreted biomolecule may comprise one or more biomarkers, molecular signatures, proteins, exosomes, liposomes, lipids, hormones, glycoproteins, lipoproteins, glycolipids, glycoproteins, peptides, hormones, cholesterol, carbohydrates, apoptosis-related substances, necrosis-related substances, nucleic acids, macromolecular assemblies, or any combination thereof. In some cases, the secreted biomolecule may comprise an engineered or recombinant biomolecule (e.g., a recombinant protein). Automated secretome assay

[0390] In some cases, an automated fluidic system can be used to transfer the biological sample. In some cases, the automated fluidic system includes a microfluidic system. In some cases, the automated fluidic system can be configured to provide a supplemented medium at various times. In some cases, the automated fluidic system can be configured to change the composition of the provided supplemented medium at various times. In some cases, the transferring step can be performed using one or more fluid connections and one or more pumps included in the loading unit. In some cases, the transferring step is performed using one or more pipettes and one or more pumps included in the loading unit.

[0391] In some cases, the device may include a compartment that contains the particles. In some cases, the compartment may be a chamber. In some cases, the chamber may be a compartment. Figures 4 and 5 show multiple compartments according to some embodiments. In some cases, the device may include a single compartment (e.g., an Eppendorf tube) for holding a volume of sample or reagent. In some cases, the device may include multiple compartments (e.g., multiple wells of a 16-well plate, a 96-well plate, a 384-well plate, a microwell plate) for holding a volume of sample or reagent. In some cases, the compartment may include a well, a channel (e.g., a microfluidic channel of a microfluidic device), or a compartment. In some cases, the compartment may include a plastic product (e.g., a plastic multiwell plate), a metal structure (e.g., a metal multiwell plate), a carbon material structure (e.g., a carbon composite multiwell plate), a gel, a glass product, or any combination thereof. In some cases, the fluidic channel or chamber may be a microfluidic or nanofluidic channel or chamber. In some cases, the compartments may be sealed (e.g., with a manipulable plastic slip or a pierceable septum) or sealable (e.g., may include a reusable cap or lid). In some cases, the compartments may be configured to hold volumes of at least 1-10 microliters (μL), at least 5-25 μL, at least 20-50 μL, at least 40-200 μL, at least 100-500 μL, at least 200 μL to 1 mL, at least 2 mL, at least 3 mL, or more. In some cases, the compartments may be configured to hold volumes of less than about 240 μL, less than about 200 μL, less than about 150 μL, less than about 100 μL, less than about 75 μL, less than about 50 μL, less than about 25 μL, less than about 10 μL, less than about 5 μL, less than about 1 μL, or less. In some cases, the compartments may be temperature controlled. In some cases, the compartment may be configured to prevent or reduce evaporation, hi some cases, the compartment may be designed to minimize the influx of ambient light.In some cases, the compartments may contain one or more compartments configured to hold a control volume. A non-limiting example of a control volume that can be used in connection with the methods and systems disclosed herein is illustrated in FIG. 4. The control volume(s) may include one or more of a process control 401, a digestion control 402, a control peptide mixture 403, or a mass spectrometry control peptide mixture 404, or any combination thereof. In some cases, the compartments may be grouped by particle, sample, control, or any combination thereof. An example is illustrated in FIG. 5, where a plurality of compartments that can be used with five types of particles (e.g., NP1, NP2, NP3, NP4, and NP5) are arranged in 8 rows and 12 columns. In some cases, each nanoparticle may occupy two columns, and up to 16 biological samples may be placed. In some cases, each biological sample is labeled X1, X2, X3, etc., up to X16. In some cases, two columns may be for control experiments, in which case each control well in the column may receive a control particle composition, a control biological sample, or both. In some cases, the control biological sample may include a reference composition of biomolecules, including a predetermined composition. In some cases, the control wells may be configured to receive protease for quality control of the protease used in the assay. In some cases, each control well may be utilized at or between certain steps of an experiment, thus allowing troubleshooting of subsequent experimental procedures. In some cases, particles may be present in the compartments, and then the biological sample may be added later. In some cases, biological sample may be present in the compartments, and then the particles may be added later. In some cases, a subset of the compartments may be grouped by particle or grouped by sample. In some cases, the multiple compartments may include rows for samples and columns for particles. In some cases, the multiple compartments may be grouped by specific particle composition.

[0392] In some cases, a compartment may contain a single particle for a single biological sample. In some cases, a compartment may contain multiple particles for a single biological sample. In some cases, a compartment may contain a single particle for multiple biological samples. In some cases, a compartment may contain multiple particles for multiple biological samples.

[0393] In some cases, one or more transfer units can be configured to transport biological sample (e.g., secretome or exosome) to a single compartment, or to distribute sample to multiple compartments, or to sequentially transfer sample from one compartment to another.For example, 5 ml of sample can be distributed evenly to 500 compartments, resulting in separate 10 μl sample volumes.In some cases, sample can be mixed with reagent in the compartment.In some cases, sample can be subjected to dilution in the compartment.

[0394] In some cases, the device may include a magnet configured to apply a magnetic field to the contents of the compartment. In some cases, the applied magnetic field can separate magnetic and non-magnetic materials in the compartment. In some cases, the device may include a shaker. In some cases, the substrate can be shaken, vibrated, or sonicated by the instrument.

[0395] In some cases, the compartment may be operably coupled to one or more transfer units, for example as shown in FIG. 8. In some cases, one or more transfer units may be temporarily coupled to the compartment for transporting a portion of the biological sample from compartment 801. In some cases, one or more transfer units may be moved into the vicinity of the compartment, contacted with the biological sample in the compartment, collected a portion of the biological sample from the compartment, and then moved away from the compartment. In some cases, one or more transfer units may be temporarily coupled to the compartment for transport to compartment 802. In some cases, one or more transfer units may be moved into the vicinity of the compartment, coupled to the compartment, and transported to the compartment.

[0396] In some cases, one or more transfer units can be coupled to the compartment by a fluid connection, and in some cases, one or more transport units can actuate a pump to transport a portion of the biological sample in the compartment from the compartment to another component of the device.

[0397] FIG. 10 illustrates an apparatus according to some embodiments. In some cases, the apparatus may include a stage 1003. In some cases, components of the apparatus may be coupled to the stage 1003. In some cases, the stage 1003 may include one or more supports 1004 coupled thereto. In some cases, the one or more supports 1004 may be configured to move the apparatus. For example, in the apparatus illustrated in FIG. 10, the supports 1004 include casters 1005. In some cases, components of the apparatus may be coupled to the one or more supports 1004. In some cases, the apparatus may include a housing 1001. In some cases, components of the apparatus may be disposed inside the housing. In some cases, the apparatus may include a display 1002 coupled thereto. In some cases, the housing may include one or more supports coupled thereto. In some cases, the apparatus may include a rail. In some cases, components of the apparatus may be movably coupled to the rail.

[0398] In some cases, the device may include one or more transfer units 1101, 1103. In some cases, the transfer units may be configured to transport liquid samples. FIG. 11 shows multiple transfer units according to some embodiments. In some cases, the transfer units may be configured to transport solid samples. In some cases, the transfer units may include a pipette 1102. In some cases, the transfer units may include multiple pipettes. In some cases, the transfer units may include a pump. In some cases, the transfer units may be movable. In some cases, the transfer units may include rails. In some cases, the transfer units may include a motor configured to move the transfer units across the rails. In some cases, the transfer units may include multiple rails such that the transfer units are movable in at least two dimensions. In some cases, the transfer units may include multiple rails such that the transfer units are movable in at least three dimensions. In some cases, the transfer units may include a robotic arm. In some cases, the transfer units may include a gripper 1104. In some cases, the gripper may be configured to transfer to one or more compartments.

[0399] FIG. 12 shows an illustration of the device components. In some cases, the device may include a filtration system 1201. In some cases, the filtration system may include a vacuum. In some cases, the filtration system may include a pump. In some cases, the device may include a magnetic separation system 1202. In some cases, the magnetic separation system may include a magnet. In some cases, the magnetic separation system may be configured to couple with one or more compartments. In some cases, the device may include a cooler 1203. In some cases, the device may include a heater, a shaker, or both 1204. In some cases, the device may include a ruler 1205. In some cases, the device may include a work surface 1206. In some cases, the device may include a work deck 1207.

[0400] In some cases, the sample storage unit may be operably coupled to one or more transfer units. In some cases, one or more transfer units may be temporarily coupled to the sample storage unit to transport a portion of the biological sample from the sample storage unit. In some cases, one or more transfer units may be moved into the vicinity of the sample storage unit, contacted with the biological sample in the sample storage unit, collected a portion of the biological sample from the sample storage unit, and then moved away from the sample storage unit, as shown, for example, in FIG. 6.

[0401] In some cases, one or more transfer units may be fluidly coupled to the sample storage unit, for example as shown in Figure 7. In some cases, one or more transport units may actuate a pump to transport a portion of the biological sample in the sample storage unit from the sample storage unit to another component of the device.

[0402] FIG. 13 shows a layout of device components according to some embodiments. In some cases, the device may include a sample storage chamber or well 1316 configured to receive and hold a biological sample. In some cases, the sample storage chamber or well may be configured to receive and hold at least 1, 2, 4, 8, 16, 32, 64, 96, 128, or 256 distinct biological samples. In some cases, the device may include a particle storage chamber or well 1317 configured to receive and hold one or more particles. In some cases, the particle storage chamber or well may be configured to receive and hold at least 1, 2, 4, 8, 16, 32, 64, 96, 128, or 256 distinct particles. In some cases, the device may include multiple plates. In some cases, the device may include a cleanup plate 1301, a sample preparation plate 1304, an intermediate plate 1305, a peptide collection plate 1313, or any combination thereof. In some cases, the device may include multiple reagent storage chambers or wells. In some cases, the device may include reagent containing chambers or wells for wash solutions 1302, cleanup reagents 1303, control diluent solutions 1306, denaturing reagents 1307, reducing reagents 1308, alkylating reagents 1309, water 1310, trypsin / lysis reagents 1315, or any combination thereof. In some cases, the device may include empty slots 1311 for additional components. In some cases, the device may include a rack for pipette tips 1314. In some cases, the device may include one or more cell culture chambers 1318.

[0403] In some cases, the device may include one or more sensors. In some cases, the one or more sensors may include an optical sensor. In some cases, the optical sensor may include a microscope. In some cases, the optical sensor may include a camera. In some cases, the optical sensor may be configured to acquire an image of the cells. In some cases, the optical sensor may be configured to acquire an image of the cell culture chamber. Proteomic analysis

[0404] As used herein, "proteome 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 of the present disclosure for assaying using one or more surfaces. In some cases, the surface may include a surface of a material with a large surface area, such as a nanoparticle, a particle, a microparticle, or a porous material. As used herein, "surface" may refer to a surface for assaying polyamino acids. When particle composition, physical properties, or uses thereof are described herein, it should be understood that in some cases, the surface of the particle may include the same composition, the same physical properties, or the same uses thereof. Similarly, when surface composition, physical properties, or uses thereof are described herein, it should be understood that the particle may include a surface that includes the same composition, the same physical properties, or the same uses thereof.

[0405] Materials for particles and surfaces 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 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 (SPIONs). In some cases, the magnetic particles can be ferromagnetic particles, ferrimagnetic particles, paramagnetic particles, superparamagnetic particles, or any combination thereof (e.g., the particles can include ferromagnetic and ferrimagnetic materials).

[0406] The present disclosure describes a panel of particles or surfaces. In some cases, the panel may include more than one distinct surface type. The panels described herein may vary the number of surface types and the diversity of surface types within a single panel. For example, the surfaces within the panel may vary based on size, polydispersity, shape and morphology, surface charge, surface chemistry and functionalization, and substrate. In some cases, the panel may be incubated with a sample and analyzed for polyamino acids, polyamino acid concentrations, nucleic acids, nucleic acid concentrations, or any combination thereof. In some cases, the polyamino acids in the sample are adsorbed onto the distinct surfaces to form one or more adsorbed layers of biomolecules. The identity of the biomolecules and the concentration of the biomolecules in the one or more adsorbed layers may depend on the physical properties of the distinct surfaces and the physical properties of the biomolecules. Thus, each surface type within a panel may have differently adsorbed biomolecules due to the adsorption of a different set of biomolecules, different concentrations of a particular biomolecule, or a combination thereof. Each surface type within a panel may have mutually exclusive adsorbed biomolecules or may have overlapping adsorbed biomolecules.

[0407] In some cases, the panels disclosed herein can be used to identify the number of distinct biomolecules disclosed herein across a wide dynamic range in a given biological sample. For example, the panels can enrich for a subset of biomolecules in a sample, thereby identifying the biomolecules across a wide dynamic range (e.g., secretome or exosome) where they are present in the sample. In some cases, enrichment can be selective, for example, enriching for biomolecules within a subset, but not enriching and / or depleting biomolecules outside the subset. In some cases, the subsets can include proteins with different post-translational modifications. For example, a first particle type in a particle panel can enrich a protein or a group of proteins with a first post-translational modification, a second particle type in a particle panel can enrich the same protein or the same group of proteins with a second post-translational modification, and a third particle type in a particle panel can enrich the same protein or the same group of proteins that lack post-translational modification. In some cases, a panel comprising any number of distinct particle types disclosed herein enriches and identifies a single protein or a group of proteins by binding to different domains, sequences, or epitopes of the protein or group of proteins.For example, a first particle type in the particle panel can enrich a protein or a group of proteins by binding to a first domain of the protein or group of proteins, and a second particle type in the particle panel can enrich the same protein or the same group of proteins by binding to a second domain of the protein or group of proteins.In some cases, a panel comprising any number of distinct particle types disclosed herein can enrich and identify biomolecules over a dynamic range of at least 5, 6, 7, 8, 9, 10, 15, or 20 orders of magnitude.In some cases, a panel comprising any number of distinct particle types disclosed herein can enrich and identify biomolecules over a dynamic range of up to 5, 6, 7, 8, 9, 10, 15, or 20 orders of magnitude.

[0408] A panel can have more than one surface type. Increasing the number of surface types in a panel can be one way to increase the number of proteins that can be identified in a given sample.

[0409] The particle or surface may comprise a polymer, which may constitute a core material (e.g., the core of the particle may comprise a particle), a layer (e.g., the particle may include a layer of polymer disposed 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 of polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, or polyamine, polyalkylene glycol (e.g., polyethylene glycol (PEG)), polyester (e.g., poly(lactide-co-glycolide) (PLGA), polylactic acid, or polycaprolactone), or copolymers of two or more polymers, such as copolymers of polyalkylene glycol (e.g., PEG) and polyester (e.g., PLGA), or any combination thereof. The polymers may include crosslinks. The multiple polymers within the particle may be phase separated or may include some degree of phase separation.

[0410] Lipids that may be used to form the particles or surfaces of the present disclosure include, for example, cationic, anionic, and neutrally charged lipids. For example, the particles and / or surfaces may be formed using lipids such as dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS), phosphatidylglycerol, cardiolipin, diacylphosphatidylethanolamine ... lysylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, di Oleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidyl dipalmitoyl oleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE),The phosphatidyl ester may be composed of any one of 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, cholesterol, and any combination thereof.

[0411] The particle panel may include a combination of particles with silica and polymer surfaces. For example, the particle panel may include SPIONs coated with a thin layer of silica, SPIONs coated with poly(dimethylaminopropyl methacrylamide) (PDMAPMA), and SPIONs coated with poly(ethylene glycol) (PEG). The particle panel consistent with the present disclosure may also include two or more particles selected from the group consisting of silica-coated SPIONs, N-(3-trimethoxysilylpropyl)diethylenetriamine-coated SPIONs, PDMAPMA-coated SPIONs, carboxyl-functionalized polyacrylic acid-coated SPIONs, amino-surface-functionalized SPIONs, polystyrene carboxyl-functionalized SPIONs, silica particles, and dextran-coated SPIONs. The particle panel consistent with the present disclosure includes carboxylic acid particles without surfactant, carboxyl-functionalized polystyrene particles, silica-coated particles, silica particles, dextran-coated particles, oleic acid-coated particles, boronized 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 A particle panel consistent with the present disclosure may also include two or more particles selected from the group consisting of particles coated with N-(3-trimethoxysilylpropyl)diethylenetriamine, particles coated with poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA), sugar phosphate functionalized polystyrene particles, amine functionalized polystyrene particles, polystyrene carboxyl functionalized particles, ubiquitin functionalized polystyrene particles, dextran coated particles, or any combination thereof.

[0412] A particle panel consistent with the present disclosure may include silica-functionalized particles, amine-functionalized particles, silicon alkoxide-functionalized particles, carboxylic acid-functionalized particles, and benzyl or phenyl-functionalized particles. A particle panel consistent with the present disclosure may include silica-functionalized particles, amine-functionalized particles, silicon alkoxide-functionalized particles, polystyrene-functionalized particles, and saccharide-functionalized particles. A particle panel consistent with the present disclosure may include silica-functionalized particles, N-(3-trimethoxysilylpropyl)diethylenetriamine-functionalized particles, PDMAPMA-functionalized particles, dextran-functionalized particles, and polystyrene carboxyl-functionalized particles. A particle panel consistent with the present disclosure may include five particles including silica-functionalized particles, amine-functionalized particles, and silicon alkoxide-functionalized particles.

[0413] The distinct surfaces or distinct particles of the present disclosure may differ in one or more physicochemical properties. The one or more physicochemical properties are selected from the group consisting of composition, size, surface charge, hydrophobicity, hydrophilicity, roughness, density, surface functionalization, surface topography, surface curvature, porosity, core material, shell material, shape, and any combination thereof. The surface functionalization may include polymer functionalization, small molecule functionalization, or any combination thereof. The small molecule functionalization may include aminopropyl functionalization, amine functionalization, boronic acid functionalization, carboxylic acid functionalization, alkyl group functionalization, N-succinimidyl ester functionalization, monosaccharide functionalization, phosphate sugar functionalization, sulfurylated sugar functionalization, ethylene glycol functionalization, streptavidin functionalization, methyl ether functionalization, trimethoxysilylpropyl functionalization, silica functionalization, triethoxypropylaminosilane functionalization, thiol functionalization, PCP functionalization, citric acid functionalization, lipoic acid functionalization, ethyleneimine functionalization. The particle panel may include a plurality of particles having a plurality of small molecule functionalizations selected from the group consisting of silica-functionalized, trimethoxysilylpropyl-functionalized, dimethylaminopropyl-functionalized, sugar phosphate-functionalized, amine-functionalized, and carboxyl-functionalized.

[0414] Small molecule functionalization may include polar functional groups. Non-limiting examples of polar functional groups include carboxyl groups, hydroxyl groups, thiol groups, cyano groups, nitro groups, ammonium groups, imidazolium groups, sulfonium groups, pyridinium groups, pyrrolidinium groups, phosphonium groups, or any combination thereof. In some embodiments, the functional groups are acidic functional groups (e.g., sulfonic acid groups, carboxyl groups, etc.), basic functional groups (e.g., amino groups, cyclic secondary amino groups (e.g., pyrrolidyl groups and piperidyl groups, etc.), pyridyl groups, imidazole groups, guanidine groups, etc.), carbamoyl groups, hydroxyl groups, aldehyde groups, etc.

[0415] The small molecule functionalization may include ionic or ionizable functional groups. Non-limiting examples of ionic or ionizable functional groups include ammonium, imidazolium, sulfonium, pyridinium, pyrrolidinium, and phosphonium groups. The small molecule functionalization may include polymerizable functional groups. Non-limiting examples of polymerizable functional groups include vinyl and (meth)acrylic groups. In some embodiments, the functional groups are pyrrolidyl acrylate, acrylic acid, methacrylic acid, acrylamide, 2-(dimethylamino)ethyl methacrylate, hydroxyethyl methacrylate, and the like.

[0416] Surface functionalization may include charge. For example, particles may be functionalized to have a net neutral surface charge, a net positive surface charge, a net negative surface charge, or a zwitterionic surface. Surface charge may be determinative of the type of biomolecule collected on the particle. Thus, optimization of particle panels may include selecting particles with different surface charges, which may increase the number of different proteins collected on the particle panel, as well as increase the likelihood that the biological state of the sample will be identified. Particle panels may include positively charged particles and negatively charged particles. Particle panels may include positively charged particles and neutral particles. Particle panels may include positively charged particles and zwitterionic particles. Particle panels may include neutral particles and negatively charged particles. Particle panels may include neutral particles and zwitterionic particles. Particle panels may include negatively charged particles and zwitterionic particles. Particle panels may include positively charged particles, negatively charged particles, and neutral particles. The particle panel may include positively charged particles, negatively charged particles, and zwitterionic particles.The particle panel may include positively charged particles, neutral particles, and zwitterionic particles.The particle panel may include negatively charged particles, neutral particles, and zwitterionic particles.

[0417] The particles may contain a single surface, e.g., a specific small molecule, or multiple surface functionalizations, e.g., multiple different small molecules. Surface functionalization may affect the composition of the biomolecular corona of the particle. Such surface functionalizations may include small molecule functionalizations or polymer functionalizations. Surface functionalizations may be coupled to particle materials such as polymers, metals, metal oxides, inorganic oxides (e.g., silicon dioxide), or to another surface functionalization.

[0418] The surface functionalization may include binding molecules. The binding molecules may be small molecules, oligomers, or macromolecules. The binding molecules may include binding specificity for a group or class of analytes (e.g., a class of sugars or proteins). The binding molecules may include moderate binding specificity for that group or class of analytes. Conversely, the binding molecules may include a lack of affinity for a group or class of analytes, making the binding of these species less favorable compared to the same particle lacking the binding molecule. For example, the binding molecules may include a negative charge distribution that repels negatively charged nucleic acids, making the binding of those binding molecules less favorable.

[0419] The binding molecules may include peptides. Peptides are a wide variety of biomolecules that may include a wide range of physical and chemical properties. Depending on their composition, sequence, and chemical modifications, peptides may be hydrophilic, hydrophobic, amphiphilic, lipophilic, lipophobic, positively charged, negatively charged, zwitterionic, neutral, chaotropic, antichaotropic, reactive, redox active, inert, acidic, basic, inflexible, flexible, or any combination thereof. Thus, peptide surface functionalization can impart a variety of physicochemical properties to the particles. The particles may include a single peptide surface functionalization or multiple peptide surface functionalizations. A single peptide surface functionalization may include multiple identical or sequence-sharing peptides uniformly bound to the particles.

[0420] The surface functionalization may include small molecule functionalization, polymer functionalization, or a combination of two or more such functionalizations. In some cases, the polymer functionalization may include a biopolymer such as a protein or a polynucleotide (e.g., a 100-mer DNA molecule). The polymer functionalization may include a protein, a polynucleotide, or a polysaccharide, or may be comparable in size to any of the above-mentioned classes of species. In some cases, the surface functionalization may include an ionizable moiety. In some cases, the surface functionalization may include a pKa of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some cases, the surface functionalization may include a pKa of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some cases, the small molecule functionalization may include an organic small molecule such as an alcohol (e.g., octanol), an amine, an alkane, an alkene, an alkyne, a heterocycle (e.g., a piperidinyl group), a heteroaromatic group, a thiol, a carboxylic acid, a carbonyl, an amide, an ester, a thioester, a carbonate, a thiocarbonate, a carbamic acid, a thiocarbamic acid, an urea, a thiourea, a halogen, a sulfate, a phosphate, a monosaccharide, a disaccharide, a lipid, or any combination thereof. For example, the small molecule functionalization may include a sugar phosphate, a sugar acid, or a sulfurylated sugar.

[0421] In some cases, the polymer functionalization may include a particular form of attachment to the particle. In some cases, the macromolecule may be tethered to the particle via a linker. In some cases, the linker may hold the macromolecule in the vicinity of the particle, thereby restricting the movement and reorientation of the macromolecule relative to the particle, or may stretch the macromolecule away from the particle. In some cases, the linker may be inflexible (e.g., a polyolefin linker) or flexible (e.g., a nucleic acid linker). In some cases, the linker may be at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nm long. In some cases, the linker may be up to about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nm long. Thus, the surface functionalization on the particle may protrude beyond the initial corona associated with the particle. In some cases, surface functionalization can be located beneath or within the biomolecular corona formed on the particle surface. In some cases, macromolecules can be tethered to a specific location, for example, the C-terminus of a protein, or to several possible sites. For example, a peptide can be covalently attached to the particle via any of its surface-exposed lysine residues.

[0422] In some embodiments, the macromolecule may be peptide-modified. In some embodiments, the macromolecule comprises a thiol or an azide. In some embodiments, the surface comprises a peptide-modified macromolecule immobilized on the surface. In some embodiments, the macromolecule is covalently coupled to the surface. In some embodiments, the macromolecule is electrostatically coupled to the surface. In some embodiments, the macromolecule is coupled to the surface by a polymerization event. In some embodiments, the polymerization event comprises a reaction with a vinyl group on the surface.

[0423] In some embodiments, the peptide-modified macromolecule can be immobilized on a surface for identification, binding, or enrichment of biomolecules (e.g., proteins). In some embodiments, the surface can include a peptide-modified macromolecule, where the peptide includes a binding site and a protein that interacts with the peptide at the binding site. In some embodiments, a biological sample can be contacted with a surface that includes a peptide-modified macromolecule, where the peptide is configured to bind to the protein, thereby releasing a plurality of biomolecules from the surface.

[0424] In some cases, the particles can be contacted with a biological sample (e.g., a biological fluid) to form a biomolecular corona. In some cases, the biomolecular corona can include at least two biomolecules that do not share a common binding motif. The particles and biomolecular corona can be separated from the biological sample by, for example, centrifugation, magnetic separation, filtration, or gravity separation. The particle types and biomolecular corona can be separated from the biological sample using several separation techniques. Non-limiting examples of separation techniques include magnetic separation, column-based separation, filtration, spin column-based separation, centrifugation, ultracentrifugation, density or gradient-based centrifugation, gravity separation, or any combination thereof. Protein corona analysis can be performed on the separated particles and biomolecular corona. Protein corona analysis can include identifying one or more proteins in the biomolecular corona, for example, by mass spectrometry. In some cases, a single particle type can be contacted with the biological sample. In some cases, multiple particle types can be contacted with the biological sample. In some cases, multiple particle types can be combined and contacted with the biological sample in a single sample volume. In some cases, multiple particle types can be contacted sequentially with the biological sample and subsequent particle types can be contacted with the biological sample after separation from the biological sample. In some cases, the biomolecules adsorbed onto the particles can have a compressed (e.g., smaller) dynamic range compared to a given original biological sample.

[0425] In some cases, the particles of the present disclosure can be used to perform sequential interrogation of a sample by incubating a first particle type with the sample to form a biomolecular corona on the first particle type, isolating the first particle type, incubating a second particle type with the sample to form a biomolecular corona on the second particle type, isolating the second particle type, and repeating the interrogation (by incubating with the sample) and isolation for any number of particle types. In some cases, the biomolecular corona on each particle type used in sequential interrogation of a sample can be analyzed by protein corona analysis. The biomolecular content of the supernatant can be analyzed after sequential interrogation with one or more particle types.

[0426] In some cases, the methods disclosed herein can identify a large number of species of unique biomolecules (e.g., proteins) in a biological sample (e.g., biological fluid). In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecules. In some cases, the surfaces disclosed herein can be incubated with a biological sample to adsorb up to 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecules. In some cases, the surface disclosed herein can be incubated with a biological sample to adsorb at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique biomolecular groups. In some cases, the surface 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 biomolecular groups. In some cases, several different types of surfaces can be used separately or in combination to identify multiple proteins in a particular biological sample. In other words, the surface can be multiplexed to bind and identify multiple biomolecules in a biological sample.

[0427] In some cases, the disclosed methods can identify a large number of unique proteoforms 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 proteoforms. In some cases, the methods can identify up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 unique proteoforms. In some cases, a surface 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 proteoforms. In some cases, the surface 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 proteoforms. In some cases, several different types of surfaces can be used separately or in combination to identify a large number of proteins in a particular biological sample. In other words, the surface can be multiplexed to bind and identify a large number of biomolecules in a biological sample.

[0428] The biomolecules collected on the particles can be subjected to further analysis. In some cases, the method can include collecting the biomolecular corona or a subset of biomolecules from the biomolecular corona. In some cases, the collected biomolecular corona or a subset of biomolecules from the collected biomolecular corona can be subjected to further particle-based analysis (e.g., particle adsorption). In some cases, the collected biomolecular corona or a subset of biomolecules from the collected biomolecular corona can be purified or fractionated (e.g., by chromatographic methods). In some cases, the collected biomolecular corona or a subset of biomolecules from the collected biomolecular corona can be analyzed (e.g., by mass spectrometry).

[0429] In some cases, the panels disclosed herein can be used to identify several proteins, peptides, protein groups, or protein classes using the protein analysis workflows described herein (e.g., protein corona analysis workflows). In some cases, protein analysis can include contacting a sample with a distinct surface type (e.g., a particle panel), forming a layer of adsorbed biomolecules on the distinct surface type, and identifying the biomolecules in the layer of adsorbed biomolecules (e.g., by mass spectrometry). The feature intensity disclosed herein can refer to the intensity of individual spikes ("features") seen in a plot of mass-to-charge ratio vs. intensity from a mass spectrometry run of a sample. In some cases, these features can correspond to randomly ionized peptides and / or protein fragments. In some cases, the data analysis methods described herein can be used to sort the feature intensities 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 two or more proteins identified by a common peptide sequence. In some cases, a protein group may refer to one protein identified using a unique identification sequence. For example, if a common peptide sequence between two proteins (protein 1: XYZZX and protein 2: XYZYZ) is assayed in a sample, the protein group may be an "XYZ protein group" with two members (protein 1 and protein 2). In some cases, if a peptide sequence is unique to only one protein (protein 1), the protein group may be a "ZZX" protein group with one member (protein 1). In some cases, each protein group may be supported by more than one peptide sequence. In some cases, the protein detected or identified according to the present disclosure may refer to a separate protein detected in a sample (e.g., separate to other proteins detected using mass spectrometry).In some cases, analysis of proteins present in distinct coronas corresponding to distinct surface types in a panel gives rise to a large number of feature intensities. In some cases, this number decreases when the feature intensities are processed into distinct peptides, further decreased when the distinct peptides are processed into distinct proteins, and further decreased when the peptides are grouped into protein groups (two or more proteins that share a distinct peptide sequence).

[0430] In some cases, the methods disclosed herein include isolating one or more particle types from a sample or from more than one sample (e.g., biological samples or samples interrogated sequentially). A magnet can be used to rapidly isolate or separate particle types from a sample. Additionally, multiple spatially isolated samples can be processed in parallel. In some cases, the methods disclosed herein provide for the isolation or separation of particle types from unbound proteins in a sample. In some cases, particle types can be separated by various means, including but not limited to magnetic separation, centrifugation, filtration, or gravity separation. In some cases, a particle panel can be incubated with multiple spatially isolated samples, where each spatially isolated sample is in a well of a well plate (e.g., a 96-well plate). In some cases, the particles in each well of the well plate can be separated from unbound proteins present in the spatially isolated samples by placing the entire plate on a magnet. In some cases, this allows the superparamagnetic particles in the particle panel to be pulled down simultaneously. In some cases, the supernatant of each sample can be removed to remove unbound proteins. In some cases, these steps (incubation, pull-down) can be repeated to effectively wash the particles and thus remove residual background unbound proteins that may be present in the sample.

[0431] In some cases, the systems and methods disclosed herein can also reveal protein classes or interactions of protein classes.In some cases, protein classes can include a set of proteins that share a common function (e.g., amine oxidase or proteins involved in angiogenesis); proteins that share a common physiological, cellular, or subcellular localization (e.g., peroxisomal or membrane proteins); proteins that share a common cofactor (e.g., heme or flavoproteins); proteins that correspond to a particular biological state (e.g., proteins related to hypoxia); proteins that contain a particular structural motif (e.g., cupin fold); functionally related proteins (e.g., part of the same metabolic pathway); or proteins that have post-translational modifications (e.g., ubiquitinated or citrullinated proteins).In some cases, protein classes can contain at least 2 proteins, 5 proteins, 10 proteins, 20 proteins, 40 proteins, 60 proteins, 80 proteins, 100 proteins, 150 proteins, 200 proteins, or more.

[0432] In some cases, proteomic data of a biological sample can be identified, measured, and quantified using a number of different analytical techniques. For example, SDS-PAGE or any gel-based separation technique can be used to generate proteomic data. In some cases, immunoassays such as ELISA can also be used to identify, measure, and quantitate 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 quantitate proteomic data.

[0433] In some cases, the assay may include protein collection of the particles, protein digestion, and mass spectrometry (e.g., MS, LC-MS, LC-MS / MS). In some cases, the digestion may include chemical digestion, for example, digestion with cyanogen bromide or 2-nitro-5-thiocyanatobenzoic acid (NTCB). In some cases, the digestion may include enzymatic digestion, for example, digestion with trypsin or pepsin. In some cases, the digestion may include enzymatic digestion with multiple proteases. In some cases, digestion may include a protease selected from the group consisting of trypsin, chymotrypsin, Glu C, Lys C, elastase, subtilisin, proteinase K, thrombin, factor X, Arg C, papain, Asp N, thermolysin, pepsin, aspartyl protease, cathepsin D, zinc metalloprotease, glycoprotein endopeptidase, proline, aminopeptidase, prenyl protease, caspase, kex2 endoprotease, or any combination thereof. In some cases, digestion may cleave peptides at random positions. In some cases, digestion may cleave peptides at specific positions (e.g., methionine) or sequences (e.g., glutamic acid-histidine-glutamic acid). In some cases, digestion may allow for the identification of similar proteins. For example, in an assay, eight distinct proteins may be resolved as a single protein group using a first digestion method, and eight separate proteins with distinct signals using a second digestion method. In some cases, digestion may produce an average peptide fragment length of 8-15 amino acids. In some cases, digestion may produce an average peptide fragment length of 12-18 amino acids. In some cases, digestion may produce an average peptide fragment length of 15-25 amino acids. In some cases, digestion may produce an average peptide fragment length of 20-30 amino acids. In some cases, digestion may produce an average peptide fragment length of 30-50 amino acids.

[0434] In some cases, the assay can rapidly generate biological samples for analysis.In some cases, the biological samples can contain proteolytic peptides.In some cases, the method of the present disclosure can generate biological samples from input biological samples (e.g., buccal or nasal smears, plasma, secretome, or tissue) in less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 12 hours, less than about 16 hours, less than about 20 hours, less than about 24 hours, or less than about 48 hours. In some cases, the methods of the disclosure can generate a biological sample starting from an input biological sample (e.g., a buccal or nasal smear, plasma, secretome, or tissue) in less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 12 hours, less than about 16 hours, less than about 20 hours, less than about 24 hours, or less than about 48 hours.

[0435] In some cases, the assay can rapidly generate and analyze proteomic data. In some cases, the method of the present disclosure can generate and obtain proteomic data starting from an input biological sample (e.g., buccal or nasal smear, plasma, or tissue) in less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 12 hours, less than about 16 hours, less than about 20 hours, less than about 24 hours, or less than about 48 hours. In some cases, the disclosed methods can generate and analyze proteomic data in less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 12 hours, less than about 16 hours, less than about 20 hours, less than about 24 hours, or less than about 48 hours starting from an input biological sample (e.g., buccal or nasal smear, plasma, or tissue). In some cases, the analysis can include identifying protein groups. In some cases, the analysis can include identifying protein classes. In some cases, the analysis can include quantifying the abundance of a biomolecule, peptide, protein, protein group, or protein class. In some cases, the analysis can include identifying the ratio of abundance of two biomolecules, peptides, proteins, protein groups, or protein classes. In some cases, the analysis can include identifying a biological state.

[0436] Examples of particle types of the present disclosure include carboxylate (citric acid) 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-benzenetetracarboxylic acid coated SPIONs, poly(vinylbenzyltrimethylammonium chloride) (PVBTMAC) coated SPIONs, carboxylate, PAA coated SPIONs, and carboxylate, PAA coated SPIONs. The surface-coated particles may be: SPIONs coated with poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), carboxylic acid microparticles, polystyrene carboxyl functionalized particles, carboxylic acid coated particles, silica particles, carboxylic acid particles with a diameter of about 150 nm, amino surfaced microparticles with a diameter of about 0.4-0.6 μm, silica amino functionalized microparticles with a diameter of about 0.1-0.39 μm, Jeffamine surfaced particles with a diameter of about 0.1-0.39 μm, polystyrene microparticles with a diameter of about 2.0-2.9 μm, silica particles, proprietary coated carboxylated particles with a diameter of about 50 nm, particles coated with a dextran-based coating with a diameter of about 0.13 μm, or silica silanol coated particles with low acidity. In some cases, the particles may lack functionalized specific binding moieties for specific binding to their surface. In some cases, the particles may lack functionalized proteins for specific binding to their surface. In some cases, the surface functionalized particle does not include an antibody or T cell receptor, a chimeric antigen receptor, a receptor protein, or a variant or fragment thereof. In some cases, the ratio of surface area to mass can be a determining factor of the particle's properties. The particle of the present disclosure can be a nanoparticle. The nanoparticle of the present disclosure can be about 10 nm to about 1000 nm in diameter.For example, the nanoparticles disclosed herein can have a diameter of at least 10 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, from 10 nm to 50 nm, from 50 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 250 nm, from 250 nm to 300 nm, from 300 nm to 350 nm, from 350 nm to 400 nm, from 400 nm to 450 nm, from 450 nm to 500 nm, from 500 nm to 550 nm, from 550 nm to 600 nm, The nanoparticles may be 600nm to 650nm, 650nm to 700nm, 700nm to 750nm, 750nm to 800nm, 800nm ​​to 850nm, 850nm to 900nm, 100nm to 300nm, 150nm to 350nm, 200nm to 400nm, 250nm to 450nm, 300nm to 500nm, 350nm to 550nm, 400nm to 600nm, 450nm to 650nm, 500nm to 700nm, 550nm to 750nm, 600nm to 800nm, 650nm to 850nm, 700nm to 900nm, or 10nm to 900nm. The nanoparticles may be less than 1000nm in diameter. The particles of the present disclosure may be microparticles. The microparticles may be particles having a diameter of from about 1 μm to about 1000 μm.For example, the microparticles disclosed herein can have a diameter of at least 1 μm, at least 10 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, 10 μm to 50 μm, 50 μm to 100 μm, 100 μm to 150 μm, 150 μm to 200 μm, 200 μm to 250 μm, 250 μm to 300 μm, 300 μm to 350 μm, 350 μm to 400 μm, 400 μm to 450 μm, 450 μm to 500 μm, 500 μm to 550 μm, 550 μm to 600 μm, μm, 600μm to 650μm, 650μm to 700μm, 700μm to 750μm, 750μm to 800μm, 800μm to 850μm, 850μm to 900μm, 100μm to 300μm, 150μm to 350μm, 200μm to 400μm, 250μm to 450μm, 3 The particle size may be from 00 μm to 500 μm, 350 μm to 550 μm, 400 μm to 600 μm, 450 μm to 650 μm, 500 μm to 700 μm, 550 μm to 750 μm, 600 μm to 800 μm, 650 μm to 850 μm, 700 μm to 900 μm, or 10 μm to 900 μm. The microparticles may be less than 1000 μm in diameter. The particles disclosed herein have a surface area to mass ratio of 3 to 30 cm. 2 / mg, 5-50cm 2 / mg, 10-60cm 2 / mg, 15-70cm 2 / mg, 20-80cm 2 / mg, 30-100cm 2 / mg, 35-120cm 2 / mg, 40-130cm 2 / mg, 45-150cm 2 / mg, 50-160cm 2 / mg, 60-180cm 2 / mg, 70-200cm 2 / mg, 80-220cm 2 / mg, 90-240cm2 / mg, 100-270cm 2 / mg, 120-300cm 2 / mg, 200-500cm 2 / mg, 10-300cm 2 / mg, 1-3000cm 2 / mg, 20-150cm 2 / mg, 25-120cm 2 / mg, or 40-85cm 2 Small particles (e.g., diameters of 50 nm or less) have significantly higher surface area to mass ratios, which may be due in part to the fact that the dependence of mass on diameter is of a higher order than the dependence of surface area on diameter. In some cases (e.g., for small particles), particles may have surface area to mass ratios of 200-1000 cm 2 / mg, 500-2000cm 2 / mg, 1000-4000cm 2 / mg, 2000-8000cm 2 / mg, or 4000-10000cm 2 In some cases (e.g., for larger particles), the particles may have a surface area to mass ratio of 1-3 cm 2 / mg, 0.5-2cm 2 / mg, 0.25-1.5cm 2 / mg, or 0.1-1cm 2 The particle may be 100% by weight / mg. The particle may include a wide range of physical properties. The particle physical properties may include composition, size, surface charge, hydrophobicity, hydrophilicity, amphiphilicity, surface functionality, surface topography, surface curvature, porosity, core material, shell material, shape, zeta potential, and any combination thereof. The particle may have a core-shell structure. In some cases, the core material may include metals, polymers, magnetic materials, paramagnetic materials, oxides, and / or lipids. In some cases, the shell material may include metals, polymers, magnetic materials, oxides, and / or lipids. Proteomics Information

[0437] In some cases, proteomic information or data may refer to information about substances that include peptide and / or protein components. In some cases, proteomic information may include primary structure information, secondary structure information, tertiary structure information, or quaternary structure information about peptides or proteins. In some cases, proteomic information may include information about protein-ligand interactions, where the ligand may include any one of 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.

[0438] In some cases, proteomic information may include information about a single cell, a tissue, an organ, a system of tissues and / or organs (such as the cardiovascular system, respiratory system, digestive system, or nervous system), or an entire multicellular organism. In some cases, proteomic information may include information about an individual (e.g., an individual human or an individual bacterium), or a population of individuals (e.g., a human diagnosed with cancer or a bacterial colony). Proteomic information may include information from various forms of life, including forms of life from Archaea, Bacteria, Eukarya, Protozoa, Chromista, Plantae, Fungi, or Animalia. In some cases, proteomic information may include information from viruses.

[0439] In some cases, proteomic information may include information related to exons and / or introns. In some cases, proteomic information may include information about variations in the primary structure, secondary structure, tertiary structure, or quaternary structure of peptides and / or proteins. In some cases, proteomic information may include information about variations in the expression of exons, including alternative splicing variations, structural variations, 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 regulatory chemical)-related information, or substrate-related information of peptides and / or proteins.

[0440] In some cases, the proteomic information may include information related to various proteoforms in a 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 splicing variants, allelic variants, post-translational modification variants, or any combination thereof. In some cases, the peptide variants or protein variants may include post-translational modifications. In some cases, the post-translational modification can be acylation, alkylation, prenylation, flavinylation, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, sumoylation, neddylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hypusine formation, beta-lysine addition, acetylation, formylation, methylation, including amidation, amide bond formation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester formation, phosphoramidate formation, adenylation, uridinylation, propionylation, pyroglutamate formation, glutathionylation, sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, glycation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, pegylation, citrullination, deamidation, eliminylation, disulfide bond formation, proteolytic cleavage, isoaspartate formation, racemization, protein splicing, chaperone-mediated folding, or any combination thereof. Composition Improvement Assay

[0441] In some cases, the method of the present disclosure may include the use of a composition-improved assay. In some cases, the non-targeted assay may be a composition-improved assay. In some cases, the composition-improved assay may improve access to a subset of biomolecules in a biological sample. In some cases, the composition-improved assay may improve detection of a subset of biomolecules in a biological sample. In some cases, the composition-improved assay may improve identification of a subset of biomolecules in a biological sample. In some cases, the subset of biomolecules may be low abundance biomolecules. In some cases, the subset of biomolecules may be rare biomolecules. In some cases, the rare biomolecules may be biomolecules that are rarely expressed by cells. In some cases, the composition-improved 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.

[0442] In some cases, the composition improvement assay may include providing one or more of the surface regions comprising one or more surface types. In some cases, the composition improvement assay may include contacting a biological sample with one or more surface regions to obtain a set of biomolecules adsorbed on the one or more surface regions. In some cases, the set of adsorbed biomolecules may include a reduced or compressed dynamic range compared to the same biomolecules in a non-adsorbed biological sample. In some cases, the concentration of low abundance biomolecules in the biological sample may be increased on one or more surface regions. In some cases, the concentration of high abundance biomolecules in the biological sample may be decreased on one or more surface regions. In some cases, the composition improvement assay may include desorbing at least a portion of the set of adsorbed biomolecules from the one or more surface regions to obtain a set of polyamino acids. In some cases, the composition improvement assay may include contacting a biological sample with one or more surface regions to capture a set of biomolecules on the one or more surface regions. In some cases, the composition improvement assay may include releasing at least a portion of the set of biomolecules from the one or more surface regions to obtain a set of polyamino acids. In some cases, the one or more surface regions are disposed on a single continuous surface. In some cases, the one or more surface regions are disposed on one or more individual surfaces. In some cases, the one or more individual surfaces are the surfaces of one or more particles. In some cases, the one or more particles may include nanoparticles. In some cases, the one or more particles may include microparticles. In some cases, the one or more particles may include porous particles. In some cases, the one or more particles may include bifunctional particles, trifunctional particles, or N-functional particles.

[0443] In some cases, the composition improvement assay may include providing a plurality of surface regions comprising a plurality of surface types. In some cases, the composition improvement assay may include contacting a biological sample with the plurality of surface regions to obtain a set of biomolecules adsorbed to the plurality of surface regions. In some cases, the composition improvement assay may include desorbing at least a portion of the set of adsorbed biomolecules from the plurality of surface regions to obtain a set of polyamino acids. In some cases, the composition improvement assay may include contacting a biological sample with the plurality of surface regions to capture the set of biomolecules on the plurality of surface regions. In some cases, the composition improvement assay may include releasing at least a portion of the set of biomolecules from the plurality of surface regions to obtain a set of polyamino acids. In some cases, the plurality of surface regions are disposed on a single contiguous surface. In some cases, the plurality of surface regions are disposed on a plurality of separate surfaces. In some cases, the plurality of separate 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 particles, trifunctional particles, or N-functional particles. non-specific binding

[0444] The surface may bind to the biomolecule by randomly selective adsorption (e.g., adsorption of the biomolecule or biomolecules upon contacting the particle with a biological sample containing the biomolecule or biomolecules, where the adsorption is randomly selective depending on factors including, for example, the physicochemical properties of the particle) or non-specific binding. Non-specific binding may refer to a class of binding interactions that excludes specific binding. Examples of specific binding may include protein-ligand binding interactions, antigen-antibody binding interactions, nucleic acid hybridization, or binding interactions between a template molecule and a target molecule, where the template molecule provides a sequence or 3D structure that favors binding to a target molecule that contains a complementary sequence or complementary 3D structure and disfavors binding to a non-target molecule(s) that does not contain a complementary sequence or complementary 3D structure.

[0445] Non-specific binding may include one or a combination of a wide variety of chemical and physical interactions and effects. Non-specific binding may include electromagnetic forces, such as electrostatic interactions, London dispersion forces, van der Waals interactions, or dipole-dipole interactions (e.g., both between permanent and induced dipoles). Non-specific binding may be mediated by covalent bonds, such as disulfide bridges. Non-specific binding may be mediated by hydrogen bonds. Non-specific binding may include solvophilic effects (e.g., hydrophobic effects), where one entity is repelled from the solvent environment and pushed toward the boundary of the solvent, such as the surface of another entity. Non-specific binding may include depletion interactions, or entropic effects, such as the rise of thermal energy above a critical solution temperature (e.g., a lower critical solution temperature). Non-specific binding may include kinetic effects, where one binding molecule may have faster binding kinetics than another binding molecule.

[0446] 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 are adsorbed to a single particle, or up to 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 are adsorbed to a single particle). The multiple targets may have similar non-specific binding affinities that fall 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 may be in contrast to specific binding, which may involve a higher binding affinity for a given target molecule than for a non-target molecule.

[0447] Biomolecules may be adsorbed to the surface by non-specific binding at various densities to the surface. In some cases, biomolecules or proteins may be adsorbed to the 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 may 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 may 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 may 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 may 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 may be adsorbed at a density of at most 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 2In some cases, the biomolecule or protein may 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 2 In some cases, the biomolecule or protein may 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 may 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 may 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 It can be adsorbed at a density of

[0448] The adsorbed biomolecules can include a variety 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 different proteins.

[0449] In some cases, the proteins in the biological sample may include a concentration of 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 proteins in the biological sample may include a concentration of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 orders of magnitude. Computer Systems

[0450] The present disclosure provides a computer system programmed to implement the methods of the present disclosure. Figure 25 shows a computer system 2501 programmed or otherwise configured to, for example, culture cells, transport biological samples, perform assays for biomolecules, run analytical instruments, capture images of cell cultures, or any combination thereof.

[0451] The computer system 2501 may coordinate various aspects of the analysis, calculations, and production of the present disclosure, such as for culturing cells, transporting biological samples, performing assays for biomolecules, running analytical instruments, capturing images of cell cultures, or any combination thereof. The computer system 2501 may be a user's electronic device or may be a computer system located remotely from the electronic device. The electronic device may be a portable electronic device.

[0452] The computer system 2501 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 2505, which may be a single-core processor or a multi-core processor, or multiple processors for parallel processing. The computer system 2501 also includes memory or memory locations 2510 (e.g., random access memory, read-only memory, flash memory), electronic storage 2515 (e.g., hard disk), communication interface 2520 (e.g., network adapter) for communicating with one or more other systems, and peripherals 2525, such as cache, other memory, data storage, and / or electronic display adapters. The memory 2510, storage 2515, interface 2520, and peripherals 2525 are in communication with the CPU 2505 through a communication bus (solid lines), such as a motherboard. The storage 2515 may be a data storage device (or data repository) for storing data. Computer system 2501 may be operatively coupled to a computer network ("network") 2530 via communication interface 2520. Network 2530 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet.

[0453] The network 2530 is, in some cases, a telecommunications and / or data network. The network 2530 may include one or more computer servers, thereby enabling distributed computing, such as cloud computing. For example, one or more computer servers may enable cloud computing through the network 2530 ("cloud") to perform various aspects of the analysis, calculation, and generation of the present disclosure, such as, for example, culturing cells, transporting biological samples, performing assays for biomolecules, running analytical instruments, capturing images of cell cultures, or any combination thereof. Such cloud computing may be provided by cloud computing platforms, such as, for example, Amazon Web Services (AWS), Microsoft Azure, Google Cloud Platform, and IBM Cloud. The network 2530 may, in some cases, implement a peer-to-peer network through the computer system 2501, thereby enabling devices coupled to the computer system 2501 to operate as clients or servers.

[0454] The CPU 2505 may include one or more computer processors and / or one or more graphic processing units (GPUs). The CPU 2505 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 2510. The instructions may be directed to the CPU 2505, which may then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 2505 may include fetch, decode, execute, and writeback.

[0455] The CPU 2505 may be part of a circuit, such as an integrated circuit. One or more other components of the system 2501 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0456] The storage device 2515 may store files such as drivers, libraries, and saved programs. The storage device 2515 may store user data, such as user settings and user programs. The computer system 2501 may in some cases include one or more additional data storage devices external to the computer system 2501, such as, for example, on a remote server in communication with the computer system 2501 over an intranet or the Internet.

[0457] The computer system 2501 may communicate with one or more remote computer systems through the network 2530. For example, the computer system 2501 may communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a telephone, a smartphone (e.g., Apple® iPhone®, Android®-enabled devices, Blackberry®), or a personal digital assistant. A user may access the computer system 2501 through the network 2530.

[0458] The computer system 2501 may be in communication with the devices described herein. The devices may include one or more sensors, illustrated in FIG. 25 as optical sensor 2560. The devices and / or optical sensor 2560 may be in communication with the computer system 2501 through a network 2530.

[0459] The methods described herein may be implemented by machine (e.g., a computer processor) executable code stored in an electronic storage location of the computer system 2501, such as memory 2510 or electronic storage device 2515. The machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by the processor 2505. In some cases, the code may be read from storage device 2515 and stored in memory 2510 for access by the processor 2505. In some situations, the electronic storage device 2515 may be omitted, in which case the machine executable instructions are stored in memory 2510.

[0460] The code may be precompiled and configured for use on a machine having a processor adapted to execute the code, or it may be compiled at run time. The code may be provided in a programming language that can be selected so that the code allows execution in a precompiled or compiled-on-the-fly fashion.

[0461] Aspects of the systems and methods provided herein (e.g., computer system 2501, etc.) may be embodied in programming. Various aspects of this technology may be considered to be "products" or "articles of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried or embodied in some type of machine-readable medium. The machine executable code may be stored in an electronic storage unit (e.g., memory (e.g., read-only memory, random access memory, flash memory), or hard disk. A "storage" type medium may include any or all tangible memory (e.g., various semiconductor memories, tape drives, disk drives, etc.) of a computer, processor, etc., or its associated modules, which may provide non-transitory storage at any point in time for the software programming. The entire or portions of the software may at times be communicated over the Internet or various other telecommunications networks. Such communications may be, for example, from one computer or processor to another, for example, via a management server, etc. The software may be loaded from a host computer to the computer platform of the application server. Thus, another type of medium that may carry software elements includes light waves, radio waves, or electromagnetic waves (e.g., those used across physical interfaces between local devices through wired and optical landline networks, and by various wireless connections). The physical elements that carry such waves (e.g., wired or wireless connections, optical connections, etc.) may also be considered media that carry software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0462] Thus, the machine-readable medium (e.g., computer executable code) may take many forms, including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include optical or magnetic disks, such as those that may be used to implement a database, such as any storage device in any computer, such as those shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer readable media thus include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium having a pattern of holes, RAM, ROM, PROM and EPROM, Flash EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which conveys such a carrier wave, or any other medium from which a computer can read code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0463] The computer system 2501 may include or be in communication with an electronic display 2535 that includes a user interface (UI) 2540, for example, culturing cells, transporting biological samples, performing assays for biomolecules, running analytical instruments, capturing images of the cell culture, or any combination thereof. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0464] The computer system 2501 may further include additional peripheral devices.

[0465] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by the central processing unit 2505. The algorithms can, for example, culture cells, transport biological samples, perform assays for biomolecules, run analytical instruments, capture images of cell cultures, or any combination thereof.

[0466] Although preferred aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are presented by way of example only. Numerous variations, changes, and substitutions will be readily apparent to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in the practice of the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby. EXAMPLES

[0467] The following examples are provided to further illustrate some embodiments of the present disclosure and are not intended to limit the scope of the disclosure, and are of an illustrative nature, it will be understood that other procedures, methodologies, or techniques known to those skilled in the art may be substituted. Example 1 Comparison of mass spectrometry performed on improved secretome composition with that performed on direct digestion

[0468] This example describes experiments performed to test analytical improvements in performing mass spectrometry on particle-enhanced secretome compositions compared to direct digestion secretome compositions.

[0469] Hela S3 cells were cultured in F-12K medium supplemented with 10% fetal bovine serum and 1% PS solution. The cells were maintained at 37°C, 95% air and 5% CO2. The cell medium was removed from the cell culture wells by aspiration. The collected medium was centrifuged and then analyzed by Proteograph™ system.

[0470] Using an Orbitrap-2 (with a waters column), mass spectrometry was performed on (a) the secretome composition improved using particles (Proteograph™), (b) the secretome from direct digestion, (c) and a control sample of the HeLa digest. For (a), the particles used were NP-1, NP-2, NP-3, NP-4, and NP-5.

[0471] Figures 16A-16B show the mass of peptides and the number of protein groups identified by this experiment, respectively. The mass of peptides obtained by direct digestion (approximately 51.7 μg) far exceeded the mass obtained from the secretome composition using particles (approximately 3.11, 1.60, 1.68, 0.24, and 2.49 μg for NP-3, NP-2, NP-1, NP-5, and NP-4, respectively). However, the number of protein groups identified using the improved secretome composition (approximately 1694, 1614, 1945, 1959, and 1724 groups for NP-3, NP-2, NP-1, NP-5, and NP-4, respectively) was much higher than the number obtained by direct digestion (approximately 351 groups). These results are consistent with the enrichment of low-abundance proteins in the secretome composition by particles.

[0472] 17A-17B show the number of peptides and protein groups identified by mass spectrometry experiments, respectively. The number of peptides and protein groups identified using improved secretome composition (about 15,994 peptides and 2961 protein groups) was much higher than that identified by direct digestion (about 1061 peptides and 351 protein groups). The results show that using Proteograph™ to improve the composition of the secretome prior to mass spectrometry can result in significantly higher protein and / or peptide coverage than performing mass spectrometry compared to direct digestion.

[0473] Figure 18 shows the number of proteins that were exclusively identified in the improved secretome composition (3160) or in the direct digest (26), as well as the number of proteins that were identified in both experiments (325). The results show that by improving the composition of the secretome using Proteograph™ prior to mass spectrometry, almost all proteins that are detected in the direct digest can be detected.

[0474] 19A-19B show the spectral counts (representing protein abundance levels; higher spectral counts indicate higher protein abundance) and the count rank order of the proteins identified in the direct digest (neat conditioned medium) and improved secretome (Protegraph Assay), respectively. Each spot in the plot represents a protein detected at a certain abundance level based on the Hela DB database. Nearly 10-fold more proteins were observed with Proteograph™ compared to the neat medium alone, and proteins were observed across the entire HeLa proteome database concentration range, spanning at least 7 orders of magnitude. The identified proteins are mapped onto a line in FIG. 19A and FIG. 19B. These figures further illustrate that mass spectrometry performed with the improved secretome composition provides broad and deep coverage of proteins in the secretome. It should be noted that low abundance cytokines and other low abundance proteins were identified in the improved secretome composition.

[0475] Figure 20 shows the number of protein groups that are identified exclusively or mutually for the particles and direct digestion tested.The results show that in some cases, a smaller number of particles (e.g., two or three) can cover the majority of proteins and / or protein groups in secretome.For example, the combination of NP-2, NP-5 and NP-1 (boxed in Figure 20) covers 92% (or 2737 groups) of the protein groups identified by the combination of all five tested particles.

[0476] Figures 21A-21C show violin plots of mass distribution (Figure 21A), hydropathic index distribution (Figure 21B), and isoelectric point distribution (Figure 21C) of the protein groups identified in the direct digestion and improved secretome composition, respectively. The results show that the distribution of protein properties of the proteins oriented in the direct digestion or improved composition is similar, thereby suggesting that using Proteograph™ to improve the secretome composition does not introduce significant bias in the proteins detected.

[0477] 22A-22F show the number of proteins identified in the improved secretome composition (NP-1-NP-5) and direct digestion ("none") for various protein keywords. The protein keywords considered were phosphorylated protein (FIG. 22A), tumor suppressor (FIG. 22B), glycoprotein (FIG. 22C), Alzheimer's disease (FIG. 22D), cytokine (FIG. 22E), and kinase (FIG. 22F). As illustrated in FIGS. 22A-22F, for each particle tested for improved secretome composition, at least several times more proteins were identified with the respective particle than with direct digestion, thereby suggesting that improving secretome composition as described herein can result in the identification of more meaningful protein biomarkers associated with human disease than direct digestion. Example 2 Evaluation of a multi-layer engineered nanoparticle-based proteomic assay for unbiased, deep, and rapid analysis of fetal bovine serum-derived cell culture media

[0478] In this study, we first compare the Proteograph™ workflow with the traditional proteomics workflow for plasma proteome analysis to demonstrate the advantages of Proteograph™ using unique nanoparticles. Plasma samples were processed using one of four workflows: Proteograph™ workflow, high pH fractionation, plasma depletion, and direct digestion of unprocessed plasma. The test workflow is illustrated in Figure 23A.

[0479] As secreted medium, 250 μL of medium containing 10% FBS was collected from HeLa cells. The conditioned medium was processed directly on a Proteograph™ automated instrument, and tryptic peptides were analyzed on a Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer with LC-MS analysis in data-dependent acquisition (DDA) mode using a 2-hour LC gradient per NP sample. The LC-MS / MS raw data files were processed using the Proteograph™ Analysis Suite, using MaxQuant and applying a 1% FDR cutoff at the protein and peptide levels.

[0480] Other label-free proteomics workflows were compared with the Proteograph™ Product Suite using 120 min of DDA analysis for each of the five nanoparticles in the Proteograph™ assay, with a total analysis time of 10 h per sample. The Proteograph™ data detected approximately 3000 cell-derived protein groups (Figures 23B and 24A) and over 16000 peptides (Figure 24B) (1% FDR at protein and peptide levels) over a seven-order dynamic range in HeLa cell culture supernatants containing FBS using MaxQuant. The Protegraph™ data allowed the identification of at least twice as many protein groups compared to the high pH fractionation and plasma depletion workflow (Figure 23B), and showed improved reproducibility coefficients of variation compared to high pH fractionation (Figure 23C).

[0481] The Proteograph™ platform provided approximately 10-fold or greater improvement in proteome coverage depth compared to results derived from direct digestion of the same conditioned medium material, allowing the identification of low-abundance cytokines in culture media. Some of these low-abundance cytokines were not robustly detected by several simple techniques or complex workflows involving depletion and fractionation. Identification of low-abundance cytokines and other low-abundance proteins was performed using the Proteograph™ Product Suite. Table 1 lists examples of low-abundance cytokines detected by Proteograph™. [Table 1]

[0482] This study demonstrates that the capabilities of the Proteograph™ platform coupled with label-free mass spectrometry enable deeper profiling of secreted proteins in cell culture media more rapidly, and that this platform may enable in-depth, unbiased, large-scale conditioned media testing to detect novel insights. Example 3 Evaluation of engineered nanoparticle-based proteomic assays for unbiased, deep, and rapid analysis of host cell proteins in formulations

[0483] In this study, we first compared the Proteograph™ workflow with a conventional proteomics workflow for plasma proteome analysis to demonstrate the advantages of the unique nanoparticle-based Proteograph™.

[0484] Samples from the National Institute of Standards and Technology (NIST 8671, humanized IgG1K mAb) were processed with the Proteograph™ workflow. One batch of samples was processed with a stock solution with IgG1K mAb at a concentration of 10 mg / mL. Another batch of samples was processed after diluting the stock solution in PBS to achieve an IgG1K mAb concentration of 1 mg / mL. 250 μL of sample was used per Proteograph™ assay. As a control, a subset within each batch was processed without the Proteograph™ assay ("direct digestion", or DD). Figure 28 shows an example of the plate layout for a single run of the Proteograph™ assay.

[0485] LC-MS was configured using a Bruker timsTOF Pro2 with data independent acquisition (DIA). Total MS analysis time per injection was 33 minutes. For experiments performed with the Proteograph™, proteins were injected at 40ng, 100ng, or 200ng of protein per μL depending on the experiment. For experiments using direct digestion, proteins were injected at 200ng of protein per μL.

[0486] Data analysis was performed using Proteograph Analysis Suite (PAS™ 1.5), using DIA-NN library-free search, and using the mouse reference proteome (UP000000589_10090). Figure 29 shows the number of protein groups identified, according to some embodiments. The number of protein groups identified using Proteograph™ was about 3 to about 5 times the number of protein groups identified using direct digestion. List of embodiments

[0487] The following list of embodiments of the invention should be considered as disclosing various features of the invention, which features may be considered specific to the particular embodiment for which they are discussed, or which may be combinable with various other features listed in other embodiments. Thus, simply because a feature is discussed under one particular embodiment does not necessarily limit the use of that feature to that embodiment.

[0488] Embodiment 1. A method for selecting a candidate therapeutic agent for a disease, comprising: (a) incubating cells in a predetermined environment containing the therapeutic agent, such that the cells produce a biological sample containing a plurality of biomolecules, wherein the biological sample is at least partially affected by the therapeutic agent; (b) contacting the biological sample with a surface to adsorb the plurality of biomolecules onto the surface; (c) releasing at least a portion of the plurality of biomolecules on the surface; (d) detecting at least a portion of the plurality of biomolecules, thereby identifying the plurality of biomolecules; and (e) selecting a therapeutic agent as a candidate therapeutic agent based at least in part on the identified plurality of biomolecules.

[0489] Embodiment 2. The method of embodiment 1, wherein the cells are infected or mutated.Embodiment 3. The method of embodiment 1, wherein the cells are cancer cells.Embodiment 4. The method of embodiment 3, wherein the cancer cells are cells obtained in a biopsy of a patient.Embodiment 5. The method of embodiment 1, wherein the particles comprise a surface.Embodiment 6. The method of embodiment 5, wherein the particles are nanoparticles.Embodiment 7. The method of embodiment 1, wherein the contacting step of (b) further comprises contacting the biological sample with a second surface to adsorb the second plurality of biomolecules onto the second surface.Embodiment 8. The method of embodiment 1, wherein the detecting step comprises mass spectrometry.Embodiment 9. The method of embodiment 1, wherein the predetermined environment comprises a predetermined solvent environment, a predetermined temperature, or both.Embodiment 10. The method of embodiment 1, wherein the predetermined environment comprises serum.Embodiment 11. The method of embodiment 1, wherein the biological sample comprises a secretome composition or exosomes.Embodiment 12. The method of embodiment 1, wherein (a) further comprises incubating the plurality of cells. Embodiment 13. The method of embodiment 12, wherein (a) further comprises incubating each cell of the plurality of cells in a plurality of predetermined environments.Embodiment 14. The method of embodiment 12, wherein each predetermined environment of the plurality of predetermined environments comprises a different therapeutic agent.Embodiment 15. The method of embodiment 12, wherein each cell of the plurality of cells comprises a different cell line.Embodiment 16. The method of embodiment 12, wherein each predetermined environment of the plurality of predetermined environments comprises a different predetermined solvent environment, a different predetermined temperature, or both.Embodiment 17. The method of embodiment 12, wherein each cell of the plurality of cells is incubated for a different period of time.

[0490] Embodiment 18. A method for monitoring cellular activity comprising: (a) incubating cells in a predetermined environment such that the cells produce a biological sample containing a plurality of biomolecules; (b) contacting the biological sample with a surface, whereby the plurality of biomolecules are adsorbed onto the surface; (c) releasing at least a portion of the plurality of biomolecules on the surface; (d) detecting at least a portion of the plurality of biomolecules, thereby identifying the plurality of biomolecules; and (e) repeating steps (a)-(d) after a predetermined amount of time, thereby monitoring cellular activity.

[0491] Embodiment 19. A method for identifying biomolecules in a biological sample, comprising: (a) incubating cells in a predetermined environment so that the cells produce a biological sample; (b) contacting the biological sample with a surface to adsorb a plurality of low abundance biomolecules in the biological sample onto the surface; (c) releasing at least a portion of the plurality of low abundance biomolecules on the surface; and (d) detecting at least a portion of the plurality of low abundance biomolecules, thereby identifying the plurality of low abundance biomolecules.

[0492] The method of embodiment 19, further comprising the step of adding a biomolecule to the biological sample prior to embodiment 20(c), wherein the biomolecule reduces the detectability of a plurality of low abundance biomolecules in the biological sample.

[0493] Embodiment 21. An apparatus for assaying a biological sample, comprising: a substrate comprising a surface; a cell culture chamber comprising cells; a loading unit operably coupled to the substrate and the cell culture chamber; and a computer readable medium comprising machine executable code which, when executed by a processor, implements a method comprising: providing a controlled environment for cells in the cell culture chamber for a predetermined duration such that the cells produce a biological sample in the cell culture chamber; transferring a portion of the biological sample from the cell culture chamber to the substrate using the loading unit, thereby contacting the portion with the surface and allowing biomolecules in the portion of the biological sample to adsorb onto the surface; and assaying at least a portion of the biomolecules to detect biomolecules in the biological sample.

[0494] Embodiment 22. A method for identifying one or more biomolecules, comprising: (a) providing a supplemented medium; (b) generating a set of biomolecules by incubating cells in the supplemented medium under conditions sufficient for the cells to generate the set of biomolecules; (c) contacting at least a portion of the supplemented medium with one or more surfaces, whereby the set of biomolecules is adsorbed; (d) removing the one or more surfaces and the set of biomolecules from at least a portion of the supplemented medium, whereby a separated sample is produced; (e) releasing the set of biomolecules from the one or more surfaces in the separated sample; and (f) detecting at least a subset of the set of biomolecules, thereby identifying one or more biomolecules.

[0495] Embodiment 23. The method of embodiment 22, wherein after the contacting step of (b), the set of biomolecules, when adsorbed onto one or more surfaces, comprises a reduced dynamic range compared to the original dynamic range of the set of biomolecules in the supplemented medium.Embodiment 24. The method of embodiment 22 or 23, wherein in (a), the step of incubating the cells in the supplemented medium is carried out for less than about 5 seconds, less than 5 minutes, less than 5 hours, or less than 5 days.Embodiment 25. The method of any one of embodiments 22 to 24, wherein the cells are infected or mutated. Embodiment 26. The method of any one of embodiments 22-25, wherein the cells are viable cells, including cancer cells, epithelial cells, bone cells, muscle cells, adipocytes, tissue cells, senescent cells, pluripotent cells, stem cells, or neural cells.Embodiment 27. The method of any one of embodiments 22-26, wherein the cells are cancer cells that are biopsied cells of a patient.Embodiment 28. The method of any one of embodiments 22-27, wherein the particles comprise a surface.Embodiment 29. The method of any one of embodiments 28, wherein the particles are nanoparticles.Embodiment 30. The method of any one of embodiments 22-29, wherein the contacting step of (b) further comprises contacting the biological sample with a second surface to adsorb the second plurality of biomolecules onto the second surface.Embodiment 31. The method of any one of embodiments 22-30, wherein the detecting step comprises mass spectrometry. Embodiment 32. The method of any one of embodiments 22-31, wherein the conditions sufficient for the cells to produce the set of biomolecules in the supplemented medium include a predetermined temperature, a predetermined pressure, a predetermined flow regime, a predetermined solvent environment, or a combination thereof.Embodiment 33. The method of embodiment 32, wherein the conditions are kept constant.Embodiment 34. The method of any one of embodiments 22-33, wherein the supplemented medium comprises in part or in whole serum, plasma, cerebrospinal fluid (CSF), synovial fluid (SF), urine, tears, gingival crevicular fluid, semen, whole blood, milk, nipple aspirate, needle aspirate, ductal lavage, vaginal fluid, nasal fluid, ear fluid, gastric fluid, pancreatic juice, trabecular fluid, lung lavage, prostatic fluid, sputum, excreta, bronchial lavage, fluid from a swab, bronchial aspirate, sweat, saliva, or any combination thereof. Embodiment 35. The method of any one of embodiments 22-34, wherein the supplemented medium comprises a cell culture supernatant, a co-culture secretome, an exosome, a tissue or cell lysate, or any combination thereof.Embodiment 36. The method of any one of embodiments 22-35, wherein the supplemented medium comprises a synthetic supplemented medium.Embodiment 37. The method of any one of embodiments 22-36, wherein the set of biomolecules comprises one or more biomarkers, molecular signatures, secreted proteins, absorbed proteins, secretomes, exosomes, or any combination thereof.Embodiment 38. The method of any one of embodiments 1 to 37, further comprising repeating (a) to (e) for a second supplemented medium comprising a second set of biomolecules, wherein the second supplemented medium is generated by incubating the cells in the supplemented medium for different lengths of time. Embodiment 39. The method of any one of embodiments 22 to 38, wherein the providing step is performed by an automated fluidic system. Embodiment 40. The method of embodiment 39, wherein the automated fluidic system comprises a microfluidic system. Embodiment 41. The method of embodiment 39 or 40, wherein the automated fluidic system provides the supplemented medium at different times. Embodiment 42. The method of any one of embodiments 22 to 41, wherein the generating step comprises active secretion of at least a portion of the set of biomolecules. Embodiment 43. The method of any one of embodiments 22 to 42, wherein the generating step comprises passive release of at least a portion of the set of biomolecules. Embodiment 44. The method of any one of embodiments 22 to 43, wherein the generating step comprises release of exosomes or liposomes by the cells. Embodiment 45. The method of any one of embodiments 22-44, wherein the generating step comprises apoptosis of the cells.Embodiment 46. The method of any one of embodiments 22-45, wherein the generating step comprises necroptosis of the cells.Embodiment 47. The method of any one of embodiments 22-46, wherein the biomolecule of the set of biomolecules is a complex.Embodiment 48. The method of any one of embodiments 22-47, wherein the biomolecule of the set of biomolecules is a protein.Embodiment 49. The method of any one of embodiments 22-48, wherein the biomolecule of the set of biomolecules is a polypeptide.Embodiment 50. The method of any one of embodiments 22-49, wherein the biomolecule of the set of biomolecules is a nucleic acid.Embodiment 51. The method of any one of embodiments 22-50, wherein the cell is part of a plurality of cells.Embodiment 52. The method of embodiment 51, wherein the plurality of cells are cells of the same type.Embodiment 53. The method of any one of embodiments 22-52, wherein the cell is from a tissue sample, an organoid, an immortalized cell line, or any combination thereof. Embodiment 54 The method of any one of embodiments 22 to 53, wherein the cell is a stem cell.Embodiment 55. The method of any one of embodiments 22-54, wherein the conditions sufficient for the cells to exchange the set of biomolecules with the supplemented medium include one or more of the following: presence or absence of organic compounds, presence or absence of inorganic compounds, presence or absence of autocrine signaling molecules, presence or absence of paracrine signaling molecules, presence or absence of antigens, presence or absence of one or more co-cultured cells, presence or absence of radiation, presence or absence of one or more toxins, presence or absence of protein aggregates, presence or absence of one or more proteins, presence or absence of active virus particles, presence or absence of inactivated virus particles, presence or absence of applied heating or cooling, presence or absence of applied mechanical stress, presence or absence of electrical stimulation, presence or absence of transposons, presence or absence of exosomes, presence or absence of liposomes, presence or absence of coated nucleic acids, presence or absence of shock, or any combination thereof. Embodiment 56. The method of embodiment 55, wherein the conditions are varied over time. Embodiment 57. The method of any one of embodiments 22-56, wherein the contacting step of (b) is performed such that a portion of the supplemented medium contacts one or more surface regions of the one or more surfaces, and the one or more surface regions do not include a specific targeting moiety.Embodiment 58. The method of any one of embodiments 1-57, further comprising the step of determining, at least in part, one or more protein-protein interactions, biomarkers, molecular signatures, biomolecules absorbed by the cells, biomolecules secreted by the cells, biomolecules dissociated from the cell surface, biomolecules cleaved or released from the surface, macromolecular complexes budding, released, cleaved from the surface, biomolecules passively released by the cells, conventional and non-conventional released proteins, apoptotic release of biomolecules, necrotic released biomolecules, post-translational modifications, cell-cell interactions, cell-cell signaling, or any combination thereof, based at least in part on the identification, wherein the molecular signature is any pattern of proteins / proteoforms indicative of a biological state.Embodiment 59. The method of embodiment 58, wherein the biomolecules secreted by the one or more cells are conventional secretions, non-conventional secretions, type 1 non-conventional secretions, or type 2 non-conventional secretions.Embodiment 60. The method of any one of embodiments 22-59, wherein the set of biomolecules comprises a biomolecular assembly.Embodiment 61. The method of embodiment 60, wherein the biomolecular assembly comprises a quaternary protein, a vesicle, or an exosome.Embodiment 62. The method of any one of embodiments 22-61, wherein the cell is a eukaryote or a prokaryote.Embodiment 63. The method of any one of embodiments 22-62, further comprising a plurality of cells comprising the cell.Embodiment 64. The method of embodiment 63, wherein the plurality of cells is comprised within a tissue, an organoid, an organism, or a plurality of organisms.Embodiment 65. The method of embodiment 63, wherein the plurality of cells comprises at least a first cell of a first type and a second cell of a second type, such that the first cell exchanges one or more biomolecules of the set of biomolecules with the second cell. Embodiment 66. The method of embodiment 65, wherein the first cell and the second cell are co-cultured.Embodiment 67. The method of embodiment 65 or 66, wherein the first cell is comprised in a feeder culture for the second cell.Embodiment 68. The method of any one of embodiments 22-67, wherein the cell is derived from an immortalized cell line.Embodiment 69. The method of embodiment 68, wherein the cell is a HeLa cell.Embodiment 70. The method of any one of embodiments 22-69, wherein the cell is comprised in a primary cell culture.Embodiment 71. The method of any one of embodiments 63-70, wherein the plurality of cells are disposed in a plurality of separate volumes, each volume comprising a different supplemented medium or incubation condition.Embodiment 72. The method of any one of embodiments 22-71, wherein the releasing step comprises the use of a protease. Embodiment 73. The method of any one of embodiments 22-72, wherein the one or more surfaces comprise at least two surfaces comprising distinct physicochemical properties, such that at least two surfaces adsorb different patterns of biomolecule abundance from the set of biomolecules.Embodiment 74. The method of any one of embodiments 1-73, further comprising the step of determining that the one or more biomolecules were produced by the cells and were not originally present in the supplemented medium.Embodiment 75 The method of any one of embodiments 22 to 74, wherein the supplemented medium comprises fetal bovine serum.

[0496] Embodiment 76. A method for monitoring cellular activity comprising: (a) incubating cells such that the cells produce a biological sample containing a plurality of biomolecules; (b) contacting the biological sample with a surface, whereby the plurality of biomolecules are adsorbed onto the surface; (c) separating the surface from the biological sample; (d) releasing at least a portion of the plurality of biomolecules on the surface; (e) detecting at least a portion of the plurality of biomolecules, thereby identifying the plurality of biomolecules; and (f) repeating (a)-(d) after a predetermined amount of time, thereby monitoring cellular activity.

[0497] Embodiment 77. The method of embodiment 76, wherein the cells generate the biological sample by one of producing, releasing, adsorbing, digesting, or modifying a biomolecule of the plurality of biomolecules.Embodiment 78. The method of embodiment 76 or 77, further comprising a step of varying incubation conditions of the cells based at least in part on the activity of the cells.Embodiment 79. The method of any one of embodiments 76-78, wherein the incubating step comprises exposing the cells to a supplemented medium, the supplemented medium obscuring detection of at least some of the plurality of biomolecules.Embodiment 80. The method of any one of embodiments 76-79, further comprising a step of analyzing the activity of the cells.Embodiment 81. The method of any one of embodiments 76-80, wherein at least some of the plurality of biomolecules comprises a dynamic range of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Embodiment 82. The method of any one of embodiments 76-81, wherein at least a portion of the plurality of biomolecules comprises at least about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 biomolecules.Embodiment 83. The method of any one of embodiments 76-82, wherein (b) comprises contacting the biological sample with a plurality of surfaces.Embodiment 84. The method of any one of embodiments 76-83, wherein the releasing step of (d) is performed using a protease that enzymatically cleaves the surface.Embodiment 85. The method of any one of embodiments 76-84, wherein the surface is disposed on a magnetic substrate and the separating step of (c) is performed using a magnetic field to separate the magnetic substrate from the biological sample.

[0498] Embodiment 86. A method for identifying low abundance biomolecules in a biological sample, comprising: (a) incubating cells in a predetermined environment so that the cells produce a biological sample; (b) contacting the biological sample with a surface, whereby a plurality of low abundance biomolecules in the biological sample are adsorbed onto the surface; (c) separating the surface from the biological sample; (d) releasing at least a portion of the plurality of low abundance biomolecules on the surface; and (e) detecting at least a portion of the plurality of low abundance biomolecules, thereby identifying the plurality of low abundance biomolecules.

[0499] Embodiment 87. The method of embodiment 86, further comprising the step of adding a biomolecule to the biological sample prior to (c), wherein the biomolecule reduces the detectability of the plurality of low abundance biomolecules in the biological sample.Embodiment 88. The method of embodiment 86 or 87, wherein the signal of the step of detecting the low abundance biomolecule is about 7 orders of magnitude higher than the signal from direct digestion of the cells in the medium.Embodiment 89. The method of any one of embodiments 86 to 88, wherein the step of incubating comprises exposing the cells to a supplemented medium, wherein the supplemented medium obscures the detection of at least a portion of the plurality of low abundance biomolecules. Embodiment 90. The method of any one of embodiments 86-89, wherein the plurality of low abundance biomolecules comprises at least about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or 50000 biomolecules.Embodiment 91. The method of any one of embodiments 86-90, wherein (b) comprises contacting the biological sample with a plurality of surfaces.Embodiment 92. The method of any one of embodiments 86-91, wherein the releasing step of (d) is performed using a protease that enzymatically cleaves the surface.Embodiment 93. The method of any one of embodiments 86-92, wherein the surface is disposed on a magnetic substrate and the separating step of (c) is performed using a magnetic field to separate the magnetic substrate from the biological sample.

[0500] Embodiment 94. An apparatus for assaying a biological sample, comprising: a substrate comprising a surface; a cell culture chamber comprising cells; a loading unit operably coupled to the substrate and the cell culture chamber; and a computer readable medium comprising machine executable code which, when executed by a processor, implements a method comprising: i. providing a controlled environment for cells in the cell culture chamber for a predetermined duration such that the cells produce a biological sample in the cell culture chamber; ii. transferring at least a portion of the biological sample from the cell culture chamber to the substrate using the loading unit, thereby contacting at least a portion with the surface and allowing biomolecules in at least a portion of the biological sample to be adsorbed onto the surface; and iii. assaying at least a portion of the biomolecules to detect biomolecules in the biological sample.

[0501] Embodiment 95. The device of embodiment 94, comprising a plurality of cell culture chambers.Embodiment 96. The device of embodiment 95, in which a first cell culture chamber of the plurality of cell culture chambers is provided with a first controlled environment and a second cell culture chamber of the plurality of cell culture chambers is provided with a second controlled environment.Embodiment 97. The device of any one of embodiments 94-96, in which the transferring step is performed using one or more fluid connections and one or more pumps included in the loading unit.Embodiment 98. The device of any one of embodiments 94-97, in which the transferring step is performed using one or more pipettes and one or more pumps included in the loading unit.

[0502] Embodiment 99. A method for identifying one or more biomolecules comprising: (a) incubating cells under conditions sufficient for the cells to produce exosomes, the exosomes comprising a plurality of biomolecules; (b) contacting the exosomes with one or more surfaces, capturing at least a portion of the exosomes; (c) removing the one or more surfaces and at least a portion of the exosomes from the cells, producing a separated sample; (d) releasing at least a portion of the exosomes from the one or more surfaces in the separated sample; and (e) detecting at least a subset of the plurality of biomolecules in at least a portion of the exosomes, thereby identifying the one or more biomolecules.

[0503] Embodiment 100. A method for monitoring cellular activity comprising: (a) incubating cells such that the cells produce a biological sample containing exosomes, the exosomes comprising a plurality of biological molecules; (b) contacting the biological sample with a surface, whereby at least a portion of the exosomes are captured on the surface; (c) releasing at least a portion of the exosomes from the surface; (d) detecting at least a portion of the plurality of biological molecules in at least a portion of the exosomes, thereby identifying the plurality of biological molecules; and (e) repeating (a)-(d) after a predetermined amount of time, thereby monitoring cellular activity.

[0504] Embodiment 101. A method for identifying low abundance biomolecules in a biological sample, comprising: (a) incubating cells in a predetermined environment such that the cells produce a biological sample containing exosomes, the exosomes containing a plurality of low abundance biomolecules; (b) contacting the biological sample with a surface, whereby at least a portion of the exosomes in the biological sample are captured on the surface; (c) releasing at least a portion of the exosomes from the surface; and (d) detecting at least a portion of the plurality of low abundance biomolecules in at least a portion of the exosomes, thereby identifying the plurality of low abundance biomolecules.

[0505] Embodiment 102. An apparatus for assaying a biological sample, comprising: a substrate comprising a surface; a cell culture chamber comprising cells; a loading unit operably coupled to the substrate and the cell culture chamber; and a computer readable medium comprising machine executable code, which, when executed by a processor, implements a method comprising: i. providing a controlled environment for a predetermined duration to cells in the cell culture chamber, such that the cells produce a biological sample in the cell culture chamber comprising exosomes, the exosomes comprising one or more biomolecules; ii. transferring at least a portion of the biological sample from the cell culture chamber to the substrate using the loading unit, thereby contacting at least a portion with the surface and adsorbing exosomes in at least a portion of the biological sample onto the surface; and iii. assaying the exosomes to detect one or more biomolecules in the biological sample.

[0506] Embodiment 103. A method for identifying a biomolecule, comprising: (a) processing one or more exosomes to release a plurality of biomolecules in the one or more exosomes into an environment external to the one or more exosomes, wherein a subset of the biomolecules in the plurality of biomolecules comprises a first distribution of relative abundance in the one or more exosomes; (b) performing a composition refinement assay on the plurality of biomolecules to increase the relative abundance for the subset of biomolecules from the first distribution to a second distribution of relative abundance for the subset of biomolecules; and (c) assaying the plurality of biomolecules to identify the subset of biomolecules.

[0507] Embodiment 104. The method of embodiment 103, wherein the processing is carried out, at least in part, by adding a lysis buffer to the one or more exosomes.Embodiment 105. The method of embodiment 103 or 104, wherein the processing is carried out, at least in part, by providing ultrasonic energy to the one or more exosomes.Embodiment 106. The method of any one of embodiments 103-105, wherein the processing is carried out, at least in part, by freezing and thawing the one or more exosomes.Embodiment 107. The method of any one of embodiments 103-106, wherein the processing is carried out, at least in part, by heating the one or more exosomes.Embodiment 108. The method of any one of embodiments 103-107, wherein the processing is carried out, at least in part, by shearing the one or more exosomes.Embodiment 109. The method of any one of embodiments 103-108, wherein the processing is carried out, at least in part, by disrupting the one or more exosomes. Embodiment 110. The method of any one of embodiments 103-109, wherein the subset of biomolecules comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 unique biomolecules. Embodiment 111. The method of any one of embodiments 103 to 110, wherein the composition improvement assay comprises contacting a plurality of biomolecules with one or more surfaces to adsorb a subset of the biomolecules to the one or more surfaces, such that when the subset of biomolecules is adsorbed to the one or more surfaces, the subset of biomolecules comprises a second distribution.Embodiment 112. The method of any one of embodiments 103 to 111, wherein the contacting increases the visibility of the subset of biomolecules in the assaying step of (c) when the subset of biomolecules are adsorbed to one or more surfaces compared to the visibility of the subset of biomolecules when the subset of biomolecules are in one or more exosomes, and the subset of biomolecules are low abundance biomolecules comprising less than about 1 mass percent of the biomolecules in the one or more exosomes.

[0508] Embodiment 113. A method for identifying a biomolecule, comprising: (a) contacting a biological sample containing one or more exosomes with a plurality of particles to non-specifically bind a subset of the one or more exosomes onto the plurality of particles, the plurality of particles comprising distinct physicochemical properties, and the one or more exosomes comprising a plurality of biomolecules; (b) processing the one or more exosomes to release the plurality of biomolecules into an environment external to the one or more exosomes; and (c) assaying the plurality of biomolecules to identify at least a subset of the biomolecules in the plurality of biomolecules.

[0509] Embodiment 114. The method of embodiment 113, wherein the processing is carried out, at least in part, by adding a lysis buffer to the one or more exosomes.Embodiment 115. The method of embodiment 113 or 114, wherein the processing is carried out, at least in part, by providing ultrasonic energy to the one or more exosomes.Embodiment 116. The method of any one of embodiments 113-115, wherein the processing is carried out, at least in part, by freezing and thawing the one or more exosomes.Embodiment 117. The method of any one of embodiments 113-116, wherein the processing is carried out, at least in part, by heating the one or more exosomes.Embodiment 118. The method of any one of embodiments 113-117, wherein the processing is carried out, at least in part, by shearing the one or more exosomes.Embodiment 119. The method of any one of embodiments 113-118, wherein the processing is carried out, at least in part, by disrupting the one or more exosomes. Embodiment 120. The method of any one of embodiments 113-119, wherein the subset of biomolecules comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 unique biomolecules. Embodiment 121. The method of any one of embodiments 113 to 120, wherein the plurality of particles increases the visibility of the subset of biomolecules in the assaying step of (c) when the subset of biomolecules is adsorbed to the plurality of particles compared to the visibility of the subset of biomolecules when the subset of biomolecules is within one or more exosomes, and the subset of biomolecules are low abundance biomolecules comprising less than about 1 mass percent of the plurality of biomolecules in the one or more exosomes.

[0510] Embodiment 122. A method for characterizing a biological preparation, comprising: (a) contacting the biological preparation with one or more surfaces, wherein a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces, the plurality of biomolecules including product biomolecules and impurities; and (b) assaying the plurality of biomolecules, wherein a difference between a composition of the plurality of biomolecules and a reference composition is determined, the difference indicating the purity or activity of the biological preparation.

[0511] Embodiment 123. The method of embodiment 122, wherein the biological preparation comprises a cell culture.Embodiment 124. The method of embodiment 123, wherein the plurality of biomolecules comprises a secretome or exosome from the cell culture.Embodiment 125. The method of embodiment 123 or 124, wherein the plurality of biomolecules comprises a portion of one or more cells of the cell culture.Embodiment 126. The method of any one of embodiments 123-125, wherein the cell culture comprises a host organism that produces the product biomolecule.Embodiment 127. The method of any one of embodiments 123-126, wherein the cell culture comprises a host organism that produces an impurity.Embodiment 128. The method of any one of embodiments 123-127, wherein the cell culture comprises a contaminating organism that produces an impurity.Embodiment 129. The method of embodiment 127, wherein the impurity comprises a host cell protein.Embodiment 130. The method of embodiment 129, wherein the host cell protein comprises a protease, lipase, or isomerase. Embodiment 131. The method of embodiment 129 or 130, wherein the host cell proteins comprise passively released proteins or actively released proteins.Embodiment 132. The method of any one of embodiments 129-131, wherein the host cell proteins comprise a portion of a host organism.Embodiment 133. The method of embodiment 132, wherein the portion comprises a cell membrane, an organelle, or both.Embodiment 134. The method of any one of embodiments 123-133, wherein the cell culture comprises a plurality of host organism species or strains.Embodiment 135. The method of any one of embodiments 123-134, wherein the cell culture comprises a plurality of contaminating species or strains.Embodiment 136. The method of any one of embodiments 123-135, wherein the biological preparation comprises a plurality of impurities.Embodiment 137. The method of any one of embodiments 123-136, wherein the biological preparation comprises a plurality of product biomolecules. Embodiment 138. The method of any one of embodiments 126 to 137, wherein the host organism comprises a prokaryotic host organism or a eukaryotic host organism.Embodiment 139. The method of embodiment 138, wherein the prokaryotic host organism comprises Escherichia coli, Streptomyces sp., Acetobacter, Lactobacillus, Thermophilic Bacillus sp., Clostridium thermocellus, Agrobacterium tumefaciens, Thermus aquaticus, Bacillus coagulans, Pseudomonas stutzeri, Acetobacter sp., Micrococcus sp., Haemophilus influenzae, or Leuconostoc mesenteroides.Embodiment 140. The method of embodiment 138, wherein the eukaryotic host organism comprises yeast, fungi, HeLa cells, stem cells, cancer cells, genetically modified cells, or algae.Embodiment 141. The method of embodiment 140, wherein the genetically modified cells comprise an exogenous nucleic acid sequence encoding a product biomolecule.Embodiment 142. The method of any one of embodiments 122 to 141, wherein the impurity is a proteoform of a biomolecule of the plurality of biomolecules. Embodiment 143. The method of any one of embodiments 122 to 142, wherein the impurity is a proteoform of the product biomolecule.Embodiment 144. The method of embodiment 143, wherein the proteoform comprises a splicing variant, an allelic variant, or a post-translational modification variant.Embodiment 145. The post-translational modification variant is acylation, alkylation, prenylation, flavinylation, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, sumoylation, neddylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hypusine formation, beta-lysine addition, acetylation, formylation, methylation, amidation, amidoyl ... 145. The method of embodiment 144, comprising a post-translational modification comprising: dopeptide bond formation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphate ester 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, eliminylation, disulfide bond formation, proteolytic cleavage, isoaspartate formation, racemization, protein splicing, chaperone mediated folding, or any combination thereof. Embodiment 146. The method of any one of embodiments 122 to 145, wherein the difference between the first log(water-octanol partition coefficient) for the impurity and the second log(water-octanol partition coefficient) for the product biomolecule is less than about 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.Embodiment 147. The method of any one of embodiments 122-146, wherein the difference between the first log(water-octanol partition coefficient) for the impurity and the second log(water-octanol partition coefficient) for the product biomolecule is greater than about 2, 1.5, 1, 0.9, 0.8, 0.7, about 0.6, about 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. Embodiment 148. The method of any one of embodiments 122-147, wherein the impurity and the product biomolecule are hydrophobic. Embodiment 149. The method of any one of embodiments 122-148, wherein the impurity and the product biomolecule are hydrophilic. Embodiment 150. The method of any one of embodiments 122-149, wherein the difference between the first pKa of the impurity and the second pKa of the product biomolecule is at most 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. Embodiment 151. The method of any one of embodiments 122-150, wherein the difference between the first pKa of the impurity and the second pKa of the product biomolecule is at least 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.Embodiment 152. The method of any one of embodiments 122-151, wherein the impurity and the product biomolecule are stereoisomers of each other.Embodiment 153. The method of any one of embodiments 122-152, wherein the impurity and the product biomolecule comprise enantiomers of each other. Embodiment 154. The method of any one of embodiments 122-153, wherein the difference between the first mass to charge ratio of the impurity and the second mass to charge ratio of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons / e-.Embodiment 155. The method of any one of embodiments 122-154, wherein the difference between the first mass-to-charge ratio of the impurity and the second mass-to-charge ratio of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons / e-.Embodiment 156. The method of any one of embodiments 122-155, wherein the impurity comprises a first histidine tag and the product biomolecule comprises a second histidine tag.Embodiment 157. The method of embodiment 156, wherein the first histidine tag and the second histidine tag comprise the same number of amino acids. Embodiment 158. The method of any one of embodiments 122-157, wherein the difference between the first molecular weight of the impurity and the second molecular weight of the product biomolecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons. Embodiment 159. The method of any one of embodiments 122-158, wherein the difference between the first molecular weight of the impurity and the second molecular weight of the product biological molecule is 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, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilodaltons.Embodiment 160. The method of any one of embodiments 122-159, further comprising purifying the impure biological preparation prior to (a) to produce a biological preparation, wherein the impure biological preparation and the biological preparation comprises an impurity.Embodiment 161. The method of embodiment 160, wherein the purifying step increases the purity or activity of the biological preparation compared to the impure biological preparation. Embodiment 162. The method of any one of embodiments 122 to 161, wherein the impurities are present in lower abundance than the product biomolecule.Embodiment 163. The method of embodiment 162, wherein the impurities are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.Embodiment 164. The method of embodiment 162 or 163, wherein the impurity is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass, or mass spectrometry signal intensity.Embodiment 165. The method of any one of embodiments 122 to 164, wherein the plurality of biomolecules comprises a reduced dynamic range on the one or more surfaces compared to the dynamic range of the plurality of biomolecules in the biological preparation.Embodiment 166. The method of embodiment 165, wherein the reduced dynamic range is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less dynamic range. Embodiment 167. The method of embodiment 165 or 166, wherein the reduced dynamic range is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less dynamic range. Embodiment 168. The method of any one of embodiments 122 to 167, wherein the biological preparation comprises a drug or a metabolite thereof. Embodiment 169. The method of embodiment 168, wherein the impurities comprise a drug. Embodiment 170. The method of embodiment 168, wherein the impurities comprise a metabolite thereof. Embodiment 171. The method of embodiment 168, wherein the product biomolecule comprises a drug. Embodiment 172. The method of embodiment 168, wherein the host cell protein comprises a drug. Embodiment 173. The method of any one of embodiments 168 to 172, wherein the drug comprises an antibody, a biological therapeutic, or a chemotherapeutic biomolecule. Embodiment 174. The method of any one of embodiments 168-173, wherein the drug comprises methamphetamine, isotretinoin, an antibiotic, an antiplatelet drug, dutasteride, an antithrombotic drug, insulin, hepatitis B immunoglobulin, growth hormone, tamsulosin, finasteride, acitretin, etretinate, or any combination thereof.Embodiment 175. The method of any one of embodiments 168-174, wherein the impurity is capable of reducing the activity of the product biomolecule, and the impurity is produced by the host organism or a contaminating organism foreign to the host organism.Embodiment 176. The method of any one of embodiments 122-167, wherein the biological preparation comprises a vaccine.Embodiment 177. The product biomolecule is an antiviral agent for the prevention of pathogenic bacteria. The method of embodiment 176, wherein the product biomolecule comprises an antigen of a virus, or a derivative thereof. Embodiment 178. The method of embodiment 176 or 177, wherein the product biomolecule comprises an immunity protein or a derivative thereof. Embodiment 179. The method of any one of embodiments 122-167, wherein the product biomolecule comprises an enzyme. Embodiment 180. The method of embodiment 179, wherein the enzyme is configured to degrade synthetic polymers, degrade oil, or catalyze ethanol production. Embodiment 181. The method of any one of embodiments 122-167, wherein the biological preparation comprises blood, plasma, platelets, clotting factors, or any combination thereof. Embodiment 182. The method of any one of embodiments 122-167, wherein the impurity comprises a biomolecule produced by a pathogen. Embodiment 183. The method of embodiment 182, wherein the pathogen comprises Hepatitis B virus, Hepatitis C virus, COVID-19, or HIV. Embodiment 184. The method of any one of embodiments 122 to 167, wherein the biological preparation comprises a human consumable product or a livestock consumable product.Embodiment 185. The method of embodiment 184, wherein the human consumable product or the livestock consumable product comprises chicken, beef, pork, vegetables, fungi, a fermentation product, or a meat substitute.Embodiment 186. The method of embodiment 184 or 185, wherein the impurities comprise bacterial biomolecules.Embodiment 187. The method of any one of embodiments 184 to 186, further comprising estimating the shelf life of the biological preparation based on the difference.Embodiment 188. The method of any one of embodiments 184 to 187, further comprising determining the suitability of the biological preparation for human or livestock consumption based on the difference.Embodiment 189. The method of any one of embodiments 184 to 188, wherein the human consumable product comprises a fermentation product.Embodiment 190. The method of embodiment 189, wherein the fermentation product comprises ethanol, acetic acid, or lactic acid. Embodiment 191. The method of embodiment 189 or 190, wherein the fermented product comprises beer or wine.Embodiment 192. The method of embodiment 189 or 190, wherein the fermented product comprises vinegar.Embodiment 193. The method of embodiment 189 or 190, wherein the fermented product comprises yogurt.Embodiment 194. The method of any one of embodiments 168 to 193, wherein impurities may increase the risk of complications when the biological preparation is administered to a subject.Embodiment 195. The method of any one of embodiments 168-193, wherein the impurity is expected to reduce the activity / stability of the product biomolecule to a human subject when administered to or ingested by a human subject.Embodiment 196. The method of any one of embodiments 168-193, wherein the impurity may harm a human subject when administered to or ingested by a human subject.Embodiment 197. The method of any one of embodiments 168-193, wherein the biological preparation comprises a host organism or a contaminating organism and may harm a human subject when administered to or ingested by a human subject.Embodiment 198. The method of any one of embodiments 196 or 197, wherein the harming comprises an immune response, blood clotting, an allergic reaction, food poisoning, or any combination thereof.Embodiment 199. The method of any one of embodiments 122-198, wherein the difference comprises a difference between the level of activity of the biological preparation and a reference activity level for the reference composition. Embodiment 200. The method of embodiment 199, wherein the level of activity is assayed by contacting a plurality of biomolecules on one or more surfaces in an activity assay. Embodiment 201. The method of embodiment 200, wherein the activity assay comprises an in vitro cell culture. Embodiment 202. The method of embodiment 200, wherein the activity assay comprises a substrate. Embodiment 203. The method of embodiment 200, wherein the activity assay comprises an immunoaffinity assay. Embodiment 204. The method of embodiment 200, wherein the activity assay comprises an avidity assay. Embodiment 205. The method of any one of embodiments 122-198, wherein the difference comprises a difference between the safety level of the biological preparation and a reference safety level for the reference composition. Embodiment 206. The method of embodiment 205, wherein the safety level is assayed by contacting a plurality of biomolecules on one or more surfaces in a safety assay. Embodiment 207. The method of embodiment 205 or 206, wherein the safety level comprises an immunogenicity level and the reference safety level comprises a reference immunogenicity level. Embodiment 208. The method of embodiment 207, wherein the immunogenicity level is assayed by contacting a plurality of biomolecules on one or more surfaces with one or more human blood samples or derivatives thereof.Embodiment 209. The method of embodiment 208, wherein the one or more human blood samples or derivatives thereof comprise one or more white blood cells.Embodiment 210. The method of any one of embodiments 122-209, wherein the difference comprises a detectable level of an impurity.Embodiment 211. The method of any one of embodiments 122-210, wherein the difference comprises a level of a product biomolecule below a reference level of the product biomolecule.Embodiment 212. The method of any one of embodiments 122-211, wherein the difference comprises a difference between the impurity level and the reference impurity level.Embodiment 213. The method of any one of embodiments 122-212, further comprising purifying the biological preparation based on the difference.Embodiment 214. The method of embodiment 213, wherein the purifying step is performed if the difference is greater than 1, 2, 3, 4, 5, or 6 times the standard deviation of the measured impurity level. Embodiment 215. The method of embodiment 213 or 214, wherein the purifying step is performed if the difference is greater than 1, 2, 3, 4, 5, or 6 times the standard error of the measurement of the impurity level, and the standard error is based on at least N assays. Embodiment 216. The reference composition is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.9, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.9 ... 216. The method of any one of embodiments 122-215, comprising a product biomolecule with a purity of 9.99, 99.991, 99.992, 99.992, 99.993, 99.994, 99.995, 99.996, 99.997, 99.998, 99.999, 99.999, 99.9991, 99.9992, 99.9993, 99.9994, 99.9995, 99.9996, 99.9997, 99.9998, or 99.9999 percent.Embodiment 217. The method of any one of embodiments 122-216, wherein the reference composition contains impurities at a purity of at most 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.9 percent. Embodiment 218. The method of embodiment 217, wherein the reference composition comprises 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, or 900 ppm of impurities.Embodiment 219. The method of embodiment 218, wherein the reference composition comprises 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, or 900 ppb of impurities. Embodiment 220. The method of any one of embodiments 216 to 219, wherein the percent purity is based on the signal intensity of the product determined from the assaying step.Embodiment 221. The method of any one of embodiments 122 to 220, wherein the difference is determined using a machine learning algorithm.Embodiment 222. The method of embodiment 221, wherein the machine learning algorithm is configured to receive one or more features representative of the composition of the plurality of biomolecules and output the difference based on the composition.Embodiment 223. The method of embodiment 221 or 222, wherein the machine learning algorithm is trained to learn a reference impurity level based on a first plurality of samples comprising a purity or activity above a predetermined threshold, a second plurality of samples comprising a purity or activity below a predetermined threshold, or both.Embodiment 224. The method of any one of embodiments 221 to 223, further comprising using a machine learning algorithm to classify the biological preparation as comprising a purity or activity above a predetermined threshold or comprising a purity or activity below a predetermined threshold based on the composition of the plurality of biomolecules.Embodiment 225. The method of any one of embodiments 122 to 224, further comprising monitoring the biological preparation by assaying a plurality of biomolecules a plurality of times to determine a plurality of differences in the plurality of times.Embodiment 226. The method of embodiment 225, further comprising determining drift in the genetic makeup of the host organism based on the plurality of differences.Embodiment 227. The method of embodiment 225, further comprising determining drift in a population of cell cultures based on the plurality of differences.Embodiment 228. The method of embodiment 225, further comprising determining contamination in a workflow for producing a biological preparation based on the plurality of differences.

[0512] Embodiment 229. A method for detecting an impurity in a biological preparation, comprising: (a) contacting the biological preparation with one or more surfaces, wherein a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces; and (b) assaying the plurality of biomolecules, wherein a biomolecular fingerprint of the biological preparation is detected, the biomolecular fingerprint comprising a signature of an impurity in the plurality of biomolecules.

[0513] Embodiment 230. An apparatus for characterizing a biological preparation, the apparatus comprising: a first chamber configured to hold a biological preparation, the biological preparation comprising a plurality of biomolecules, the plurality of biomolecules including product biomolecules and impurities; a second chamber comprising one or more surfaces; a loader operably coupled to the first chamber and the second chamber, the loader configured to transfer the biological preparation between the first chamber and the second chamber; and a computer-readable medium for measuring purity or activity of a biological preparation comprising machine-executable code that, when executed by a processor, implements a method comprising using the loader to contact the biological preparation from the first chamber with one or more surfaces in the second chamber, wherein the plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces.

[0514] Embodiment 231. The device of embodiment 230, wherein the method further comprises assaying the plurality of biomolecules to determine a difference between the composition of the plurality of biomolecules and a reference composition, the difference being indicative of purity or activity of the biological preparation.Embodiment 232. The device of embodiment 230 or 231, further comprising a first separator operably coupled to the second chamber, the first separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the biological preparation.Embodiment 233. The device of embodiment 232, wherein the first separator comprises a magnet.Embodiment 234. The device of any one of embodiments 230-233, further comprising a second separator operably coupled to the second chamber, the second separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the one or more surfaces.Embodiment 235. The device of any one of embodiments 230-234, further comprising one or more purifiers for purifying the biological preparation. Embodiment 236. The device of embodiment 235, wherein the method further comprises purifying the biological preparation using one or more purifiers based on the quality of the product.Embodiment 237. The device of embodiment 235 or 236, wherein the one or more purifiers comprise a plurality of purifiers.Embodiment 238. The device of embodiment 237, wherein the plurality of purifiers are provided in an arrangement such that the biological preparation purified in one of the plurality of purifiers is fed to another of the plurality of purifiers.Embodiment 239. The device of embodiment 238, wherein the processing conditions of the plurality of purifiers are based on the quality of the product.Embodiment 240. The device of any one of embodiments 235-239, wherein the method further comprises purifying the biological preparation using one or more purifiers based on the difference to improve the purity or activity of the biological preparation.Embodiment 241. The device of any one of embodiments 230-240, further comprising a third chamber operably coupled to the loader, the chamber comprising an in vitro cell culture or a human blood sample or a derivative thereof. Embodiment 242. The device of any one of embodiments 230 to 241, wherein the method further comprises a step of authenticating or rejecting the biological preparation based on the differences.Embodiment 243. The device of any one of embodiments 230 to 242, further comprising a mass spectrometer for performing an assay to determine the difference.Embodiment 244. The device of any one of embodiments 230 to 243, wherein the first chamber comprises an incubator.Embodiment 245. The device of embodiment 244, wherein the first chamber comprises a heater or cooler operably connected to the incubator.Embodiment 246. The device of embodiment 245, wherein the method comprises controlling the temperature of the first chamber using the heater or cooler.Embodiment 247. The device of any one of embodiments 244 to 246, wherein the first chamber is pressurized.Embodiment 248. The device of any one of embodiments 244 to 247, wherein the device comprises a plurality of chambers, including the first chamber, each chamber of the plurality of chambers operably connected to a loader and a second chamber, and each chamber of the plurality of chambers comprises a portion of the biological preparation. Embodiment 249. The device of any one of embodiments 244 to 248, wherein each chamber of the plurality of chambers comprises a host organism of a different species or strain configured to produce at least a portion of the biological preparation.Embodiment 250. The device of any one of embodiments 230 to 249, further comprising one or more particles comprising one or more surfaces and one or more supports.Embodiment 251. The device of embodiment 250, wherein the one or more supports comprise a paramagnetic material.Embodiment 252. The device of embodiment 251, wherein the paramagnetic material comprises a superparamagnetic material.Embodiment 253. The device of any one of embodiments 230 to 252, wherein the one or more surfaces comprise a plurality of surface types.Embodiment 254. The device of embodiment 253, wherein the one or more particles comprise a plurality of particles comprising a plurality of surface types.Embodiment 255. The device of any one of embodiments 250 to 254, wherein the one or more particles comprise one or more microparticles.Embodiment 256. The device of any one of embodiments 250 to 255, wherein the one or more particles comprise one or more nanoparticles. Embodiment 257. The device of any one of embodiments 250 to 256, wherein the one or more particles include one or more porous particles.Embodiment 258. The device of any one of embodiments 230 to 257, wherein the one or more surfaces are configured to reduce the dynamic range of the plurality of biomolecules in the biological preparation when the plurality of biomolecules are adsorbed to the one or more surfaces.Embodiment 259. The device of any one of embodiments 230 to 258, wherein the impurities are present in lower abundance than the product biomolecules.Embodiment 260. The device of embodiment 259, wherein the impurities are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass or mass spectrometry signal intensity.Embodiment 261. The device of embodiment 259 or 260, wherein the impurities are at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than the product biomolecule in terms of counts, mass or mass spectrometry signal intensity. Embodiment 262. The apparatus of any one of embodiments 258 to 261, wherein the dynamic range of the plurality of biomolecules is reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude.Embodiment 263. The apparatus of any one of embodiments 258 to 262, wherein the reduced dynamic range is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less than the dynamic range.

[0515] Embodiment 264. An apparatus for detecting impurities in a biological preparation, comprising: a plurality of chambers operably connected to each other, the plurality of chambers comprising one or more surfaces; one or more fluid transfer devices operably coupled to the plurality of chambers; and a computer-readable medium for detecting impurities in a biological preparation comprising machine-executable code that, when executed by a processor, implements a method comprising contacting a biological preparation with one or more surfaces using the one or more fluid transfer devices, wherein a plurality of biomolecules in the biological preparation are adsorbed onto the one or more surfaces.

[0516] Embodiment 265. The device of embodiment 264, wherein the method further comprises assaying a plurality of biomolecules to detect a biomolecular fingerprint of the biological preparation, the biomolecular fingerprint comprising a signature of a contaminating biomolecule in the plurality of biomolecules.Embodiment 266. The device of embodiment 264 or 265, further comprising a first separator operably coupled to the plurality of chambers, the first separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the biological preparation.Embodiment 267. The device of embodiment 266, wherein the first separator comprises a magnet.Embodiment 268. The device of any one of embodiments 264-267, further comprising a second separator operably coupled to the second chamber, the second separator configured to separate the plurality of biomolecules adsorbed on the one or more surfaces from the one or more surfaces.Embodiment 269. The device of any one of embodiments 264-268, further comprising one or more purifiers for purifying the biological preparation. Embodiment 270. The device of embodiment 269, wherein the method further comprises purifying the biological preparation using one or more purifiers based on the biomolecular fingerprint.Embodiment 271. The device of embodiment 269 or 270, wherein the one or more purifiers comprise a plurality of purifiers.Embodiment 272. The device of embodiment 271, wherein the plurality of purifiers are provided in an arrangement such that the biological preparation purified in one of the plurality of purifiers is fed to another of the plurality of purifiers.Embodiment 273. The device of embodiment 272, wherein the processing conditions of the plurality of purifiers are based on the biomolecular fingerprint.Embodiment 274. The device of any one of embodiments 269 to 273, wherein the method further comprises purifying the biological preparation using one or more purifiers to reduce the signature of contaminating biomolecules based on the biomolecular fingerprint.Embodiment 275. The device of any one of embodiments 264 to 274, further comprising a third chamber operably coupled to the loader, the chamber comprising an in vitro cell culture or a human blood sample or a derivative thereof.Embodiment 276. The device of any one of embodiments 264 to 275, wherein the method further comprises authenticating or rejecting the biological preparation based on the biomolecular fingerprint.Embodiment 277. The device of any one of embodiments 264 to 276, further comprising a mass spectrometer for performing an assay for determining the biomolecular fingerprint.Embodiment 278. The device of any one of embodiments 264 to 277, wherein the plurality of chambers comprises an incubator.Embodiment 279. The device of embodiment 278, wherein the plurality of chambers comprises a heater or cooler operably connected to the incubator.Embodiment 280. The device of embodiment 279, wherein the method comprises controlling the temperature of the plurality of chambers using a heater or cooler.Embodiment 281. The device of any one of embodiments 278 to 280, wherein the plurality of chambers is pressurized.Embodiment 282. The device of any one of embodiments 264 to 281, wherein each chamber of the plurality of chambers comprises a host organism of a different species or strain configured to produce at least a portion of the biological preparation. Embodiment 283. The device of any one of embodiments 264 to 282, further comprising one or more particles comprising one or more surfaces and one or more supports.Embodiment 284. The device of embodiment 283, wherein the one or more supports comprise a paramagnetic material.Embodiment 285. The device of embodiment 284, wherein the paramagnetic material comprises a superparamagnetic material.Embodiment 286. The device of any one of embodiments 264 to 285, wherein the one or more surfaces comprise a plurality of surface types.Embodiment 287. The device of embodiment 286, wherein the one or more particles comprise a plurality of particles comprising a plurality of surface types.Embodiment 288. The device of any one of embodiments 283 to 287, wherein the one or more particles comprise one or more microparticles.Embodiment 289. The device of any one of embodiments 283 to 288, wherein the one or more particles comprise one or more nanoparticles.Embodiment 290. The device of any one of embodiments 283 to 289, wherein the one or more particles comprise one or more porous particles.Embodiment 291. The device of any one of embodiments 283 to 290, wherein the one or more surfaces are configured to reduce the dynamic range of the plurality of biomolecules in the biological preparation when the plurality of biomolecules is adsorbed to the one or more surfaces.Embodiment 292. The device of any one of embodiments 264 to 291, wherein the contaminating biomolecule is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than another biomolecule in the plurality of biomolecules in terms of count, mass or mass spectrometry signal intensity.Embodiment 293. The device of any one of embodiments 264 to 292, wherein the contaminating biomolecule is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude less abundant than another biomolecule in the plurality of biomolecules in terms of count, mass or mass spectrometry signal intensity. Embodiment 294. The device of any one of embodiments 264 to 293, wherein the dynamic range of a plurality of biomolecules is reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 orders of magnitude.

[0517] Embodiment 295. A computer-implemented method for generating a quality metric for a biological preparation, comprising: (a) receiving a plurality of mass spectrometry data sets for a plurality of polyamino acids in the biological preparation, the plurality of polyamino acids including at least one product biomolecule and a plurality of impurities; (b) generating a plurality of polyamino acid identifications and a plurality of polyamino acid abundances for the plurality of polyamino acids based on the plurality of mass spectrometry data sets; and (c) processing the plurality of polyamino acid identifications to output a quality metric for the biological preparation.

[0518] Embodiment 296. The computer-implemented method of embodiment 295, wherein the plurality of polyamino acid abundances indicates relative abundances between the plurality of polyamino acids.Embodiment 297. The computer-implemented method of embodiment 295 or 296, wherein the plurality of polyamino acid identifications comprises at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, or 50000 polyamino acid identifications.Embodiment 298. The computer-implemented method of any one of embodiments 295-297, wherein the plurality of polyamino acids comprises a dynamic range of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 in the biological preparation. Embodiment 299. The computer-implemented method of any one of embodiments 295-298, wherein the plurality of polyamino acid abundances comprises a dynamic range of less than about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Embodiment 300. The computer-implemented method of any one of embodiments 295-299, wherein the plurality of impurities comprises a proteoform of a polyamino acid of the plurality of polyamino acids. Embodiment 301. The computer-implemented method of embodiment 300, wherein the proteoform comprises a splicing variant, an allelic variant, or a post-translational modification variant.Embodiment 302. The post-translational modification variant is acylation, alkylation, prenylation, flavinylation, amination, deamination, carboxylation, decarboxylation, nitrosylation, halogenation, sulfurylation, glutathionylation, oxidation, oxygenation, reduction, ubiquitination, sumoylation, neddylation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol anchor formation, lipoylation, heme functionalization, phosphorylation, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol functionalization, hypusine formation, beta-lysine addition, acetylation, formylation, methylation, amidation, amide bond formation, bromine ... 302. The computer-implemented method of embodiment 301, comprising a post-translational modification comprising thiryla...

Claims

1. A method for identifying one or more biomolecules produced by a cell, comprising: (a) providing a supplemented medium; (b) producing a set of biomolecules by incubating cells in said supplemented medium under conditions sufficient for said cells to produce said set of biomolecules; (c) contacting at least a portion of the supplemented medium with one or more particles to adsorb at least a portion of the set of biomolecules; (d) removing the adsorbed portion of the one or more particles and the set of biomolecules from the at least part of the supplemented medium to produce a separated sample; (e) releasing the adsorbed portion of the set of biomolecules from the one or more particles; (f) detecting at least a subset of the set of biomolecules using mass spectrometry, wherein the at least a subset of the set of biomolecules comprises biomolecules produced by the cells and not originally present in the supplemented medium, thereby identifying one or more biomolecules produced by the cells; A method comprising:

2. 2. The method of claim 1, wherein after the contacting step of (c), the set of biomolecules, when adsorbed onto the one or more particles, comprises a reduced dynamic range compared to the original dynamic range of the set of biomolecules in the supplemented medium.

3. The method of claim 1 , wherein the cells are infected or mutated.

4. 10. The method of claim 1, wherein the cell is a viable cell, including a cancer cell, an epithelial cell, a bone cell, a muscle cell, an adipocyte, a tissue cell, a senescent cell, a pluripotent cell, a stem cell, or a neural cell.

5. The method of any one of claims 1 to 4, wherein the particles are nanoparticles.

6. 5. The method of claim 1, wherein the contacting step of (c) further comprises contacting the biological sample with second particles to adsorb a second plurality of biomolecules onto the second particles.

7. 5. The method of any one of claims 1 to 4, wherein the conditions sufficient for the cells to produce the set of biomolecules in the supplemented medium comprise a predetermined temperature, a predetermined pressure, a predetermined flow regime, a predetermined solvent environment, or a combination thereof.

8. 5. The method of any one of claims 1 to 4, wherein the supplemented medium comprises cell culture supernatant, co-culture secretome, exosomes, tissue or cell lysate, or any combination thereof.

9. The method of any one of claims 1 to 4, wherein the supplemented medium comprises a synthetic supplemented medium.

10. 5. The method of any one of claims 1 to 4, wherein the set of biomolecules comprises one or more biomarkers, molecular signatures, secreted proteins, absorbed proteins, secretomes, exosomes, or any combination thereof.

11. A method according to any one of claims 1 to 4, further comprising the step of repeating (a) to (e) for a second supplemented medium and a second set of biomolecules, wherein, if necessary, the second supplemented medium is generated by incubating the cells in the supplemented medium for different lengths of time.

12. 5. The method of claim 1, wherein the generating step comprises the release of exosomes or liposomes by the cells.

13. The method according to any one of claims 1 to 4, wherein a biomolecule of the set of biomolecules is a polypeptide.

14. The method of any one of claims 1 to 4, wherein the conditions are varied over time.

15. The method of any one of claims 1 to 4, further comprising a plurality of cells comprising said cell, and further comprising said plurality of cells being contained in a tissue, an organoid, an organism, or multiple organisms.

16. 16. The method of claim 15, wherein the plurality of cells comprises at least a first cell of a first type and a second cell of a second type, such that the first cell exchanges one or more biomolecules of the set of biomolecules with the second cell.

17. 17. The method of claim 16, wherein the first cell and the second cell are co-cultured.

18. 17. The method of claim 16, wherein the first cells are contained in a feeder culture for the second cells.

19. The method of any one of claims 1 to 4, wherein the cells are comprised in a primary cell culture.

20. 16. The method of claim 15, wherein the plurality of cells are disposed in a plurality of separate volumes, each volume comprising a different supplemented medium or incubation condition.

21. The method of any one of claims 1 to 4, wherein the releasing step comprises the use of a protease.

22. 5. The method of any one of claims 1 to 4, wherein the one or more particles comprise at least two particles that comprise distinct physicochemical properties such that the at least two particles adsorb different patterns of biomolecule abundance from the set of biomolecules.

23. 5. The method of any one of claims 1 to 4, wherein the set of biomolecules comprises a dynamic range of concentrations of at least about 6, 7, 8, 9, 10 or more.

24. 5. The method of any one of claims 1-4, wherein particles of the one or more particles are disposed on a magnetic substrate, and the removing step of (d) comprises separating the magnetic substrate from the at least a portion of the supplemented medium using a magnetic field.

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