Compositions and methods for detecting proteins in protein corona

Abstract

This specification provides a method for detecting proteins and / or their proteoforms. The method comprises adding one or more small molecule compounds and one or more protein binders to a biological sample. A protein corona is formed on the surface of the protein binder, and the protein and / or its proteoform in the protein corona is detected by antibody-based or proteomics techniques. This specification also provides a method for detecting one or more biomarkers or patterns of one or more biomarkers associated with a disease or a state on the health spectrum, and a method for diagnosing or predicting disease in a subject. Compositions comprising one or more small molecule compounds, one or more protein binders, and one or more biological samples are also provided.

Description

Detailed Description of the Invention

[0001] [Cross - Reference to Related Applications] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 471,578, filed on June 7, 2023, and U.S. Provisional Application No. 63 / 603,325, filed on November 28, 2023. Each of the above patent applications is incorporated herein by reference in its entirety.

[0002] [Statement Regarding Government Support] This invention was made with government support under Grant No. DK131417 from the U.S. National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in this invention.

[0003] [Field] The present disclosure relates to a method of adding small - molecule compounds to biological samples for improving proteomic analysis of the protein corona on the surface of protein binders such as nanoparticles (NPs).

[0004] [Background] This section provides background information related to the present disclosure, which is not necessarily prior art.

[0005] The quest for comprehensive analysis of the plasma proteome has become extremely important for the advancement of disease diagnosis and monitoring, as well as biomarker discovery. However, due to the predominance of high - abundance proteins in plasma, challenges such as the identification of low - abundance proteins remain. The combined amount of the seven most abundant proteins accounts for 85% of the total protein mass. These high - abundance proteins, especially peptides derived from albumin, tend to dominate the mass spectrum, resulting in the hindrance of the detection of low - abundance proteins.

[0006] To address this challenge, techniques such as affinity removal, Protein Equalizer™ technology, and electrolyte fractionation have been developed to improve the detection of low-abundance proteins by reducing the concentration of these high-abundance proteins. Furthermore, a wide range of techniques have been developed to increase the throughput and depth of protein detection and identification, from advanced acquisition modes to methods for concentrating low-abundance proteins or peptides for liquid chromatography-mass spectrometry (LC-MS / MS) analysis. For example, affinity removal strategies utilize affinity chromatography columns with specific ligands that bind to high-abundance proteins such as albumin, immunoglobulins, and haptoglobin. However, the cost and effort associated with such removal strategies hinder their application to large cohorts. As another example, salting-out (also known as salt-induced precipitation) selectively precipitates high-abundance proteins using reagents (e.g., ammonium sulfate), leaving low-abundance proteins in the supernatant. However, these methods can introduce a bias that also precipitates low-abundance proteins, so robust additional strategies are needed in biomarker discovery research to avoid missing low-abundance proteins that have high diagnostic potential.

[0007] 〔overview〕 This section provides a general overview of the disclosure and does not constitute a comprehensive disclosure of its entire scope or all its features.

[0008] In certain embodiments, the Disclosure provides a method for detecting proteins and / or their proteoforms in a biological sample, such as human plasma. The Disclosure also provides a method for detecting one or more biomarkers, or patterns of one or more biomarkers, associated with a disease, such as a neoplastic or neurological disease. Furthermore, the Disclosure provides a method for diagnosing a disease in a subject.

[0009] In some embodiments, one or more small molecule compounds may be added to a biological sample along with one or more protein binders (such as nanoparticles). In some embodiments, the method may include the step of adding one or more small molecule compounds to at least two biological samples, each sample derived from a different subject diagnosed with a certain disease. The protein binder has a surface capable of binding proteins, thereby forming a protein corona on the surface of the protein binder. Thus, a complex containing the protein corona and the protein binder may be formed. In some embodiments, one or more small molecule compounds may be biomolecules such as lipids, metabolites, nutrients, or plant-derived molecules. Furthermore, in some embodiments, one or more small molecule compounds may be a combination of different small molecule compounds. In some embodiments, the small molecule compounds may be phosphatidylcholine (PdtCho) or a derivative thereof.

[0010] In some embodiments, different proteins and / or their proteoforms may be detected in the protein corona. In some embodiments, one or more biomarkers or patterns of one or more biomarkers may be detected in the protein corona. In some embodiments, proteins and / or biomarkers may be detected, for example, by antibody-based technology or proteomics technology. In some embodiments, an increase in the number of proteins and / or their proteoforms detected in the protein corona may be observed compared to a biological sample without the addition of one or more protein binders and one or more small molecule compounds, or compared to a biological sample containing one or more protein binders but without the addition of one or more small molecule compounds.

[0011] In further embodiments, the disclosure provides compositions comprising one or more small molecule compounds, one or more protein binders, and one or more biological samples. In some embodiments, the small molecule compound may be phosphatidylcholine. Additionally or alternatively, in some embodiments, the protein binder may be nanoparticles.

[0012] Further applicable areas will become apparent from the descriptions contained herein. The descriptions and examples in this summary are for illustrative purposes only and do not limit the scope of this disclosure.

[0013] [Brief explanation of the drawing] This patent or application document includes at least one color drawing. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon payment of the required fees and request.

[0014] The drawings described herein illustrate selected embodiments and do not represent all possible embodiments, nor do they limit the scope of this disclosure.

[0015] Figure 1 is an exemplary overview of the experimental process described herein. After exposing small molecular weight compounds or combinations of small molecular weight compounds to human plasma, nanoparticles (NPs) were incubated with human plasma, purified and isolated, and used to analyze the protein corona profile on the NP surface using SDS-PAGE and LC-MS / MS.

[0016] Figure 2 includes SDS-PAGE images analyzing protein corona-coated NPs in the presence of eight individual small molecule compounds and two combinations of small molecule compounds. The small molecule compounds were analyzed at concentrations of 0, 10, 100, and 1000 μg / ml. Each combination of small molecule compounds contains four small molecule compounds at a given concentration. For example, combination 1 (10 μg / ml) contains glucose (10 μg / ml), triacylglycerol (10 μg / ml), diacylglycerol (10 μg / ml), and phosphatidylcholine (10 μg / ml). Controls 1 and 2 correspond to SDS-PAGE results for all uncoated (bare) small molecule compounds (1,000 μg / ml) in the absence of human plasma. Further details are as follows: 1: Glucose, 2: Triacylglycerol, 3: Diacylglycerol, 4: Phosphatidylcholine, 5: Phosphatidylethanolamine, 6: L-α-Phosphatidylinositol, 7: Inosine 5'-monophosphate, 8: Vitamin B complex, 9: Combination of low molecular weight compounds 1, 10: Combination of low molecular weight compounds 2.

[0017] Figures 3A and 3B are bar graphs showing the analysis of dynamic light scattering (DLS) (Figure 3A) and zeta potential (Figure 3B) for uncoated NPs and untreated protein corona-coated NPs.

[0018] Figures 4A to 4C are transmission electron microscope (TEM) images of NPs. Figures 4A and 4B are TEM images of uncoated polystyrene NPs, and Figure 4C is a TEM image of protein corona-coated NPs. The polydispersity index (PDI) of uncoated NPs and protein corona-coated NPs was 0.023 and 0.214, respectively.

[0019] Figure 5 is a bar graph showing the number of quantified proteins in plasma, untreated protein corona, and protein corona in the presence of small molecule compounds and combinations of small molecule compounds (mean ± standard deviation of 3 technical replicates). The cumulative number of unique proteins identified under all conditions is also shown. For fair comparison, data analysis was performed separately for each category (plasma, untreated protein corona, and protein corona in the presence of each small molecule compound or combination of small molecule compounds). The experiment was performed in 3 technical replicates, and the protein abundances under each condition were averaged.

[0020] Figure 6 is a box plot showing the distribution of normalized intensities of quantified protein in plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules (center line, median; upper and lower limits of the box include 50%; upper and lower quartiles are 75% and 25%, respectively; maximum is the maximum value excluding outliers; minimum is the minimum value excluding outliers; outliers are values ​​greater than 1.5 times the upper and lower quartiles). The experiment was performed in three technical replicates, and the protein abundance under each condition was averaged.

[0021] Figure 7 is a heatmap showing the hierarchical clustering of all proteins (1793 proteins in total) quantified in all samples (plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules). The experiment was performed in three technical iterations, and the protein abundances under each condition were averaged.

[0022] Figure 8 is a heatmap showing the clustering of 117 proteins common to all samples (plasma, untreated protein corona, and protein corona in the presence of small molecule compounds and combinations of small molecule compounds). The experiment was performed in three technical iterations, and the protein abundances under each condition were averaged.

[0023] Figure 9 is a heatmap showing the correlation of plasma proteome profiles using the Pearson correlation coefficient at 10 - 1000 μg / ml for 117 proteins common to all samples (plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules).

[0024] Figure 10 includes two charts showing that different combinations of small molecules can enrich or decrease specific proteins. The charts show the number of unique proteins that were quantified in a given group but not in plasma or uncoated NPs.

[0025] Figures 11A - 11D show the enriched and decreased proteins in combinations 1 (Figures 11A and 11B) and 2 (Figures 11C and 11D) of small molecules compared to the untreated protein corona. For all decreased and enriched proteins, pathway analysis was performed respectively (Figures 11B and 11D). Significance was calculated using Welch's two - sample t - test for unequal variances.

[0026] Figure 12 includes a scatter plot showing the enrichment and decrease of specific proteins (only the shared ones) by spike - addition of small molecules to the NP protein corona compared to the abundance of proteins in the untreated NP protein corona. Only the results for the highest concentration (1000 μg / ml) of each small molecule are shown.

[0027] Figure 13 includes a bar graph showing pathway enrichment in KEGG processes and biological processes for all proteins that were significantly enriched and decreased, cumulating all concentrations for each small molecule.

[0028] Figure 14 includes an integrated enrichment plot in KEGG and biological processes for all small molecules and combinations of small molecules compared to the untreated protein corona, cumulating all concentrations.

[0029] Figure 15 includes a bar graph classifying quantified protein coronas of various small molecules and combinations of small molecules according to their physiological function.

[0030] Figure 16 is a graph showing the effect of adding spikes of small molecule compounds or combinations of small molecule compounds on the dynamic range of the proteome. The dynamic range (quantitative) of proteomics analysis for different samples is shown.

[0031] Figures 17A to 17C are graphs comparing the abundance and ranking of proteins (albumin - Figure 17A, serotransferrin - Figure 17B, haptoglobin - Figure 17C) between untreated plasma and protein corona profiles. Each graph shows the results based on the presence or absence of the addition of small molecule compounds or combinations of small molecule compounds.

[0032] Figures 18A–18C include charts showing protein abundances after incubation with NP and phosphatidylcholine (PtdCho). Figure 18A shows a stream diagram or alluvial diagram (including only proteins shared with plasma) showing a significant decrease in abundant plasma proteins, particularly albumin, after incubation of plasma with NP and PtdCho. Figure 18B shows the total number of proteins identified in plasma samples incubated with NP, comparing treatment with and without the addition of various concentrations of PtdCho (mean ± standard deviation of 3 technical replicates). Figure 18C is a stream diagram showing the decrease pattern in abundant plasma proteins, particularly albumin, in response to NP addition, indicating that this decrease is enhanced with increasing PtdCho concentration (including only proteins shared with plasma).

[0033] [Detailed explanation] A. Introduction In recent years, nanoparticles (NPs) have attracted attention for their ability to enhance biomarker discovery through the analysis of naturally formed protein / biomolecular coronas (i.e., layers of biomolecules, primarily proteins, that form on the surface of NPs when exposed to plasma or other biological fluids). NP protein coronas can contain the inherent ability to enrich low-abundance proteins, easily reducing the complexity of the proteome in LC-MS / MS analysis.

[0034] Discovering biomarkers using single NPs has limitations in achieving deep proteome coverage, typically only detecting a few hundred proteins. To improve proteome coverage and quantify more plasma proteins, protein corona sensor arrays or multiple NPs with distinct physicochemical properties can be used. This approach increases proteome coverage by leveraging the unique protein corona formed on each NP, but it has the drawback of requiring the analysis of multiple NP samples and testing of many NP types to achieve increased proteome coverage.

[0035] While it is known that protein coronas form on NP surfaces upon exposure to biological fluids, this disclosure provides a novel method for detecting low-abundance proteins by adding small molecule compounds. By adding a small molecule compound or a combination of small molecule compounds to a biological fluid along with a surface-containing protein binder (such as an NP), distinct protein corona patterns can be generated, significantly expanding the dynamic range of the plasma proteome that can be captured and detected by LC-MS / MS. The inventors unexpectedly discovered that the addition of small molecule compounds such as phosphatidylcholine (PtdCho) can significantly increase proteome coverage. Therefore, this technique can be seamlessly integrated with existing LC-MS / MS workflows, further enhancing the depth of plasma proteomic analysis for biomarker discovery.

[0036] The disclosed methods may also enable the early detection of conditions on the health spectrum, such as diseases or disorders. This may prevent or delay problems caused by diseases or conditions and improve patient prognosis (e.g., extending patient life and / or improving quality of life). For example, the disclosed methods may help prevent or reduce the risk of developing conditions such as obesity, heart disease, liver disease, kidney disease, depression, and cancer. Since various types of cancer can alter the composition of plasma even in their early stages, one approach for early detection is molecular blood analysis for one or more biomarkers. This disclosure provides novel methods for improving the ability to detect cancer (e.g., in its early stages).

[0037] Exemplary embodiments are provided so that the disclosure may be sufficient and its scope may be fully conveyed to those skilled in the art. Numerous specific details, such as examples of particular compositions, components, devices, and methods, are described so as to provide a full understanding of the embodiments of the disclosure. It will be apparent to those skilled in the art that it is not necessary to adopt specific details, that the exemplary embodiments may be carried out in many different forms, and that none of them should be construed as limiting the scope of the disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known techniques are not described in detail.

[0038] B. Definition The terms used herein are intended solely to describe and not limit to specific exemplary embodiments. Where used herein, the singular forms “a,” “an,” and “the” may also include the plural form unless otherwise specified. The terms “comprise,” “comprising,” “including,” and “having” are inclusive and therefore identify the presence of a described feature, element, composition, process, integer, operation, and / or component, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. The open-ended term “comprising” should be understood as a non-limiting term used to describe and claim the various embodiments described herein; however, in certain embodiments, the term may be understood as a more restrictive and limiting term, such as “consisting of” or “consisting essentially of.” Accordingly, with respect to any given embodiment that enumerates compositions, materials, components, elements, features, integers, operations, and / or processing steps, the Disclosure specifically includes embodiments comprising or substantially comprising the enumerated compositions, materials, components, elements, features, integers, operations, and / or processing steps. In the case of “comprising,” this alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and / or processes; on the other hand, in the case of “substantially comprising,” any additional compositions, materials, components, elements, features, integers, operations, and / or processes that substantially affect the basic and novel properties are excluded from such embodiment, but any compositions, materials, components, elements, features, integers, operations, and / or processes that do not substantially affect the basic and novel properties may be included in such embodiment.

[0039] Any method, step, process, and operation described herein should not be construed as necessarily having to be performed in a specific order described or illustrated unless the order of execution is specifically specified. Furthermore, unless otherwise indicated, additional or alternative steps may be employed.

[0040] In these claims and / or in this specification, when used in combination with the term “comprising,” the use of the terms “a” or “an” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.” Thus, the terms “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise. For example, a reference to “a compound” may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more compounds.

[0041] The use of the term "at least one" is understood to include not only one, but any quantity greater than one, such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more, thirty or more, forty or more, fifteen or more, twenty or more, forty or more, fifty or more, one hundred or more, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term it is accompanied by; furthermore, the quantity 100 / 1000 is not considered limiting, and satisfactory results may be obtained with higher upper limits. In addition, the use of the expression "at least one of X, Y, and Z" is understood to include X only, Y only, and Z only, as well as any combination of X, Y, and Z. The use of ordinal terms ("first," "second," "third," "fourth," etc.) is used solely to distinguish two or more items and does not imply, for example, that one item has a sequence, order, or importance to another item, or an additive order, etc.

[0042] The use of the term “or” in this claim means “and / or” inclusive, unless it explicitly refers to only one of the options, or unless such options are mutually exclusive. For example, the condition “A or B” is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0043] When used herein, any expression such as “one embodiment,” “embodiment,” “several embodiments,” “an example,” “for example,” or “example” means that a particular element, feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment. Where the expression “in several embodiments” or “an example” appears in various parts of this specification, they do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should not be construed as limiting the scope of these claims.

[0044] Throughout this disclosure, the term “approximately” is used to indicate that variations in error inherent in a composition / apparatus / device, a method used to determine a value, or variations existing among the subjects of study are included in the value. For example, without intent to limit, when the term “approximately” is used, a specified value may vary from a particular value by plus or minus 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Such variations are appropriate for carrying out the disclosed method and are within the range understood by those skilled in the art. In particular, with respect to a given quantity, number, or percentage, “approximately” means to include a deviation of plus or minus 10% (±10%). For example, approximately 5% includes values ​​between 4.5% and 5.5%, i.e., 4.5, 4.6, 4.7, 4.8, 4.9, 5, 4.1, 5.2, 5.3, 5.4, or 5.5, etc. Therefore, unless otherwise specified, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired characteristics intended to be obtained by the subject matter disclosed herein.

[0045] As used herein, the term “or any combination thereof” refers to all permutations and combinations of the items listed before the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, explicitly included are combinations that involve repetition of one or more items or terms. For example, BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0046] As will be understood by those skilled in the art, for any purpose, the entire scope disclosed herein includes all possible sub-scopes and all combinations thereof. Furthermore, as will be understood by those skilled in the art, the scope includes the individual components.

[0047] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. In particular, this disclosure utilizes the ordinary art in the field of wet granulation and pectin-containing materials.

[0048] As used herein, "protein corona" (often abbreviated as "PC") refers to a biomolecular shell formed on the surface of nanoparticles (NPs) during their interaction with biological fluids, and which changes over time. The term "biomolecular corona" refers to a group of different biomolecules capable of binding to a surface-containing protein binder (such as nanoparticles). The term "biomolecular corona" encompasses the term "protein corona" as used in the art, and refers to proteins, lipids, and other plasma components that bind to a protein binder, such as nanoparticles, when the binder comes into contact with biological fluids. In this specification, the term "protein corona" encompasses both soft protein coronas and hard protein coronas as referred to in the art. For example, Milani, et al., "Reversible versus Irreversible Binding of Transferrin to Polystyrene Nanoparticles: Soft and Hard Corona," ACS NANO, 2012, 6(3), pp. 2532-2541;Mirshafiee, et al., "Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake," Biomaterials, vol. 75, Jan. 2016 pp. 295-304, Mahmoudi, et al., "Emerging understanding of the protein corona at the nano-bio interfaces," Nanotoday, 11(6) Dec. 2016, pp. 817-832, and Mahmoudi, et al., "Protein-Nanoparticle Interactions: Opportunities and Challenges," Chem. Rev., 2011, 111(9), pp. See 5610-5637 (this entire section is incorporated by reference).As described in this art, the adsorption curve shows the formation of a strongly bound monolayer up to a saturation point (in the geometrically defined ratio of protein to nanoparticles). Beyond this saturation point, a secondary, weakly bound layer is formed. The first layer is irreversibly bound (hard corona), while the secondary layer (soft corona) exhibits dynamic exchange. High-affinity adsorbed proteins form what is known as a "hard" corona, which consists of tightly bound proteins that do not easily detach. On the other hand, low-affinity adsorbed proteins form what is known as a "soft" corona, which consists of loosely bound proteins. Soft and hard coronas can also be defined based on exchange time. Hard coronas typically exhibit much longer exchange times, usually on the order of several hours. See, for example, M. Rahman, et al., Protein-Nanoparticle Interactions, Spring Series in Biophysics 15, 2013 (the reference incorporates this entire work).

[0049] As used herein, “protein binding agent” is any agent that binds to, interacts with, or attracts one or more proteins in a biological sample and has a surface for protein binding. In some embodiments, the protein binding agent may be called a protein interacting agent, a protein attractant, and / or a protein corona-forming agent. In some embodiments, the protein binding agent may be a nanoscale material and / or a microscale material.

[0050] As used herein, “nanoscale material” or “nanomaterial” refers to a material whose single unit size (in at least one direction) is between 1 nanometer (nm) and 999 nm. Non-limiting examples of nanomaterials include nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, and nanotubes.

[0051] As used herein, “microscale material” or “micromaterial” refers to a material whose single unit size (in at least one direction) is between 1,000 nm and 100,000 nm. Non-limiting examples of micromaterials include microparticles, microrods, microspheres, and microbeads.

[0052] Examples of suitable nanoscale and / or microscale materials include, but are not limited to, organic materials, inorganic materials, or combinations thereof. In some embodiments, the materials are micelles, liposomes, iron oxide, graphene, silica, protein-based materials, polystyrene, silver, and gold materials such as colloidal gold, quantum dots, palladium, platinum, titanium, and combinations thereof. In some embodiments, the nanoparticles are liposomes. Those skilled in the art can select and prepare suitable nanoscale and / or microscale materials.

[0053] As used herein, "low molecular weight compound" refers to a molecule with a molecular weight of less than 5 kilodaltons (kDa). For the purposes of this disclosure, molecular weight may be calculated using the following formula: Molecular weight (in dalton units (Da) or unified atomic mass units (u)) = Σ((atomic weight of the element)) n × (Number of atoms of that element) n The low molecular weight compounds may be synthetic or natural products. In other words, the low molecular weight compounds may be synthesized or naturally occurring. The low molecular weight compounds described in the methods herein are also called protein recruiters because they promote the recruitment of different proteins (and other types of biomolecules) onto material surfaces (such as nanoparticles). In some embodiments, the low molecular weight compounds are also called "high-abundance protein binders" or "high-abundance protein interactors".

[0054] As used herein, "high-abundance protein" refers to one of the seven most abundant proteins present in human plasma (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen). These proteins together account for 85% of the total protein content in human plasma.

[0055] As used herein, "low-abundance proteins" refers to any protein present in human plasma, and these are not included in the seven high-abundance proteins.

[0056] As used herein, “biomolecules” are molecules produced by living organisms and essential for one or more biological processes. Biomolecules may be synthetically produced to mimic the function of naturally occurring biomolecules. Suitable examples of biomolecules include, but are not limited to, macromolecules (proteins, carbohydrates, lipids, nucleic acids, etc.) and smaller molecules (vitamins, hormones, etc.). Biomolecules may also be referred to herein as biological materials. Accordingly, small molecule compounds as used herein may be considered “small molecule biomolecules” if they meet the definition of a small molecule compound (a molecule with a molecular weight of less than 5 kilodaltons (kDa)) and the definition of a biomolecule (a molecule produced by a living organism and essential for one or more biological processes, or a molecule synthetically produced to mimic the function of a naturally occurring biomolecule).

[0057] As used herein, “metabolites” refers to metabolic intermediates or final products. This is intended to include all naturally occurring small molecules, synthetic small molecules, and biological small molecules, such as amino acids, alcohols, polyols, alkaloids, organic acids, sugars (e.g., glucose), and nucleotides (e.g., inosine 5'-monophosphate and guanosine 5'-monophosphate).

[0058] As used herein, “lipids” refers to a group of organic compounds, including all natural, synthetic, and biological fatty compounds. These fatty compounds include glycerolipids (e.g., triacylglycerols (also known as triglycerides, TG or TAG), diacylglycerols (also known as diglycerides, DG or DAG, such as 1,2-diacylglycerol and 1,3-diacylglycerol)), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, L-α-phosphatidylinositol), sterol lipids, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL), fatty acids, prenolipids, and sphingolipids.

[0059] As used herein, “nutrient” refers to substances that organisms utilize for survival, growth, and reproduction. In some cases, metabolites may also be considered nutrients (such as amino acids, fatty acids, vitamins (e.g., B vitamin complexes), minerals, and choline).

[0060] As used herein, "plant-derived molecule" refers to any molecule derived from plants. Suitable plant-derived molecules include low molecular weight compounds (such as auxins, gibberellic acid, alkaloids, and phenylpropanoids); plant-derived biological agents (such as anticancer biological agents); and plant-derived pharmaceuticals (phytopharmaceutical drugs) that have properties for human health problems (such as allergies and inflammation).

[0061] As used herein, the term “endogenous” refers to substances (such as nucleic acids, proteins, enzymes, and small molecules) produced within a host organism (e.g., a human), and / or substances that spontaneously arise or exist within a host organism. Thus, endogenous substances refer to substances produced by a host organism and present within the host organism. In some embodiments, endogenous substances may be encoded by the genome of the host organism. In some embodiments, said substances may be encoded by autonomously replicating plasmids possessed by the host organism. In some embodiments, endogenous substances refer to substances that were present in a biological sample of the host organism when it was first isolated from nature, i.e., substances that are inherently present (native) to the organism. For example, “endogenously produced” substances may be expressed / produced by the host organism’s own mechanisms. In other words, the host organism has not been genetically modified to produce said substances. In other words, a host organism may endogenously produce natural or non-natural substances.

[0062] In contrast, as used herein, “exogenous” substances refer to substances (such as nucleic acids, proteins, enzymes, and small molecules) that are not encoded or produced by a host organism (human or non-human, e.g., mouse, rat, or pig), and are therefore added from outside the host organism to a biological sample taken from the host organism or a host organism. For example, “exogenously added” may refer to the addition of a substance such as a small molecule to a host organism or a biological sample taken from a host organism. A nucleic acid sequence encoding a variant (i.e., mutant) polypeptide is an example of an exogenous nucleic acid sequence when added to a host organism or host organism cells. Such an exogenous nucleic acid sequence may encode a polypeptide or enzyme that is endogenous or naturally present in the cell. Such encoded polypeptides or enzymes may be considered “exogenously expressed.” For example, additional copies of an endogenous gene may be introduced into the cell to achieve overexpression of that gene (e.g., using a vector such as a plasmid). Such additional copies of endogenous genes may be considered “exogenous” (e.g., exogenous genes or exogenous nucleic acid sequences) because the additional copies are introduced into the cell from outside the cell. “Exogenous genes” or “exogenous nucleic acid sequences” may also refer to native (or endogenous) genes or nucleic acid sequences that exhibit dysregulation (e.g., up-expression, repression, or attenuation) by being operably linked to regulatory elements, or that are otherwise modified or altered. Exogenous nucleic acid sequences or exogenous genes can also be used to express or overexpress heterologous polypeptides or enzymes within a cell. Therefore, exogenous nucleic acid sequences or exogenous genes may encode polypeptides (e.g., enzymes) that are naturally present in the cell, naturally endogenous in the cell, or heterologous to the cell.

[0063] As used herein, the term “native” refers to the form of a composition, such as a small molecule compound, isolated from nature, or to a composition in its natural state, without intentionally introduced mutations in its structural sequence and without engineering modifications (e.g., changing a developmentally regulated gene into a constitutively expressed gene). Also as used herein, “native” refers to “wildtype” or “wild-type,” where the composition exists in both its typical sequence, quantity, and relative quantity as it naturally occurs in an organism. Wild-type organisms may be used as controls and / or references in determining cellular function. For example, native molecules (such as lipids, genes, nucleic acid sequences, polypeptides, or enzymes) are typically endogenous in cells; that is, they are molecules present in or produced by cells. Exogenous nucleic acid sequences or exogenous genes may encode native polypeptides or enzymes, for example, when additional copies of native genes or nucleic acid sequences are added to a cell from outside the cell, or when native genes or nucleic acid sequences are dysregulated or modified. Such exogenous nucleic acid sequences or exogenous genes may be operably linked, for example, to regulatory elements that are not naturally present in the cell or are not endogenous in that cell.

[0064] As used herein, the term “non-natural” refers to nucleic acid sequences, amino acid sequences, polypeptide sequences, enzymes, and / or small molecule compounds that do not naturally exist in a host. Heterogenes and polypeptides are considered “non-natural.” Nucleic acid sequences or amino acid sequences that are removed from host cells and introduced or reintroduced into host cells through laboratory manipulation are also considered “non-natural.” Synthetic or partially synthetic genes introduced into host cells are “non-natural.” Non-natural genes also include genes that are endogenous and / or naturally occurring in a host microorganism and are operably linked to one or more heterogeneous regulatory sequences that have been recombined into the host genome. Genes that naturally exist under the control of heterogeneous regulatory sequences are considered “non-natural.” In some embodiments, organisms containing non-natural genes may be used as controls and / or references to organisms having additional and / or different variations from wild-type organisms.

[0065] As used herein, “sample” refers to a biological sample or complex biological sample obtained from a subject. Suitable biological samples include, but are not limited to, systemic circulating blood, plasma, serum, lung lavage fluid, cell lysates, menstrual blood, urine, processed tissue samples, amniotic fluid, cerebrospinal fluid, tears, saliva, and semen. In certain embodiments, the sample is whole blood, plasma, or serum sample.

[0066] As used herein, the term "health spectrum" covers a broad range of health conditions, including complete well-being, minor health problems, chronic diseases, and serious illnesses. The health spectrum adopts a holistic and dynamic view of health, emphasizing prevention and the continuity of health conditions. It relates to overall well-being and the factors influencing it, including whether these factors contribute to optimal health or disease. The health spectrum emphasizes preventive medicine, lifestyle improvements, and overall health maintenance. In contrast, "disease / disorder" focuses on specific pathological conditions affecting health. Disease / disorder employs more specific and diagnostic approaches, focusing on the identification and treatment of specific conditions. It aims to identify specific diseases or functional impairments and address them effectively, focusing on the pathological aspects of health. Disease / disorder emphasizes medical interventions, treatment plans, and symptom management for specific conditions.

[0067] Disease / disability is one example of a health status spectrum. Other conditions include prediagnostic states, where there is a risk of developing a disease or disability, but the condition has not yet reached the stage of a diagnosed disease.

[0068] C. Method [Methods for detecting proteins and other biomolecules in biological samples] (General method) In one embodiment, the disclosure provides a method for detecting biomolecules (such as proteins and / or their proteoforms) in a biological sample, the method comprising the step of adding one or more small molecule compounds and one or more protein binders to the biological sample. The protein binder has a surface capable of binding proteins, thereby forming a protein corona on the surface of the protein binder. Thus, a complex comprising the protein corona and the protein binder is generated. The number of proteins and / or their proteoforms in the protein corona may then be detected by antibody-based techniques or proteomics techniques.

[0069] One or more low molecular weight compounds and one or more protein binders may be added to the biological sample in any order. For example, in some embodiments, one or more low molecular weight compounds may be added to the biological sample, and then one or more protein binders may be added to the biological sample containing one or more low molecular weight compounds. Alternatively, in some embodiments, one or more protein binders may be added to the biological sample, and then one or more low molecular weight compounds may be added to the biological sample containing one or more protein binders. Alternatively, in some embodiments, one or more low molecular weight compounds and one or more protein binders may be added to the biological sample simultaneously or almost simultaneously.

[0070] After adding one or more low molecular weight compounds and / or protein binders to a biological sample, the sample containing the one or more low molecular weight compounds and / or protein binders may be incubated to allow a protein corona to form on the surface of the protein binders. For example, in some embodiments, the biological sample may be incubated with one or more low molecular weight compounds. In some embodiments, the sample may be incubated with one or more protein binders. In some embodiments, the sample may be incubated with one or more low molecular weight compounds and one or more protein binders. In some embodiments, the biological sample may be incubated for at least 10 seconds to about 24 hours. For example, biological samples are tested for at least approximately 10 seconds, at least approximately 15 seconds, at least approximately 20 seconds, at least approximately 25 seconds, at least approximately 30 seconds, at least approximately 40 seconds, at least approximately 50 seconds, at least approximately 60 seconds, at least approximately 90 seconds, at least approximately 2 minutes, at least approximately 3 minutes, at least approximately 4 minutes, at least approximately 5 minutes, at least approximately 6 minutes, at least approximately 7 minutes, at least approximately 8 minutes, at least approximately 9 minutes, at least approximately 10 minutes, at least approximately 15 minutes, at least approximately 20 minutes, at least approximately 25 minutes, at least approximately 30 minutes, at least approximately 45 minutes, at least approximately 50 minutes, and at least approximately 6 The sample may be incubated for 0 minutes, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours, including any time and increments in between. In certain embodiments, the biological sample may be incubated for about 1 hour. In some embodiments, the biological sample may be incubated one or more times.For example, a biological sample may be incubated once, twice, three times, four times, or five times.

[0071] The incubation temperature can be determined by those skilled in the art and includes temperatures such as about 4°C to about 40°C, about 4°C to about 20°C, about 10°C to about 15°C, about 10°C to about 40°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 25°C, about 30°C, about 35°C, about 37°C, etc. In some embodiments, this method may be carried out at room temperature (e.g., about 37°C; e.g., about 35°C to about 40°C).

[0072] In some embodiments, the biological sample may be diluted. In some embodiments, the biological sample may be diluted with a suitable buffer such as phosphate-buffered saline (PBS), Tris-HCl buffer, ammonium bicarbonate buffer, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, PIPES (1,4-piperazine diethanesulfonic acid) buffer, or EPPS (4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid) buffer. In certain embodiments, the biological sample may be diluted with PBS. For example, a biological sample may be diluted to a final concentration of approximately 25% to 85%, 30% to 80%, 35% to 75%, 40% to 70%, 45% to 65%, 50% to 60%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. In certain embodiments, a biological sample may be diluted to a final concentration of approximately 55% using PBS.

[0073] (Biological sample) In some embodiments, the biological sample may include, but is not limited to, biological fluids such as systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysates, menstrual blood, urine, processed tissue samples, amniotic fluid, cerebrospinal fluid, tears, saliva, and semen. In certain embodiments, the biological sample may be whole blood, plasma, or serum samples. In other particular embodiments, the biological sample may be plasma samples. In some embodiments, the biological sample may contain one or more biological samples. For example, the biological sample may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more identical or different biological samples.

[0074] In some embodiments, biological fluids or complex biological samples may be prepared by methods and kits known in the art. For example, some biological samples (such as menstrual blood, blood samples, and semen) may first be centrifuged at low speed to remove cell debris, thrombi, and other cellular components that may interfere with the methods described herein. For example, in other embodiments, tissue samples may be processed. For example, tissue samples may be finely chopped or homogenized, and the tissue may be broken down by enzymatic treatment and / or cell debris may be removed by centrifugation to allow for the analysis and extraction of molecules within the tissue sample. Suitable methods for separating and / or properly preparing and preserving blood samples are known in the art and may include, but are not limited to, the addition of anticoagulants.

[0075] In some embodiments, a method for detecting proteins in a biological sample may include a step of removing one or more proteins from the biological sample. For example, the step of removing one or more proteins from a biological sample may include passing the biological sample through a resin-based depletion column or spin column. In some embodiments, passing the biological sample through a resin-based depletion column or spin column may reduce the complexity of the biological sample for analysis by antibody-based or proteomics techniques. For example, the complexity of the biological sample may be reduced for top-down, middle-down, and bottom-up proteomics analysis. In some embodiments, a depletion column or spin column may be used to reduce the complexity of biological samples (such as serum and plasma) that contain high concentrations of albumin and immunoglobulins. For example, a depletion column or spin column may be used to remove high-abundance proteins (such as albumin and IgG) from a biological sample. In some embodiments, a suitable depletion column or spin column may be a High Select® Depletion Spin Column (Thermo Scientific®). In some embodiments, the protein depletion method may be used for drug delivery and / or imaging. In some embodiments, the protein removal method (e.g., including removal columns or spin columns) may include particles such as lipid NPs having a low molecular weight compound (e.g., PtdCho) on its surface. Thus, in some embodiments, proteins such as albumin may be attracted to and / or bind to the surface of NPs coated with the low molecular weight compound. This may prolong the blood circulation time of NPs coated with the low molecular weight compound and / or allow for rapid removal of NPs coated with the low molecular weight compound by the immune system.

[0076] (Detection of biomolecules (e.g., proteins)) The methods described herein may be used to detect biomolecules in biological samples. Biomolecules that may be detected by the disclosed methods may include small molecules (lipids, fatty acids, glycolipids, sterols, monosaccharides, vitamins, hormones, neurotransmitters, metabolites, etc.), monomers (amino acids, monosaccharides, isoprene, nucleotides, etc.), oligomers (oligopeptides, oligosaccharides, terpenes, oligonucleotides, etc.), and polymers (polypeptides, proteins and / or their proteoforms, polysaccharides, polyterpenes, polynucleotides, nucleic acids, etc.). In some embodiments, the methods described herein may be used to detect proteins and / or their proteoforms. In some embodiments, the proteoforms of proteins may be different forms of proteins produced from the genome with various biological variations (sequence mutations, splicing isoforms, post-translational modifications, etc.), which may alter the primary structure and composition at the whole protein level. In some embodiments, the proteoforms may possess different biological functions.

[0077] In some embodiments, the methods described herein may be used to detect multiple unique biomolecules in a biological sample. For example, the methods may be used to detect multiple different proteins and / or their proteoforms in a biological sample. Thus, the methods described herein may be used to detect one type of protein and / or its proteoform, and / or the methods may be used to detect one or more types of proteins and / or their proteoforms. In some embodiments, the methods may be used to detect the amount of one type of protein present in a biological sample and / or in the protein corona. In some embodiments, the methods may be used to detect the amount of one or more types of proteins present in a biological sample and / or in the protein corona. In some embodiments, the methods may be used to detect the number of distinct proteins present in a biological sample and / or in the protein corona.

[0078] (Low molecular compound) The methods described herein utilize combinations of low molecular weight compounds and protein binders to detect biomolecules (such as proteins) in a biological sample. As described herein, “one or more low molecular weight compounds” covers “combinations of low molecular weight compounds” (i.e., combinations of one or more low molecular weight compounds). In some embodiments, the methods include the step of adding one or more low molecular weight compounds or combinations of low molecular weight compounds to a biological sample. In some embodiments, the methods may include the step of adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and so on, to a biological sample. In some embodiments, the methods may include the step of adding one or more combinations of low molecular weight compounds to a biological sample. In some embodiments, the combination of low molecular weight compounds may include different types of low molecular weight compounds such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the method may include the step of adding one or more combinations of low molecular weight compounds to a biological sample. In some embodiments, the method may include the step of adding combinations of low molecular weight compounds such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 to a biological sample. In some embodiments, the number of low molecular weight compounds or combinations of low molecular weight compounds added to the biological sample may be any number that reaches a desired concentration of the low molecular weight compounds or combinations of low molecular weight compounds in the biological sample.

[0079] In some embodiments, low molecular weight compounds or combinations of low molecular weight compounds may be added to a biological sample at concentrations of approximately 1 pg / ml to approximately 1 g / ml. For example, in some embodiments, low molecular weight compounds or combinations of low molecular weight compounds may be added at concentrations of approximately 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, and 400 μg / ml. / ml, approximately 500μg / ml, approximately 600μg / ml, approximately 700μg / ml, approximately 800μg / ml, approximately 900μg / ml, approximately 1,000μg / ml, approximately 2,000μg / ml, approximately 3,000μg / ml, approximately 4,000μg / ml, approximately 5,000μg / ml, approximately 6,000μg / ml, approximately 7,000μg / ml, approximately 8,000μg / ml, approximately 9,000μg / ml, approximately 10,000μg / ml, approximately 11,000μg / ml, approximately 12,000μg / ml, approximately 13,000μg / ml, approximately 14,000μg / ml, approximately 15, 000μg / ml, approximately 16,000μg / ml, approximately 17,000μg / ml, approximately 18,000μg / ml, approximately 19,000μg / ml, approximately 20,000μg / ml, approximately 10μg / ml to approximately 10,000μg / ml, approximately 20μg / ml to approximately 10,000μg / ml ml, about 30μg / ml to about 10,000μg / ml, about 40μg / ml to about 10,000μg / ml, about 50μg / ml to about 10,000μg / ml, about 60μg / ml to about 10,000μg / ml, about 70μg / ml to about 10,000μg / ml, about 80 μg / ml ~ approx. 10,000 μg / ml, approx. 90 μg / ml ~ approx. 10,000 μg / ml, approx. 100 μg / ml ~ approx. 10,000 μg / ml, approx. 200 μg / ml ~ approx. 9,000 μg / ml, approx. ml~about 7,000μg / ml, about 500μg / ml~about 6,000μg / ml, about 600μg / ml~about 5,000μg / ml, about 700μg / ml~about 4,000μg / ml, about 800μg / ml~about 3,000μg / ml, about 900μg / ml~about 2,000μg / ml, approximately 950μg / ml to approximately 1,500μg / ml, approximately 10μg / ml to approximately 1,000μg / ml, approximately 20μg / ml to approximately 1,000μg / ml, approximately 30μg / ml to approximately 1,000 μg / ml, approximately 40μg / ml to approximately 1,000μg / ml, approximately 50μg / ml to approximately 1,000μg / ml, approximately 60μg / ml to approximately 1,000μg / ml, approximately 70μg / ml to approximately 1,000μg / m l, about 80 μg / ml to about 1,000 μg / ml, about 90 μg / ml to about 1,000 μg / ml, about 10 μg / ml to about 900 μg / ml, about 10 μg / ml to about 800 μg / ml, about 10 μg / ml ml~about 700μg / ml, about 10μg / ml~about 600μg / ml, about 10μg / ml~about 500μg / ml, about 10μg / ml~about 400μg / ml, about 10μg / ml~about 300μg / ml, Approximately 10μg / ml to approximately 200μg / ml, approximately 10μg / ml to approximately 100μg / ml, approximately 10μg / ml to approximately 50μg / ml, approximately 1,000μg / ml to approximately 20,000μg / ml, approximately 2,000μ g / ml ~ approx. 19,000μg / ml, approx. 3,000μg / ml ~ approx. 18,000μg / ml, approx. 4,000μg / ml ~ approx. 17,000μg / ml, approx. 5,000μg / ml ~ approx. 16,000μ It may be added to the biological sample at concentrations of g / ml, approximately 6,000 μg / ml to approximately 15,000 μg / ml, approximately 7,000 μg / ml to approximately 14,000 μg / ml, approximately 8,000 μg / ml to approximately 13,000 μg / ml, approximately 9,000 μg / ml to approximately 12,000 μg / ml, approximately 9,000 μg / ml to approximately 11,000 μg / ml, or approximately 9,500 μg / ml to approximately 10,500 μg / ml. In some embodiments, approximately 10 μg / ml of the low molecular weight compound or combination of low molecular weight compounds may be added to the biological sample. In some embodiments, approximately 1,A low molecular weight compound or combination of low molecular weight compounds at a concentration of 000 μg / ml may be added to the biological sample. In some embodiments, one or more low molecular weight compounds or combinations of low molecular weight compounds may be added to the biological sample at various concentrations. In some embodiments, these various concentrations may range from about 1 pg / ml to about 1 g / ml.

[0080] In some embodiments, the low molecular weight compounds have molecular weights of approximately less than 5 kDa, approximately 4.9 kDa, approximately 4.8 kDa, approximately 4.7 kDa, approximately 4.6 kDa, approximately 4.5 kDa, approximately 4.4 kDa, approximately 4.3 kDa, approximately 4.2 kDa, approximately 4.1 kDa, approximately 4.0 kDa, approximately 3.9 kDa, approximately 3.8 kDa, approximately 3.7 kDa, approximately 3.6 kDa, approximately 3.5 kDa, approximately 3.4 kDa, approximately 3.3 kDa, approximately 3.2 kDa, approximately 3.1 kDa, approximately 3.0 kDa, approximately 2.9 kDa, and approximately 2. The molecular weights may be 8 kDa, approximately 2.7 kDa, approximately 2.6 kDa, approximately 2.5 kDa, approximately 2.4 kDa, approximately 2.3 kDa, approximately 2.2 kDa, approximately 2.1 kDa, approximately 2.0, approximately 1.9, approximately 1.8, approximately 1.7, approximately 1.6, approximately 1.5, approximately 1.4, approximately 1.3, approximately 1.2, approximately 1.1 kDa, approximately 1.0 kDa, approximately 0.9 kDa, approximately 0.8 kDa, approximately 0.7 kDa, approximately 0.6 kDa, approximately 0.5 kDa, approximately 0.4 kDa, approximately 0.3 kDa, approximately 0.2 kDa, or approximately 0.1 kDa. In other words, a low molecular weight compound may be a molecule with a molecular weight of less than approximately 5 kDa.

[0081] As used herein, a low molecular weight compound may be considered a “low molecular weight biomolecule” if it has a molecular weight of less than 5 kilodaltons (kDa), is produced by an organism, and is essential for one or more biological processes (or is synthetically produced to mimic the function of a naturally occurring biomolecule). In some embodiments, the low molecular weight compound may be synthetically produced (i.e., artificial). Alternatively, in some embodiments, the low molecular weight compound may be naturally occurring. In some embodiments, the low molecular weight compound may be endogenous to the host organism from which the biological sample is taken. In other embodiments, the low molecular weight compound may be exogenous to the host organism from which the biological sample is taken. Additionally or alternatively, the low molecular weight compound may be native or non-native to the host organism. For example, the low molecular weight compound may be native or non-native to the host organism from which the biological sample is taken, and may be, for example, a lipid, protein, or nucleic acid, and may be exogenously added to the biological sample.

[0082] In some embodiments, low-molecular-weight compounds may promote the accumulation of different proteins (and other types of biomolecules) on the surface of protein binders such as nanoparticles. In some embodiments, low-molecular-weight compounds may have the ability to alter protein coronas. For example, low-molecular-weight compounds may have the ability to alter protein coronas formed on the surface of protein binders such as nanoparticles. In some embodiments, low-molecular-weight compounds may have the ability to remove high-abundance plasma proteins (such as albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen) from biological samples. Therefore, in some embodiments, low-molecular-weight compounds may be called "high-abundance protein removers." In some embodiments, low-molecular-weight compounds may have the ability to remove at least one, at least two, at least three, at least four, at least five, at least six, or all seven high-abundance plasma proteins. In certain embodiments, low-molecular-weight compounds may have the ability to remove albumin.

[0083] In some embodiments, small molecule compounds may physically or chemically interact with one or more proteins in a biological sample. Therefore, in some embodiments, small molecule compounds may be referred to as “protein interactors.” In some embodiments, small molecule compounds may bind to proteins in a biological sample. In some embodiments, small molecule compounds may bind to albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and / or fibrinogen. In certain embodiments, small molecule compounds may bind to one or more abundant protein types. Therefore, in some embodiments, binding to abundant proteins may reduce the number of abundant proteins in the biological sample. In some embodiments, small molecule compounds may alter the conformation of one or more proteins in a biological sample.

[0084] In some embodiments, the low molecular weight compound may be a metabolite and / or derivative thereof. In some embodiments, the metabolite may be a naturally occurring low molecular weight compound, a synthetic low molecular weight compound, or a biological low molecular weight compound (such as amino acids, alcohols, polyols, alkaloids, organic acids, sugars (e.g., glucose), and nucleotides (e.g., inosine 5'-monophosphate and guanosine 5'-monophosphate)).

[0085] In some embodiments, the low molecular weight compound may be a lipid and / or a derivative thereof. In some embodiments, the lipid may be a group of organic compounds including natural fatty compounds, synthetic fatty compounds, or biological fatty compounds (glycerolipids (e.g., triacylglycerol (also called triglycerides, TG or TAG), diacylglycerol (also called diglycerides, DG or DAG; such as 1,2-diacylglycerol and 1,3-diacylglycerol)), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, L-α-phosphatidylinositol), sterol lipids, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), fatty acids, prenolipids, and sphingolipids).

[0086] In some embodiments, the low molecular weight compound may be a nutrient and / or a derivative thereof. In some embodiments, the metabolite may also be considered a nutrient (such as amino acids, fatty acids, vitamins (e.g., B vitamin complex), minerals, and choline).

[0087] In some embodiments, the low molecular weight compound may be a plant-derived molecule and / or a derivative thereof. For example, it may be a low molecular weight compound (such as auxin, gibberellic acid, alkaloid, or phenylpropanoid), a plant-derived biological agent (such as an anti-cancer biological agent), or a plant-derived pharmaceutical (such as one with properties for human health problems such as allergies or inflammation).

[0088] In some embodiments, the low molecular weight compound may be a metabolite and / or derivative thereof, a lipid and / or derivative thereof, a nutrient and / or derivative thereof, a plant-derived molecule and / or derivative thereof, or a combination thereof.

[0089] In some embodiments, the low molecular weight compound may be triacylglycerol and / or derivatives, diacylglycerol (such as 1,2-diacylglycerol and 1,3-diacylglycerol) and / or derivatives, glycerophospholipid and / or derivatives, glucose and / or derivatives, inosine 5'-monophosphate and / or derivatives, vitamin B complex and / or derivatives, phosphatidylcholine and / or derivatives, phosphatidylethanolamine and / or derivatives, phosphatidylserine and / or derivatives, phosphatidic acid and / or derivatives, phosphatidylinositol and / or derivatives, phosphatidylglycerol and / or derivatives, cardiolipin and / or derivatives, L-α-phosphatidylinositol and / or derivatives, or a combination thereof.

[0090] For use herein, combinations of low molecular weight compounds are also intended. For example, in some embodiments, the low molecular weight compounds may be combinations of triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives, and glycerophospholipids and / or its derivatives. Additionally or alternatively, the low molecular weight compounds may be combinations of glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and vitamin B complex and / or its derivatives. Additionally or alternatively, the low molecular weight compounds may be combinations of triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; glycerophospholipids and / or its derivatives, glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and vitamin B complex and / or its derivatives. Additionally or alternatively, the low molecular weight compounds may be combinations of glucose and / or its derivatives, triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; and phosphatidylcholine and / or its derivatives. Additionally or alternatively, the low molecular weight compounds may be combinations of phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and vitamin B complex and / or its derivatives.

[0091] (Protein binder) As described herein, this method uses a combination of low-molecular-weight compounds and protein binders to detect biomolecules (such as proteins) in a biological sample. In some embodiments, the method includes the step of adding one or more protein binders to the biological sample. In some embodiments, the addition of protein binders to the biological sample may result in the formation of a colloidal suspension.

[0092] In some embodiments, one or more protein binders may be added to the biological sample at concentrations of approximately 1 pg / ml to approximately 1 g / ml. Therefore, in some embodiments, one or more protein binders may be added at concentrations of approximately 1 μg / ml, approximately 2 μg / ml, approximately 3 μg / ml, approximately 4 μg / ml, approximately 5 μg / ml, approximately 6 μg / ml, approximately 7 μg / ml, approximately 8 μg / ml, approximately 9 μg / ml, approximately 10 μg / ml, approximately 20 μg / ml, approximately 30 μg / ml, approximately 40 μg / ml, approximately 50 μg / ml, approximately 60 μg / ml, approximately 70 μg / ml, approximately 80 μg / ml, approximately 90 μg / ml, approximately 100 μg / ml, approximately 200 μg / ml, approximately 300 μg / ml, approximately 400 μg / ml, approximately 500μg / ml, approximately 600μg / ml, approximately 700μg / ml, approximately 800μg / ml, approximately 900μg / ml, approximately 1,000μg / ml, approximately 2,000μg / ml, approximately 3,000μg / ml, approximately 4,000μg / ml, approximately 5,000μg / ml, approximately 6,00 0μg / ml, approximately 7,000μg / ml, approximately 8,000μg / ml, approximately 9,000μg / ml, approximately 10,000μg / ml, approximately 11,000μg / ml, approximately 12,000μg / ml, approximately 13,000μg / ml, approximately 14,000μg / ml, approximately 15,000 μg / ml, approximately 16,000 μg / ml, approximately 17,000 μg / ml, approximately 18,000 μg / ml, approximately 19,000 μg / ml, approximately 20,000 μg / ml, approximately 10 μg / ml to approximately 10,000 μg / ml, approximately 20 μg / ml to approximately 10,000 μg / ml , about 30μg / ml to about 10,000μg / ml, about 40μg / ml to about 10,000μg / ml, about 50μg / ml to about 10,000μg / ml, about 60μg / ml to about 10,000μg / ml, about 70μg / ml to about 10,000μg / ml, about 80μ g / ml~about 10,000μg / ml, about 90μg / ml~about 10,000μg / ml, about 100μg / ml~about 10,000μg / ml, about 200μg / ml~about 9,000μg / ml, about 300μg / ml~about 8,000μg / ml, about 400μg / m l ~ about 7,000 μg / ml, about 500 μg / ml ~ about 6,000 μg / ml, about 600 μg / ml ~ about 5,000 μg / ml, about 700 μg / ml ~ about 4,000 μg / ml, about 800 μg / ml ~ about 3,000 μg / ml, about 900 μg / ml ~ about 2,000μg / ml, approximately 950μg / ml to approximately 1,500μg / ml, approximately 10μg / ml to approximately 1,000μg / ml, approximately 20μg / ml to approximately 1,000μg / ml, approximately 30μg / ml to approximately 1,000 μg / ml, approximately 40μg / ml to approximately 1,000μg / ml, approximately 50μg / ml to approximately 1,000μg / ml, approximately 60μg / ml to approximately 1,000μg / ml, approximately 70μg / ml to approximately 1,000μg / m l, about 80 μg / ml to about 1,000 μg / ml, about 90 μg / ml to about 1,000 μg / ml, about 10 μg / ml to about 900 μg / ml, about 10 μg / ml to about 800 μg / ml, about 10 μg / ml ml~about 700μg / ml, about 10μg / ml~about 600μg / ml, about 10μg / ml~about 500μg / ml, about 10μg / ml~about 400μg / ml, about 10μg / ml~about 300μg / ml, Approximately 10μg / ml to approximately 200μg / ml, approximately 10μg / ml to approximately 100μg / ml, approximately 10μg / ml to approximately 50μg / ml, approximately 1,000μg / ml to approximately 20,000μg / ml, approximately 2,000μ g / ml ~ approx. 19,000 μg / ml, approx. 3,000 μg / ml ~ approx. 18,000 μg / ml, approx. 4,000 μg / ml ~ approx. 17,000 μg / ml, approx. 5,000 μg / ml ~ approx. 16,000 μg It may be added to the biological sample at concentrations of approximately 6,000 μg / ml to 15,000 μg / ml, approximately 7,000 μg / ml to 14,000 μg / ml, approximately 8,000 μg / ml to 13,000 μg / ml, approximately 9,000 μg / ml to 12,000 μg / ml, approximately 9,000 μg / ml to 11,000 μg / ml, or approximately 9,500 μg / ml to 10,500 μg / ml. In some embodiments, one or more protein binders (approximately 1,000 μg / ml (0.1 mg / ml)) may be added to the biological sample. In some embodiments, one or more protein binders (approximately 2,000 μg / ml (0.2 mg / ml)) may be added to the biological sample.

[0093] A protein binder may bind to, interact with, or attract one or more proteins and / or their proteoforms in a biological sample. Therefore, protein binders may also be called protein interactors and / or protein attractants. A protein binder may be any agent or material that provides a surface for protein binding. In other words, a protein binder may have a surface on which proteins can bind and form a protein corona.

[0094] The protein binder may be an inorganic agent, a metal-based agent, a metal oxide-based agent, a polymer-based agent, a lipid-based agent, a carbon-based agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof. Additionally or alternatively, the protein binder may contain one or more nanoscale or microscale materials.

[0095] Nanoscale materials may be any material whose single unit size (in at least one direction) is in the range of approximately 1 nm to approximately 999 nm. For example, the nanoscale material (or nanomaterial) may be approximately 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 999 nm. In certain embodiments, the nanoscale material may be nanoparticles of approximately 80 nm.

[0096] A microscale material may be any material whose single unit size (in at least one direction) is in the range of approximately 1,000 nm to approximately 100,000 nm. For example, a microscale material (or micromaterial) may be approximately 1,000 nm, 2,000 nm, 3,000 nm, 4,000 nm, 5,000 nm, 6,000 nm, 7,000 nm, 8,000 nm, 9,000 nm, 10,000 nm, 20,000 nm, 30,000 nm, 40,000 nm, 50,000 nm, 60,000 nm, 70,000 nm, 80,000 nm, 90,000 nm, or 100,000 nm.

[0097] Therefore, the protein binder may be any material whose single unit size (in at least one direction) is in the range of approximately 1 nm to approximately 100,000 nm. For example, the protein binder may be approximately 1 nm, approximately 50 nm, approximately 100 nm, approximately 500 nm, approximately 1,000 nm, approximately 5,000 nm, approximately 10,000 nm, approximately 50,000 nm, approximately 100,000 nm, approximately 50 nm to approximately 100,000 nm, approximately 100 nm to approximately 100,000 nm, approximately 500 nm to approximately 100,000 nm, approximately 1,000 nm to approximately 100,000 nm, approximately 5,000 nm to approximately 100,000 nm, approximately 10,000 nm to approximately 100 1,000nm, approximately 50,000 to approximately 100,000nm, approximately 1nm to approximately 50,000nm, approximately 1nm to approximately 10,000nm, approximately 1nm to approximately 10,000nm, approximately 1nm to approximately 5,000nm, approximately 1nm to approximately 1,000nm, approximately 1nm to approximately 500nm, approximately 1nm to approximately 100nm, approximately 1nm to approximately 50nm, approximately 50nm to approximately 50,000nm, approximately 100nm to approximately 10,000nm, or approximately 500nm to approximately 5,000nm.

[0098] Non-limiting examples of nanoscale materials include nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, and nanotubes. Non-limiting examples of microscale materials include microparticles, microrods, microspheres, and microbeads. One or more protein binders may comprise one or more nanoscale materials, one or more microscale materials, or a combination thereof. For example, in some embodiments, one or more protein binders may comprise a combination of one or more nanoparticles and one or more nanodiscs. In certain embodiments, one or more protein binders may comprise one or more nanoparticles.

[0099] The protein binder may be made from organic materials, inorganic materials, or combinations thereof. In some embodiments, the material may be micelles, liposomes, iron oxide, graphene, silica, protein-based materials, polystyrene, silver and gold materials, quantum dots, palladium, platinum, titanium, or combinations thereof.

[0100] In some embodiments, the multiple protein binders may comprise at least 2 to at least 1000 protein binders, which may be identical or different. In some embodiments, the number of protein binders added to the biological sample may be any number that reaches a desired concentration of protein binders in the biological sample. In some embodiments, the number of protein binders may be about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1,000 per biological sample.

[0101] In some embodiments, the protein binders used in this method may all be of the same type. For example, in some embodiments, the protein binder added to the biological sample may be polystyrene nanoparticles. In other embodiments, the protein binders may be of different types. For example, the protein binder added to the biological sample may be a mixture of polystyrene nanoparticles and silica microbeads. Any combination of protein binders having a surface for binding proteins may be used herein. The choice of protein binder is not particularly important as long as a protein corona can be formed on its surface. Those skilled in the art can determine one or more suitable materials that can form a protein corona on their surface.

[0102] In some embodiments, the protein binder may have a polydispersity index (PDI) of about 0.01 to about 10. Therefore, the protein binders are approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.02-0.6, 0.03-0.5, 0.04-0.4, 0.05-0.3, 0.06-0.2, 0.07-0.1, 0.08-0.09, 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, and 0.01. ~approximately 0.09, approximately 0.01~approximately 0.08, approximately 0.01~approximately 0.07, approximately 0.01~approximately 0.06, approximately 0.01~approximately 0.05, approximately 0.01~approximately 0.04, approximately 0.01~approximately 0.03, approximately 0.01~approximately 0.02, approximately 0.02~approximately 0.7, approximately 0.03~approximately 0.7, approximately 0.04~approximately 0.7, approximately 0. The PDI may be approximately 0.5 to 0.7, approximately 0.06 to 0.7, approximately 0.07 to 0.7, approximately 0.08 to 0.7, approximately 0.09 to 0.7, approximately 0.1 to 0.7, approximately 0.2 to 0.7, approximately 0.3 to 0.7, approximately 0.4 to 0.7, approximately 0.5 to 0.7, or approximately 0.6 to 0.7. A PDI of approximately 0.01 is sometimes called simple variance.

[0103] In some embodiments, the PDI of the protein binder may be about 0.01 to about 0.7. In certain embodiments, the PDI of the protein binder may be about 0.7. In another embodiment, the PDI of the protein binder may be about 0.3. In yet another embodiment, the PDI of the protein binder may be about 0.2.

[0104] One or more protein binders have a surface capable of binding proteins to a biological sample, causing a protein corona to form on the surface of one or more protein binders. This may produce a complex containing a protein corona and one or more protein binders. Therefore, protein binders may also be referred to herein as protein corona-forming agents or protein corona-attracting agents.

[0105] (Protein Corona) The disclosed method includes the step of adding a protein binder to a biological sample to generate a protein corona. In some embodiments, when one or more protein binders are added to the biological sample, the protein corona may spontaneously form on the protein binder. In some embodiments, after adding one or more protein binders to the biological sample, the protein corona may form on the protein binder by incubating the biological sample containing one or more protein binders. Therefore, in some embodiments, the protein binders and the biological sample may be incubated for a time sufficient to form a protein corona on the surface of one or more protein binders. In certain embodiments, the protein corona may form around nanoparticles.

[0106] The protein corona may primarily consist of proteins, but in some embodiments, as described herein, the protein corona may also consist of non-protein molecules (such as lipids and other biological sample components) that can be bound to the protein binder. In this case, the protein corona may be called a biomolecular corona. In some embodiments, the proteins in the protein corona may be present in the same proportion as the proteins in the untreated biological sample (i.e., the biological sample before the addition of low-molecular-weight compounds and / or protein binders). In some embodiments, the proteins in the protein corona may be present in a different proportion than the proteins in the untreated biological sample. In some embodiments, the protein corona profile may differ between different subjects and / or different biological samples. In some embodiments, the protein corona profile may be identical between different subjects and / or different biological samples.

[0107] In some embodiments, the protein corona may be formed in different patterns and have different compositions depending on the size, shape, composition, charge, and surface functional groups of the protein and / or protein binder. In some embodiments, the properties of the protein corona may change due to different environmental factors (such as temperature, pH, shear stress, composition of the immersion medium, and exposure time). In some embodiments, the composition of the protein corona may change according to biochemical and physicochemical surface interactions with the protein binder. In some embodiments, the protein corona may include a hard corona and / or a soft corona. The hard corona may include high-affinity proteins that can irreversibly bind to the surface of the protein binder and / or other proteins in the protein corona. The soft corona may include low-affinity proteins that reversibly bind to the surface of the protein binder and / or other proteins in the protein corona. In some embodiments, proteins in the soft corona may be replaced or detached over time. In some embodiments, larger, lower-affinity proteins may initially aggregate on the surface of the protein binder and be replaced over time by smaller, higher-affinity proteins (i.e., corona "hardening").

[0108] Additionally or alternatively, the composition of the protein corona may change when one or more low molecular weight compounds are added to a biological sample along with one or more protein binders, as described herein. For example, the composition of the protein corona may change when phosphatidylcholine is added to a biological sample along with one or more protein binders. For example, phosphatidylcholine itself may bind to certain proteins based on its properties, and therefore the types and / or number of proteins that can bind to the protein binders may change. For example, a low molecular weight compound may reduce the amount of one or more proteins that are suspended in the biological sample. Therefore, in some embodiments, the types and / or number of proteins that can bind to the protein binders may change due to one or more low molecular weight compounds in the biological sample. In some embodiments, phosphatidylcholine may bind to high-abundance proteins such as albumin, and therefore the protein corona composition may have a reduced proportion of high-abundance proteins such as albumin.

[0109] In some embodiments, protein corona may be used to detect the types and / or amounts of one or more protein types present in a biological sample.

[0110] (Method for preparing a protein detection complex) Methods for preparing a complex (containing the protein corona and a protein binder) for detecting the protein and / or its proteoform in the protein corona are also described herein. In some embodiments, the complex may be isolated from the remainder of the biological sample. For example, isolation may be performed by centrifugation (e.g., gradient centrifugation), size exclusion chromatography, magnetic separation, field flow fractionation, etc. In some embodiments, the complex may be washed and resuspended. In some embodiments, the protein derived from the complex may be reduced, alkylated, and / or digested. In some embodiments, the protein and / or proteoform present in the protein corona may be detected by antibody-based techniques. For example, the protein and / or its proteoform may be detected using Western blotting, enzyme immunosorbent assay (ELISA), or OLINK® (olink.com). Additionally or alternatively, the protein and / or its proteoform may be detected by proteomics techniques. For example, the protein and / or its proteoform may be detected by top-down, middle-down, or bottom-up proteomics (e.g., LC-MS / MS), or a combination thereof. In some embodiments, top-down, middle-down, or bottom-up proteomics may be based on data-dependent acquisition (DDA) or data-independent acquisition (DIA). In some embodiments, DDA or DIA may include unlabeled or label-based techniques (e.g., tandem mass tag (TMT) labeling, dimethyl labeling). In some embodiments, one or more protein binders may be used in the protein corona sensor array. In some embodiments, multiple biological samples may be tested and compared.

[0111] In some embodiments, proteins and / or their proteoforms may be detected in hard protein corona, soft protein corona, or a combination of hard and soft protein corona. In some embodiments, proteins present in the protein corona may be detected after the biological sample has been incubated with one or more small molecule compounds and one or more protein binders. In some embodiments, the composition of the protein corona may change over time. For example, in some embodiments, the protein corona composition after 10 minutes of incubation may differ from the protein corona composition after 20 minutes of incubation. In some embodiments, molecules other than proteins bound to the protein binder may be detected.

[0112] (Increased protein detection) In some embodiments, the methods described herein may detect more proteins and / or their proteoforms compared to using a biological sample that does not contain one or more protein binders and / or one or more small molecule compounds. In other words, the methods may result in an increase in the number of proteins detected compared to using a biological sample that does not contain one or more protein binders and / or one or more small molecule compounds or combinations of small molecule compounds. In some embodiments, the methods may detect more proteins and / or their proteoforms compared to using a biological sample that contains one or more protein binders but does not contain one or more small molecule compounds or combinations of small molecule compounds. For example, in some embodiments, when using the disclosed method, the number of proteins 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 0.1-20, 0.2-20, 0.3-20, 0.4-20, 0.5-20, 0.6-20, 0.7-20, 0.8-20, 0. The increase may be 9-20, 1-20, 2-19, 3-18, 4-17, 5-16, 6-15, 7-14, 8-13, 9-12, 10-11, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1 to 9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 times.In some embodiments, when using the disclosed method, compared to a biological sample containing one or more protein binders but not containing one or more small molecule compounds or combinations of small molecule compounds, the number of proteins and / or their proteoforms 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 0.1-20, 0.2-20, 0.3-20, 0.4-20, 0.5-20, 0.6-20, 0.7-20, 0 0.8~20, 0.9~20, 1~20, 2~19, 3~18, 4~17, 5~16, 6~15, 7~14, 8~13, 9~12, 10~11, 1~19, 1~18, 1~17, 1~16, 1~15, 1~14, 1~13, 1~12, 1~11, 1~10, 1 to 9, 1~8, 1~7, 1~6, The increase may be 1-5, 1-4, 1-3, 1-2, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 times. In certain embodiments, the number of proteins and / or their proteoforms may increase by at least 8 times compared to a biological sample that does not contain one or more protein binders and one or more small molecule compounds. In certain embodiments, the number of proteins and / or their proteoforms may increase by at least 3 times compared to a biological sample that does not contain one or more protein binders and one or more small molecule compounds. In some embodiments, the number of proteins and / or their proteoforms may increase by at least 2 times compared to a biological sample that contains one or more protein binders without the addition of one or more small molecule compounds.

[0113] In some embodiments, the disclosed method may increase the detection of low-abundance proteins. For example, one or more low-molecular-weight compounds added to the biological sample may bind to one or more high-abundance proteins, such as albumin. Thus, in some embodiments, fewer high-abundance proteins present in the biological sample may bind to the protein binder. Thus, in some embodiments, more low-abundance proteins may attach to the protein-binding molecule and be detected by the disclosed method. Therefore, in some embodiments, the step of adding one or more low-molecular-weight compounds to the biological sample together with one or more protein binders when detecting proteins in protein corona may reduce the detection of one or more high-abundance proteins by at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0114] In some embodiments, multiple types of protein binders may be used to detect one or more proteins and / or their proteoforms. In some embodiments, different protein binders may attract different protein types. In some embodiments, using one or more types of protein binders may increase the number of proteins detected in a biological sample. In some embodiments, when one or more protein binders are used, each type of protein binder may have distinct physicochemical properties. Therefore, in some embodiments, the protein corona formed around the protein binders may differ between different protein binders.

[0115] Therefore, this disclosure provides a method for increasing the number of proteins detected in a biological sample compared to other known methods.

[0116] Furthermore, compositions used for detecting proteins and / or their proteoforms are also described herein. The compositions may comprise one or more low molecular weight compounds described herein, one or more protein binders described herein, and one or more biological samples described herein.

[0117] In addition to the process of detecting proteins in a biological sample, this disclosure also provides a method for detecting biomarkers in a biological sample.

[0118] [Biomarker detection methods] In another embodiment, the Disclosure provides a method for detecting one or more biomarkers or patterns of one or more biomarkers in a biological sample. In some embodiments, the one or more biomarkers or patterns of one or more biomarkers may be associated with a health spectrum condition such as a disease or disorder. In some embodiments, the method may include the step of adding one or more low molecular weight compounds described herein to at least two biological samples, each of which may be derived from a different subject diagnosed with a certain disease. The method may also include the step of adding one or more protein binders (which may all be of the same type or a combination of different types) having a protein-binding surface as described herein. In some embodiments, a partial corona is formed on the surface of the protein binder to produce a complex comprising the protein corona and one or more protein binders.

[0119] The method may also include a step of detecting one or more biomarkers or patterns of one or more biomarkers in the protein corona by antibody-based technology or proteomics technology, as described herein. In some embodiments, the biomarker pattern may include multiple types of biomarkers. In some embodiments, the biomarker pattern may contain specific amounts of one or more biomarkers in the biological sample. In some embodiments, individual organisms or biological samples may have different biomarker patterns. For example, like biomarkers, biomarker patterns may be used for the detection and / or diagnosis of disease or disorder in a subject.

[0120] A biomarker may be a measurable indicator of some biological state or condition. In some embodiments, biomarkers may be upregulated or downregulated according to different disease types or disease stages. In some embodiments, biomarkers may be molecular, physiological, histological, and / or radiological biomarkers. Additionally or alternatively, biomarkers may be predictive, prognostic, or diagnostic. In some embodiments, predictive biomarkers may help optimize the ideal treatment. Examples of predictive biomarkers include HER2 / neu mutations in breast cancer or EGFR1 mutations in non-small cell lung cancer. In some embodiments, diagnostic biomarkers may be traceable substances introduced into an organism as a means of investigating a part of health. In other embodiments, diagnostic biomarkers may be substances whose detection indicates a particular disease state. For example, the presence of an antibody may suggest an infection. For example, a diagnostic biomarker may be prostate-specific antigen (PSA), which may be used as a measure of prostate size, and rapid changes may suggest cancer.

[0121] In some embodiments, multiple protein binder types may be used to detect one or more biomarkers and / or one or more biomarker patterns. In some embodiments, different protein binders may attract different types of biomarkers. In some embodiments, using one or more types of protein binders may increase the number of biomarkers detected in the biological sample.

[0122] In some embodiments, a state on the health spectrum may be any health state, including complete well-being, mild health problems, chronic diseases, and serious illnesses. A state on the health spectrum refers to overall well-being and the factors that influence it, which may result in optimal health or contribute to disease. In some embodiments, the health spectrum may include states that the subject may have that are not yet considered to be diseases or disabilities requiring medical treatment. For example, in some embodiments, a state on the health spectrum may include predisposition to disease or disability. In other words, the presence of a biomarker may indicate that the subject has a state on the health spectrum and / or is at risk of developing a disease / disability. In some embodiments, the biomarker for disease / disability may be identical to the predisposition to disease or disability. Additionally or alternatively, the biomarkers for disease / disability may be different for the same disease or disability. Alternatively, a health spectrum state may be a disease or disability. Examples of health spectrum conditions include, but are not limited to, predisposition, risk of developing, or diagnosis of obesity, heart disease, liver disease, kidney disease, depression, and cancer.

[0123] In some embodiments, a disease may be any condition that adversely affects all or part of the structure or function of an organism and is not directly caused by any external injury. In some embodiments, diseases to which a biomarker may be associated may be neoplastic diseases, cardiovascular diseases, metabolic diseases, infectious diseases, inflammatory diseases, congenital and genetic diseases, degenerative diseases, neurological diseases, and combinations thereof. For example, in some embodiments, a disease may be a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof. In some embodiments, the disease may be a neurological disorder selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0124] Suitable biomarkers for Alzheimer's disease include, but are not limited to, the amyloid-beta (Aβ) 42 / 40 ratio, phosphorylated tau (p-tau), serum neuronal filament light chains (NfL), and glial fibrillary acidic proteins (GFAP).

[0125] Appropriate cancer biomarkers include, for example, AHSG (α2-HS-glycoprotein), AKR7A2 (aflatoxin B1 aldehyde reductase), AKT3 (PKBγ), ASGR1 (ASGPR1), BDNF, BMP1 (BMP-1), BMPER, C9, CA6 (carbonic anhydrase VI), CAPG (CapG), carcinoembryonic antigen, CDH1 (cadherin-1), CHRDL1 (Chordin-like protein 1), CKB-CKM- (CK-MB), and CLI. C1 (intracellular chloride channel 1), CMA1 (chymase), CNTN1 (contactin-1), COL18A1 (endostatin), CRP, CTSL2 (cathepsin V), DDC (dopa decarboxylase), EGFR (ERBB1), FGA-FGB-FGG (D-dimer), FN1 (fibronectin FN1.4), GHR (growth hormone receptor), GPI (glucose phosphate isomerase), HMGB1 (HMG-1), HNRNPAB (hnRNP A / B), HP (haptoglobin, mixed type), HSP90AA1 (HSP90α), HSPA1A (HSP70), IGFBP2 (IGFBP-2), IGFBP4 (IGFBP-4), IL12B-IL23A (IL-23), ITIH4 (inter-α-trypsin inhibitor heavy chain H4), KIT (SCFsR), KLK3-SERPINA3 (PSA-ACT), L1CAM (NCAM-L1), LRIG3, MMP12 (MMP-12), MMP7 (MMP-7), NME2 (NDP kinase B), PA2G4 (ErbB3 binding protein Ebp1), PLA2G7 (LpPLA2 / PAFAH), PLAUR (suPAR), PRKACA (PRKAC-α), PRKCB (PKC-β-II), PROK1 (EG-VEGF), PRSS2 (Trypsin-2), PTN (Pleiotrophin), SERPINA1 (α1-Antitrypsin), STC1 (Staniocalcin-1), STX1A (Syntaxin 1A), TACSTD2 (GA733-1 protein), TFF3 (Trefoil's factor 3), TGFBI (βIGH3), TPI1 (Triose phosphate isomerase), TPT1 (Fortirin) YWHAG (14-3-3 protein γ), YWHAH (14-3-3 protein η), prostate cancer biomarkers (e.g., p63 protein, PSA, Pro-PSA, Pro2PSA, PHI, PCA3, TMPRSS3:ERG, PCMT, MTEN), breast cancer markers (e.g., epidermal growth factor receptor 2 (HER2) oncogene), melanoma biomarker BRAF, lung cancer biomarkers EML4-ALK, A2ML1, BAX, C10orf47, C lorfl62, CSDA, EIFC3, ETFB, GABARAPL2, GUKl, GZMH, HIST1H3B, HLA-A, HSP90AA1, NRGN, PRDX5, PTMA, RABACl, RABAGAP1L, RPL22, SAP18, SEPW1, SOX1, EGFR, EGFRvIII, apolipoprotein A1, apolipoprotein CIII, myoglobin, tenascin C, MSH6, claudin 3, claudin 4, caveolin 1, Coagulation factor III, CD9, CD36, CD37, CD53, CD63, CD81, CD136, CD147, Hsp70, Hsp90, Rabl3, Desmocolin-1, EMP-2, CK7, CK20, GCDF15, CD82, Rab-5b, Annexin V, MFG-E8, HLA-DR, miR200 microRNA, MDC, NME-2, KGF, PIGF, Flt-3L, HGF, MCP1, SAT-1, MIP-1-b, GCLM, OPG, TNFBiomarkers for breast cancer include, but are not limited to, RII, VEGF-D, ITAC, MMP-10, GPI, PPP2R4, AKR1B1, Amy1A, MIP-1b, P-cadherin, and EPO. Biomarkers for breast cancer include, but are not limited to, circulating tumor cells (EpCAM, CD45, cytokeratin 8, 18+, 19+), ER / PR, HER-2 / neu, CA15-3, CA27, 29, etc. Biomarkers for colorectal cancer include, but are not limited to, EGFR, KRAS, UGT1A1, fibrin / fibrinogen degradation products (DR-70), and human hemoglobin (fecal occult blood), etc. Biomarkers associated with leukemia / lymphoma include, but are not limited to, CD20 antigen, CD30, FIP1L1-PDGFRα, PDGFR, Philadelphia chromosome (BCR / ABL), PML / RARα, TPMT, and UGT1A1. Biomarkers associated with lung cancer include, but are not limited to, ALK, EGFR, and KRAS. Biomarkers associated with ovarian cancer include, but are not limited to, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, and CA-125. Biomarkers associated with hepatocellular carcinoma include, but are not limited to, AFP-L3%. Biomarkers associated with gastrointestinal stromal tumors include, but are not limited to, c-Kit. Biomarkers associated with pancreatic cancer include, but are not limited to, CA19-9. Biomarkers are publicly known in this field, for example, W, Herberman R B. Tumor markers and immunodiagnosis. In: Bast RC Jr., Kufe DW, Pollock RE, et al., editors. Cancer Medicine. 6th ed. Hamilton, Ontario, Canada: BC Decker Inc., 2003; Andriole G, Crawford E, Grubb R. et al. Mortality results from a randomized prostate-cancer screening trial. NewEngland Journal of Medicine 2009; 360(13):1310-1319;Schroder FH, Hugosson J, Roobol MJ, et al. Screening and prostate-cancer mortality in a randomized European study. New England Journal of Medicine 2009; 360(13):1320-1328;Buys SS, Partridge E, Black A. et al. Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA 2011; 305(22):2295-2303;Cramer DW, Bast RC Jr, Berg CD, et al. Ovarian cancer biomarker performance in prostate, lung, colorectal, and ovarian cancer screening trial specimens. Cancer Prevention Research 2011; 4(3):365-374; Sparano JA, Gray RJ, Makower DF, et al. Prospective validation of a 21-gene expression assay in breast cancer. New England Journal of Medicine 2015; First published online Sep. 28, 2015. doi: 10.1056 / NEJMoa1510764, and clinicalproteomicsjournal.biomedcentral.com / articles / 10.1186 / 1559-0275-10-13 / tables / 1, which are incorporated in their entirety by reference.

[0126] Biomarkers associated with cardiovascular disease may include, but are not limited to, physiological biomarkers based on measurements of lipid profiles, glucose and hormone concentrations, as well as concentrations of important biological molecules (such as serum ferritin, triglyceride to HDLp (high-density lipoprotein) ratio, lipofolin-cholesterol ratio, lipid-lipofolin ratio, LDL cholesterol concentration, HDLp and apolipoprotein concentrations, lipofolin and LTPs ratio, sphingolipids, omega-3 index, and ST2 concentration). Biomarkers suitable for cardiovascular disease are described, for example, in van Holten et al. “Ciculating Biomarkers for Predicting Cardiovascular Disease Risk; a Systemic Review and Comprehensive Overview of Meta-Analyses” PLoS One, 2013 8(4): e62080 (the entire work is incorporated by reference).

[0127] Biomarkers associated with neurological disorders may include, but are not limited to, Aβ1-42, t-τ and p-τ181, and α-synuclein, as well as other biomarkers. See, for example, Chintamaneni and Bhaskar, “Biomarkers in Alzheimer's Disease: A Review,” ISRN Pharmacol. 2012. 2012: 984786. Published online 2012 Jun. 28 (the entire work is incorporated by reference).

[0128] Furthermore, compositions used for detecting one or more biomarkers or patterns of one or more biomarkers are also described herein. Such compositions may include one or more low molecular weight compounds, one or more protein binders, and one or more biological samples.

[0129] [Methods for diagnosing diseases or identifying health conditions] In another embodiment, a method for diagnosing a disease or identifying a condition on another health spectrum, such as a predisposition to disease in a subject, is disclosed herein. This method may include the steps of adding one or more small molecule compounds described herein to a biological sample (derived from a subject) as described herein, and further adding one or more protein binders described herein to the biological sample as described herein. The protein binders have a surface capable of binding proteins, and a protein corona is formed on the surface of one or more protein binders. This may form a complex comprising the protein corona and one or more protein binders. This method includes the steps of detecting one or more disease-related biomarkers or one or more biomarker patterns in the protein corona by antibody-based technology or proteomics technology as described herein.

[0130] As mentioned above, the "health spectrum" covers a range of health conditions, including full well-being, mild health problems, chronic diseases, and serious illnesses. For example, examples of conditions on the health spectrum include both mental and physical conditions ranging from the pre-disease stage to serious illnesses, and include, but are not limited to, obesity, heart disease, liver disease, kidney disease, depression, and cancer.

[0131] As described above, the disease may be a neoplastic disease, cardiovascular disease, metabolic disease, infectious disease, inflammatory disease, congenital and hereditary disease, degenerative disease, neurological disease, or a combination thereof. For example, in some embodiments, the disease may be a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, or a combination thereof. In some embodiments, the disease may be a neurological disorder selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0132] In some embodiments, the disclosed method may be used to provide predispositions to disease onset (e.g., likelihood or prognosis). In some embodiments, the disclosed method may be used to detect early onset of disease (i.e., early diagnosis of disease). For example, in some embodiments, apolipoproteins, which are indicators of cardiovascular and neurodegenerative disorders, may be detected. Thus, the method may detect protein categories that are important in the onset and progression of disease.

[0133] As described herein, disease / disorder biomarkers may be the same and / or different with respect to predisposition to the disease / disorder. Therefore, in some embodiments, the same biomarkers detected in the diagnosis of a disease / disorder may be detected to identify predisposition to the disease / disorder. In some embodiments, the biomarker concentration or amount in the sample may be lower when identifying predisposition to a disease compared to when diagnosing the disease. In some embodiments, the diagnosis of a disease, or the identification of other health spectrum conditions (such as predisposition to a disease or health spectrum condition), may help prevent or reduce the risk of developing conditions such as obesity, heart disease, liver disease, kidney disease, depression, or cancer. Therefore, in some embodiments, subjects diagnosed with a disease or health spectrum condition, or identified as having a predisposition to a disease or health spectrum condition, may take measures to prevent or delay the progression of the disease or condition.

[0134] This specification also describes compositions used for the diagnosis of diseases. Such compositions may comprise one or more low molecular weight compounds, one or more protein binders, and one or more biological samples (in any order).

[0135] [Examples] The following embodiments are illustrative and do not limit the disclosure in any way.

[0136] [Example 1: Addition of various low molecular weight compounds and combinations of low molecular weight compounds alters the protein corona of nanoparticles] An overview of the process is shown in Figure 1. Generally, after exposing small molecular weight compounds to human plasma, nanoparticles (NPs) were incubated with human plasma, purified, and isolated. The obtained NPs were used to analyze the protein corona profile on the NP surface using SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS / MS).

[0137] (material) Healthy human plasma proteins were obtained from Innovative Research®, Inc. (Novi, MI) (diluted to a final concentration of 55% using phosphate-buffered saline (PBS, 1X)).

[0138] The commercially available additive-free polystyrene nanoparticles (NPs) have an average size of approximately 80 nm and were provided by Polysciences®, Inc. (Warrington, PA).

[0139] Low molecular weight compounds were sourced from Millipore Sigma [phosphatidylcholine (PtdCho), phosphatidylethanolamine (PE), L-α-phosphatidylinositol (PtdIns), inosine 5'-monophosphate (IMP)], Abcam® [triacylglycerol], Fisher Scientific® [diacylglycerol and glucose], and VWR® [vitamin B complex], and diluted with PBS (1X) to the desired concentrations. Eight distinct groups of low molecular weight compounds (i.e., glucose, triacylglycerol, diacylglycerol, phosphatidylcholine (PtdCho), phosphatidylethanolamine (PE), L-α-phosphatidylinositol (PtdIns), inosine 5'-monophosphate (IMP), and vitamin B complex) were evaluated to determine their effects on the protein corona formed around polystyrene nanoparticles. The selection of these small molecule compounds is based on their ability to interact with a wide range of proteins, which significantly influences the composition of the protein corona surrounding NPs. For example, components of the vitamin B complex can interact with a wide range of proteins (such as albumin, hemoglobin, myoglobin, pantothenate permease, acyl carrier proteins, lactoferrin, prions, β-amyloid precursors, and niacin-responsive repressors). Furthermore, to evaluate the collective effects of these molecules, two combinations of small molecule compounds were analyzed. Combination 1 of the small molecule compounds is a mixture of glucose, triacylglycerol, diacylglycerol, and PtdCho, while combination 2 of the small molecule compounds is a mixture of PE, PtdIns, IMP, and the vitamin B complex.

[0140] (Protein corona formation on NP surfaces in the presence of low molecular weight compounds) To investigate the formation of protein coronas in the presence of small molecule compounds, a commercially available pool of healthy human plasma was first diluted to 55% with PBS, and individual small molecule compounds or combinations of small molecule compounds were incubated at 37°C for 1 hour at various concentrations (i.e., 10, 100, and 1,000 μg / ml). This allowed the small molecule compounds to interact with the biological matrix. The combinations of small molecule compounds included four types of small molecule compounds (combination 1: glucose, triacylglycerol, diacylglycerol, and PtdCho; combination 2: PE, PtdIns, IMP, and vitamin B complex) at given concentrations. For example, combination 1 (10 μg / ml) contained glucose (10 μg / ml), triacylglycerol (10 μg / ml), diacylglycerol (10 μg / ml), and PtdCho (10 μg / ml). In addition, for example, combination 2 (1,000 μg / ml) contained PE (1,000 μg / ml), PtdIns (1,000 μg / ml), IMP (1,000 μg / ml), and vitamin B complex (1,000 μg / ml). Polystyrene NPs were then added to the solution containing plasma and low molecular weight compounds to adjust the final concentration of NPs to 0.2 mg / ml. The solution was then incubated at 37°C for 1 hour with stirring. These methods resulted in the formation of a separate protein corona around the NPs. All experimental trials were designed so that the concentrations of NPs, human plasma, and low molecular weight compounds were 0.2 mg / ml, 55%, and 10, 100, and 1,000 μg / ml, respectively. To remove unbound proteins in the plasma or proteins loosely attached to the NP surface, the protein-nanoparticle complexes were centrifuged at 14,000 xg for 20 minutes. The NP precipitate was washed three times with cold PBS, centrifuged under the same conditions, and the final precipitate was collected for further analysis. In the PtdCho concentration studies, various concentrations of PtdCho (i.e., 100, 500, 1,000, and 10,000 μg / ml) were used in the above protocol for sample preparation for mass spectrometry.

[0141] (Evaluation of NP characteristics) Dynamic light scattering (DLS) and zeta potential analysis were performed to measure the size distribution and surface charge of NPs before and after protein corona formation. A Zetasizer® ZS90 nano series DLS instrument (Malvern Panalytical®) was used for these measurements. A 632 nm wavelength helium-neon (He-Ne) laser was used at room temperature for size distribution measurement. Transmission electron microscopy (TEM) was performed using a JEM-2200FS field emission electron microscope (JEOL® Ltdt.) operating at 200 kV. This instrument was equipped with an in-column energy filter and an Oxford Instruments energy-dispersive X-ray spectroscopy (EDXS) system. Uncoated nanoparticles (20 μl) were deposited on a copper grid and used for imaging. For protein corona-coated NPs, the sample (20 μl) was negatively stained with 1% uranyl acetate (20 μl), washed with deionized water (DI), and then deposited on a copper grid for imaging.

[0142] Figures 3A and 3B show the results of dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM) analysis for untreated polystyrene NPs and NPs coated with protein corona. Untreated polystyrene NPs exhibited excellent monodispersity, with an average size of 78.8 nm, a polydispersity index of 0.026, and a surface charge of -30.1 ± 0.6 mV (Figures 3A and 3B). Upon protein corona formation, the average size of the NPs expanded to 113 nm, and the surface charge shifted to -10 mV ± 0.4 mV (Figures 3A and 3B). TEM analysis further confirmed the changes in NP size and morphology before and after protein corona formation. The polydispersity index (PDI) of uncoated NPs and protein corona-coated NPs was 0.023 and 0.214, respectively (Figures 4A-4C).

[0143] (Early protein corona profile) The initial protein corona profile of NPs in the presence of small molecular weight compounds and combinations of small molecular weight compounds was studied using SDS-PAGE analysis (Figure 2). For SDS-PAGE analysis, protein corona-coated NPs (20 μl) were mixed with 2x Laemmli sample buffer (20 μl), heated at 85°C for 7 minutes, and then loaded onto a precast gel. After gel electrophoresis, the gel was fixed with a solution containing 10% acetic acid and 40% ethanol and stained overnight with Coomassie blue stain (50 mL). After staining, the gel was washed and scanned. The results showed that the intensity of the protein band differed at certain concentrations of specific small molecular weight compounds. For example, the protein corona profile changed significantly in the presence of vitamin B complex molecules (Figure 2). More specifically, in the presence of high concentrations of vitamin B complex molecules, a new protein band intensity (approximately 50 kDa) appeared in the protein corona profile that was not observed under normal protein corona conditions or under low concentrations of vitamin B complex molecules. In the presence of high concentrations of triacylglycerol, some protein bands disappeared or their intensity decreased, indicating that small molecules and their concentrations have a decisive impact on the protein corona profile of NPs. To investigate how spike addition of different concentrations of small molecules may affect the molecular composition of the protein corona, the protein corona composition was also measured using LC-MS / MS.

[0144] (LC-MS / MS sample preparation for screening and concentration series experiments) Initial treatment: First, the protein corona-coated NPs were washed with PBS and then resuspended in PBS (30 μl) enriched with 15 mM phosphate (pH 7.4). The total bound protein content was estimated to be approximately 1 μg per sample by the Micro BCA® assay (Thermo Scientific®). The samples were reduced with 2 mM dithiothreitol (DTT) and incubated at 50°C for 45 minutes with shaking at 700 rpm. Subsequently, the proteins were alkylated with 8 mM iodoacetamide (IAA) at room temperature in the dark. A LysC / trypsin enzyme mixture was added at a concentration of 0.02 μg / μl, and the samples were incubated overnight at 37°C.

[0145] The sample was centrifuged at 16,000xg for 20 minutes at room temperature to precipitate NPs. The supernatant containing the peptide digest was collected, vacuum-dried, and desalted using Pierce® C18 Spin Tips (Thermo Scientific®, catalog number: 84850) according to the manufacturer's instructions. The sample was vacuum-dried again and stored at -80°C until LC-MS / MS analysis.

[0146] LC-MS / MS Analysis: Dried samples were reconstituted using 25 μl of liquid chromatography (LC) loading buffer (3% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA)) containing peptide (1 μg), and analyzed by LC-MS / MS. A 60-minute gradient was applied in label-free quantitative (LFQ) mode, with three injections of 5 μl of aliquots. A control sample (55% human plasma) was prepared using 200 μl of loading buffer containing peptide (8 μg) and analyzed similarly. An UltiMate® 3000 RSLCnano (Thermo Scientific®) high-performance liquid chromatography system was used with predefined column, solvent, and gradient settings. Data-dependent analysis (DDA) was performed using specific MS (MS) settings. 1 (also known as) and MS / MS (MS 2The scan was performed using a scan setting (also known as ), and the data was then analyzed using Proteome Discoverer® 2.4 (Thermo Scientific®) (applying the protocol detailed in Ashkarran et al., 2022). The PdtChos concentration series experiment was performed using the same protocol, and the samples were analyzed using a 120-minute gradient.

[0147] (Protein corona and small molecule compounds enable detailed profiling of the plasma proteome.) To investigate how spike addition of low-molecular-weight compounds at different concentrations can affect the molecular composition of protein coronas, samples were subjected to high-resolution LC-MS / MS analysis. While 218 intrinsic proteins were quantified by analysis of plasma alone, analysis of protein coronas formed on polystyrene NPs significantly increased the depth of plasma proteome sampling, enabling the quantification of 681 intrinsic proteins. Furthermore, by including low-molecular-weight compounds, plasma proteome sampling was further deepened, making it possible to quantify a range of intrinsic proteins from 397 to 897, depending on the low-molecular-weight compounds added to the plasma before corona formation. Comparing the analysis using protein coronas (both with and without low-molecular-weight compounds) with analysis of plasma alone (Figure 5), the number of quantifiable proteins increased significantly (approximately a threefold increase).

[0148] Interestingly, the concentration of small molecule compounds did not significantly affect the number of proteins quantified. As glucose and diacylglycerol concentrations increased, only a slight, gradual decrease in the number of quantified proteins was observed. Cumulatively, the incorporation of small molecule compounds and combinations of small molecule compounds into NP protein corona resulted in a substantial increase in protein quantification, with a total of 1793 proteins being quantified. This represents an 8.25-fold increase compared to plasma alone. Specifically, the addition of small molecule compounds resulted in the quantification of 1573 additional proteins compared to plasma alone, and 1037 more than the untreated protein corona. Particularly noteworthy is the fact that simply adding PtdCho (1000 μg / ml) as a spike increased the number of quantified proteins to 897 (4.1 times that of plasma alone and 1.3 times that of the untreated corona). This observation led to a detailed investigation into the effect of PtdCho on plasma proteome coverage. Details are described below. This analysis focuses on the multiplicative change in quantified proteins compared to plasma and untreated corona, rather than the absolute number of quantified proteins. This is primarily because the number of quantified proteins is highly dependent on the mass spectrometry workflow. For example, when the same corona-coated polystyrene NP was analyzed by various mass spectrometry centers, the number of quantified proteins varied from 235 to over 1000. Therefore, this multiplicative change analysis is more conservative and less susceptible to biases in the mass spectrometry workflow.

[0149] Figure 6 shows the distribution of normalized protein intensity in each sample. The median value in the plasma group was significantly higher than in the other sample groups, but no significant difference was observed in the overall distribution. In general, the proteome obtained from protein corona profiles in the presence of small molecule compounds showed good correlation (Pearson correlation coefficient greater than 0.6 when compared with many small molecule compounds), confirming that it faithfully reflects the relative expression of proteins after treatment with different small molecule compounds (Figure 9).

[0150] (Low-molecular-weight compounds diversify the composition of protein corona.)We investigated whether the addition of small molecule compounds altered the types and numbers of proteins detected by LC-MS / MS. Indeed, each small molecule compound and combination of small molecule compounds generated a distinct proteomic fingerprint distinct from that of the untreated protein corona or other small molecule compounds (Figure 7). Spike addition of small molecule compounds allowed for the detection of diverse protein groups in plasma. Interestingly, even the same small molecule compound or combination produced unique characteristic patterns at varying concentrations. Similar analyses were performed for 117 proteins shared across samples (Figure 8). The Venn diagram in Figure 10 shows the number of unique proteins quantified across the entire concentration range in each group. These results suggest that spike addition of small molecule compounds to human biological fluids diversifies the range of quantifiable proteins in the protein corona profile, effectively expanding proteomic coverage for low-abundance proteins. Such enrichment or depletion of specific subsets of proteins could be useful in disease-focused biomarker discovery. This property can also be used in assay design to promote the enrichment of known biomarkers by using a given small molecule compound. Representative examples are shown in Figures 11A-11B and 11C-11D, respectively, comparing enriched and depleted proteins with untreated protein corona in combinations 1 and 2 of small molecule compounds. In certain cases, enrichment or depletion was dramatic, ranging from several orders of magnitude. Enriched or depleted proteins in combinations 1 and 2 of small molecule compounds were mapped to KEGG pathways and biological processes within STRINGdb (string-db.org) (Figures 11A and 11C). While most enriched pathways were common, some pathways were specifically enriched in a given small molecule compound or combination of small molecule compounds. For example, systemic lupus erythematosus (SLE) was enriched only in the higher-level pathway of combination 2 of small molecule compounds (PE, PtdIns, IMP, and vitamin B complex). Therefore, small molecule compounds may have the potential to facilitate the discovery of biomarkers for specific diseases or to measure the abundance of known biomarkers in disease detection.

[0151] Similar analyses were performed for all small molecule compounds, and the volcano plots at the highest concentration (i.e., 1000 μg / ml) for each molecule are shown in Figure 12. Pathway analysis was also performed for all proteins that showed significant changes at all concentrations for the small molecule compounds (Figure 13). To facilitate comparison, the results of integrated enrichment analysis with untreated protein corona for all samples are shown in Figure 14.

[0152] To clarify how small molecule compounds affect the protein composition and functional categories in protein corona and to demonstrate their potential for early disease diagnosis (since proteins enriched in the corona are important in conditions such as cardiovascular and neurodegenerative diseases), we used bioanalysis to classify identified proteins based on blood-related functions (i.e., complement activation, immune response, coagulation, acute phase reactions, and lipid metabolism) (Figure 15). Different diseases may have various associations with each protein category. In the analysis, apolipoproteins were the major protein types observed in small molecule compound-treated protein corona, and their type and abundance were highly dependent on the type and concentration of the small molecule compound used. Similarly, the enrichment of other specific protein categories on the NP surface was also affected by the type and concentration of the small molecule compound used. For example, the coagulation factor antithrombin III plays a significant role in the composition of protein corona in all small molecule compounds tested, but this effect is observed only at their highest concentrations. At low concentrations or in untreated protein corona, this significant involvement is not evident. This ability of small molecules to alter the protein composition on NPs highlights the potential of small molecules for early diagnosis of health spectrum conditions and / or diseases (e.g., apolipoproteins in cardiovascular and neurodegenerative disorders), given the critical importance of these protein categories in the onset and progression of disease / conditions.

[0153] (PtdCho increases proteome coverage by reducing the amount of plasma proteins abundant.) To understand whether the quantification of more proteins in the protein corona profile was due to a lower dynamic range of proteins available for NP binding in human plasma, we plotted the minimum protein abundance against the maximum protein abundance in plasma alone and in plasma treated with small molecule compounds in Figure 16. Plasma alone showed the largest dynamic range, suggesting that quantifying low-abundance proteins is most difficult from plasma alone. On the other hand, the addition of small molecule compounds reduced the dynamic range of plasma proteins, resulting in the detection of more peptides and quantification of low-abundance proteins via the NP protein corona.

[0154] In particular, albumin, which accounts for over 81% of plasma samples, was significantly reduced to an average of 29% in the protein corona, regardless of whether it was modified with small molecule compounds. This reduction was most pronounced when treated with PtdCho (1000 μg / ml), where albumin levels fell to approximately 17% of plasma proteins (Figure 17A). Despite these changes, albumin remained the most abundant protein in all samples. Similar decreasing trends were observed for the second and third most abundant proteins, serotransferrin (TF) (Figure 17B) and haptoglobin (HB) (Figure 17C), accounting for approximately 3.9% and 3.6% of the plasma protein abundance, respectively. The ranking of the abundance of these proteins in each sample is shown at the top of the corresponding circles in Figures 17A to 17C. From this analysis, it is clear that the protein corona, whether in its natural form or modified with small molecule compounds, can significantly reduce the combined abundance of the top three proteins from approximately 90% to about 29%. The most significant reduction was observed using PtdCho (1000 μg / ml), which reduced the cumulative abundance of the top three proteins from 90% to less than 17%. PtdCho treatment also effectively reduced the level of IGHA1, a fourth abundant plasma protein. This significant reduction in the abundance of this frequently occurring plasma protein explains the significant increase in the number of intrinsic proteins detected from NP corona samples treated with PtdCho. These results suggest that high concentrations of PtdCho can be used strategically to enable more comprehensive plasma protein sampling, particularly by specifically targeting and reducing the most abundant plasma proteins, such as albumin.

[0155] The stream (or alluvial) plot in Figure 18A shows the overall change in protein abundance observed in plasma when protein corona was incubated with different concentrations of PtdCho. To verify this finding, fresh samples were prepared by stepwise treatment with PtdCho at concentrations ranging from 100 to 10,000 μg / ml. As shown in Figure 18B, 957 proteins could be quantified in protein corona treated with PtdCho (1000 μg / ml), but the number of quantified proteins did not increase at lower concentrations or with additional PtdCho administration. The stream plot in Figure 18D shows a specific decrease in albumin and other abundant proteins in plasma upon addition of PtdCho, indicating that other proteins at lower concentrations can be detected more reliably.

[0156] [LC-MS analysis using data-independent acquisition method (DIA)] To confirm that the improvement in proteome coverage by PtdCho treatment is independent of the LC-MS platform used, new samples of plasma, untreated protein corona, and protein corona treated with PtdCho (1000 μg / ml) were prepared and analyzed using data-independent acquisition (DIA) LC-MS.

[0157] To remove unbound or loosely bound proteins, the sample was centrifuged at 14,000xg for 20 minutes. The recovered NP pellet was washed three times with cold PBS and centrifuged under the same conditions. The sample was resuspended in PBS (20 μl), reduced with 2 mM dithiothreitol (DTT) (final concentration) for 45 minutes, and then alkylated with 8 mM iodoacetamide (IAA) (final concentration) in the dark for 45 minutes. Subsequently, 0.02 μg / μl LysC (5 μl) was added and treated for 4 hours, followed by treatment overnight with the same concentration and volume of trypsin. The sample was then centrifuged at 16,000xg for 20 minutes at room temperature to remove NPs, cleaned up using Pierce® C18 spin tips (Thermo Scientific®; catalog number 84850), and vacuum-dried.

[0158] Dried peptides were resuspended in a 0.1% formic acid aqueous solution and analyzed by LC-MS / MS using a Vanquish® Neo UHPLC system (Thermo Scientific®) and an Orbitrap Exploris® 480 Mass Spectrometer (Thermo Scientific®) equipped with a custom column heater set to 60°C. The peptides were degraded using a reversed-phase high-performance liquid chromatography (RP-HPLC) column (75 μM x 30 cm). The column was packed in-house with C18 resin (ReproSil®-Pur 120 C18-AQ, 1.9 μm; Dr. Maisch GmbH) at a flow rate of 0.2 μL / min. The following gradient was used for peptide separation: Buffer B was increased from 4% to 10% (7.5 min), up to 35% (67.7 min), up to 50% (15 min), and up to 95% (1 min). Subsequently, buffer B was maintained at 95% (10 minutes), then reduced to 5% (1 minute), and then maintained at 5% (4 minutes). Buffer A was water containing 0.1% formic acid, and buffer B was water containing 80% acetonitrile (ACN) and 0.1% formic acid.

[0159] The mass spectrometer was operated in DIA mode with a cycle time of 3 seconds. 1The scan was acquired in centroid mode using Orbitrap Exploris™, with a resolution of 120,000 full-width half-medium (FWHM) (at 200 m / z), a scan range of 390-910 m / z, a normalized AGC target set to 300%, and the maximum ion implantation time mode set to Auto. The MS2 scan was acquired in centroid mode using Orbitrap Exploris™, with a resolution of 15,000 FWHM (at 200 m / z), a precursor mass range of 400-900, a quadrapole isolation window of 7 m / z (with a 1 m / z window overlap), a defined initial mass of 120 m / z, a normalized AGC target set to 3000%, and a maximum implantation time of 22 ms. The collision energy was set to 28%, and the peptide was fragmented by high-energy collision dissociation (HCD). One microscan was acquired for each spectrum.

[0160] The acquired raw data files were individually searched using the directDIA workflow of Spectronaut® (Biognosys v18.6). The search targets were the Homo sapiens database (consisting of 20,360 protein sequences downloaded from UniProt on February 22, 2022) and 392 commonly observed contaminants. Default settings were used.

[0161] 322 proteins were identified in plasma alone, 1011 proteins in untreated protein corona samples, and 1436 proteins (a 1.4-fold increase compared to untreated corona) in protein corona treated with PtdCho. These findings not only demonstrate PtdCho's ability to improve plasma proteome coverage but also facilitate detailed profiling of the plasma proteome related to protein corona formed on a single type of NP surface. Since the percentage of proteins quantified through PtdCho spike addition is approximately 1.4 times higher than with NP corona alone, PtdCho can be incorporated into any LC-MS workflow aimed at facilitating proteome profiling of plasma (and any other biological sample containing albumin or high-abundance proteins). More optimized plasma proteomics pipelines or high-end mass spectrometers such as Orbitrap Astral® (Thermo Scientific®) are expected to quantify even more proteins than those reported in this study.

[0162] [Data Analysis] First, the data was normalized by the total protein intensity in each technical iteration. Data analysis was performed using R project version 4.1.0. Missing values ​​were replaced with the constant 10^-10 for all conditions.

[0163] (Statistics and Reproducibility) All measurements involved three analyses of a given dispensed solution. DIA analysis was performed in one replicate.

[0164] [Additional Embodiments] Embodiment 1: A composition for detecting multiple unique / different proteins or their proteoforms in a biological sample such as a plasma sample, the composition comprising: (1) Triacylglycerol and / or derivatives thereof; (2) Diacylglycerols (1,2-diacylglycerol, 1,3-diacylglycerol, etc.) and / or derivatives thereof; (3) Glycerophospholipids and / or derivatives thereof; (4) Triacylglycerol and / or derivatives thereof, diacylglycerol and / or derivatives thereof, and combinations thereof with glycerophospholipids and / or derivatives thereof; (5) Glucose and / or its derivatives; (6) Inosine 5'-monophosphate (also called inosinic acid or IMP) and / or derivatives thereof; (7) Vitamin B complex (also known as B complex) and / or derivatives thereof; (8) Glucose and / or derivatives thereof, inosine 5'-monophosphate and / or derivatives thereof, and combinations of vitamin B complex and / or derivatives thereof; (9) combinations of triacylglycerol and / or derivatives, diacylglycerol and / or derivatives, glycerophospholipids and / or derivatives, glucose and / or derivatives, inosine 5'-monophosphate and / or derivatives, and vitamin B complex and / or derivatives; (10) Phosphatidylcholine and / or derivatives thereof; (11) Phosphatidylethanolamine and / or its derivatives; (12) Phosphatidylserine and / or derivatives thereof; (13) Phosphatidic acid and / or derivatives thereof; (14) Phosphatidylinositol and / or derivatives thereof; (15) Phosphatidylglycerol and / or derivatives thereof; (16) Cardiolipin and / or derivatives thereof; (17) L-α-phosphatidylinositol and / or derivatives thereof; (18) glucose and / or derivatives, triacylglycerol and / or derivatives, diacylglycerol and / or derivatives; and combinations of phosphatidylcholine and / or derivatives; (19) combinations of phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and vitamin B complex and / or its derivatives; or, (20) Any one combination of (1) to (19).

[0165] Embodiment 2: The composition according to Embodiment 1, further comprising one or more protein binders.

[0166] Embodiment 3: The composition according to Embodiment 1 or 2, further comprising one or more biological samples.

[0167] Embodiment 4: The composition according to any one of Embodiments 1 to 3, wherein one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0168] Embodiment 5: The composition according to any one of Embodiments 1 to 4, wherein one or more protein binders comprise one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0169] Embodiment 6: The composition according to any one of Embodiments 1 to 5, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0170] Embodiment 7: The composition according to any one of Embodiments 1 to 6, wherein the protein binder is of the same type.

[0171] Embodiment 8: The composition according to any one of Embodiments 1 to 7, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0172] Embodiment 9: The composition according to any one of Embodiments 1 to 8, wherein one or more low molecular weight compounds are phosphatidylcholine.

[0173] Embodiment 10: The composition according to Embodiment 9, wherein approximately 1 pg / ml to approximately 1 g / ml, for example, approximately 1000 μg / ml of phosphatidylcholine is added to the biological sample.

[0174] Embodiment 11: The composition according to any one of Embodiments 1 to 10, wherein one or more low molecular weight compounds are added to a biological sample at varying concentrations.

[0175] Embodiment 12: The composition according to Embodiment 11, wherein the various concentrations range from approximately 1 pg / ml to approximately 1 g / ml.

[0176] Embodiment 13: A method for detecting proteins and / or their proteoforms in a biological sample, comprising the following steps: A step of adding one or more low-molecular-weight compounds to a biological sample; A step of adding one or more protein binders having a surface capable of binding proteins to a biological sample, forming a protein corona on the surface of one or more protein binders, and generating a complex containing the protein corona and another protein binder; and, A process for detecting the number of proteins and / or their proteoforms in a protein corona using antibody-based technology or proteomics technology.

[0177] Embodiment 14: The method according to Embodiment 13, wherein the step of detecting proteins and / or their proteoforms in a protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

[0178] Embodiment 15: The method according to Embodiment 13 or 14, wherein one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0179] Embodiment 16: The method according to any one of Embodiments 13 to 15, wherein one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0180] Embodiment 17: The method according to any one of Embodiments 13 to 16, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0181] Embodiment 18: The method according to any one of Embodiments 13 to 17, wherein one or more protein binders are of the same type.

[0182] Embodiment 19: The method according to any one of Embodiments 13 to 18, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0183] Embodiment 20: The method according to any one of Embodiments 13 to 19, wherein one or more low molecular weight compounds include a biomolecule.

[0184] Embodiment 21: The method according to any one of Embodiments 13 to 20, wherein one or more low molecular weight compounds include metabolites, lipids, nutrients, plant-derived molecules, or a combination thereof.

[0185] Embodiment 22: One or more low molecular weight compounds: Triacylglycerols and / or derivatives thereof; Diacylglycerols such as 1,2-diacylglycerol and 1,3-diacylglycerol, and / or derivatives thereof; Glycerophospholipids and / or derivatives thereof; Triacylglycerols and / or their derivatives, diacylglycerols and / or their derivatives, and combinations of glycerophospholipids and / or their derivatives; Glucose and / or its derivatives; Inosine 5'-monophosphate and / or derivatives thereof; Vitamin B complex and / or derivatives thereof; Glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; Triacylglycerols and / or derivatives, diacylglycerols and / or derivatives; glycerophospholipids and / or derivatives, glucose and / or derivatives, inosine 5'-monophosphate and / or derivatives, and combinations of vitamin B complex and / or derivatives; Phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives; Phosphatidylserine and / or its derivatives; Phosphatidic acid and / or its derivatives; Phosphatidylinositol and / or its derivatives; Phosphatidylglycerol and / or its derivatives; Cardiolipin and / or its derivatives; L-α-phosphatidylinositol and / or derivatives thereof; Glucose and / or its derivatives, triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; and combinations of phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; or, Those combinations, The method according to Embodiment 21, including the method described above.

[0186] Embodiment 23: The method according to any one of Embodiments 13 to 22, wherein one or more low molecular weight compounds are phosphatidylcholine.

[0187] Embodiment 24: The method according to Embodiment 23, wherein a biological sample is given approximately 1 pg / ml to approximately 1 g / ml of phosphatidylcholine, for example, approximately 1000 μg / ml.

[0188] Embodiment 25: The method according to any one of Embodiments 13 to 24, wherein a biological sample is passed through a resin-based removal column or spin column.

[0189] Embodiment 26: Compared to a biological sample that does not contain one or more protein binders and one or more low molecular weight compounds, an increase in the number of proteins and / or their proteoforms is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders and without one or more small molecule compounds, the increase in the number of proteins and / or their proteoforms is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 13 to 25.

[0190] Embodiment 27: The method according to any one of Embodiments 13 to 26, wherein one or more low molecular weight compounds are added to a biological sample at varying concentrations.

[0191] Embodiment 28: The method according to Embodiment 27, wherein the various concentrations range from approximately 1 pg / ml to approximately 1 g / ml.

[0192] Embodiment 29: The method according to any one of Embodiments 13 to 28, further comprising the step of preparing a complex for detecting proteins and / or their proteoforms in a protein corona.

[0193] Embodiment 30: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to Embodiment 29, including the method described in Embodiment 29.

[0194] Embodiment 31: The method according to any one of Embodiments 13 to 30, wherein one or more small molecule compounds interact with one or more intrinsic / different proteins or their proteoforms in a biological sample.

[0195] Embodiment 32: The method according to any one of Embodiments 13 to 31, wherein one or more low molecular weight compounds improve the detection of low-abundance proteins.

[0196] Embodiment 33: The method according to Embodiment 23, wherein phosphatidylcholine binds to albumin.

[0197] Embodiment 34: The method according to any one of Embodiments 13 to 33, wherein one or more protein binders (approximately 1 pg / ml to approximately 1 g / ml) are added to the biological sample.

[0198] Embodiment 35: A method for detecting one or more disease-related biomarkers, or a pattern of one or more biomarkers, comprising the following steps: A step of adding one or more low-molecular-weight compounds to each of at least two biological samples derived from different subjects diagnosed with a certain disease; A step of adding one or more protein binders having a surface capable of binding proteins to a biological sample, forming a protein corona on the surface of one or more protein binders, and generating a complex containing the protein corona and one or more protein binders; and, A process for detecting one or more biomarkers, or patterns of one or more biomarkers, in a protein corona using antibody-based technology or proteomics technology.

[0199] Embodiment 36: The method according to Embodiment 35, wherein the step of detecting one or more biomarkers, or a pattern of one or more biomarkers, in the protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

[0200] Embodiment 37: The method according to Embodiment 35 or 36, wherein one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0201] Embodiment 38: The method according to any one of Embodiments 35 to 37, wherein one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0202] Embodiment 39: The method according to any one of Embodiments 35 to 38, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0203] Embodiment 40: The method according to any one of Embodiments 35 to 39, wherein the protein binder is of the same type.

[0204] Embodiment 41: The method according to any one of Embodiments 35 to 40, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0205] Embodiment 42: The method according to any one of Embodiments 35 to 41, wherein one or more low molecular weight compounds include a biomolecule.

[0206] Embodiment 43: The method according to any one of Embodiments 35 to 42, wherein one or more low molecular weight compounds include metabolites, lipids, nutrients, plant-derived molecules, or a combination thereof.

[0207] Embodiment 44: One or more low molecular weight compounds: Triacylglycerols and / or derivatives thereof; Diacylglycerols such as 1,2-diacylglycerol and 1,3-diacylglycerol, and / or derivatives thereof; Glycerophospholipids and / or derivatives thereof; Triacylglycerols and / or their derivatives, diacylglycerols and / or their derivatives, and combinations of glycerophospholipids and / or their derivatives; Glucose and / or its derivatives; Inosine 5'-monophosphate and / or derivatives thereof; Vitamin B complex and / or derivatives thereof; Glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; Triacylglycerols and / or derivatives, diacylglycerols and / or derivatives; glycerophospholipids and / or derivatives, glucose and / or derivatives, inosine 5'-monophosphate and / or derivatives, and combinations of vitamin B complex and / or derivatives; Phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives; Phosphatidylserine and / or its derivatives; Phosphatidic acid and / or its derivatives; Phosphatidylinositol and / or its derivatives; Phosphatidylglycerol and / or its derivatives; Cardiolipin and / or its derivatives; L-α-phosphatidylinositol and / or derivatives thereof; Glucose and / or its derivatives, triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; and combinations of phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; or, Those combinations, The method according to Embodiment 43, including the method described in Embodiment 43.

[0208] Embodiment 45: The method according to any one of Embodiments 35 to 44, wherein one or more low molecular weight compounds are phosphatidylcholine.

[0209] Embodiment 46: The method according to Embodiment 45, wherein a biological sample is given approximately 1 pg / ml to approximately 1 g / ml of phosphatidylcholine, for example, approximately 1000 μg / ml.

[0210] Embodiment 47: The method according to any one of Embodiments 35 to 46, wherein a biological sample is passed through a resin-based removal column or spin column.

[0211] Embodiment 48: Compared to a biological sample that does not contain one or more protein binders and one or more small molecule compounds, an increase in the number of biomarkers or biomarker patterns is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders and without one or more small molecule compounds, the increase in the number of biomarkers or biomarker patterns is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 35 to 47.

[0212] Embodiment 49: The method according to any one of Embodiments 35 to 48, wherein one or more low molecular weight compounds are added to a biological sample at varying concentrations.

[0213] Embodiment 50: The method according to Embodiment 49, wherein the various concentrations range from approximately 1 pg / ml to approximately 1 g / ml.

[0214] Embodiment 51: The method according to any one of Embodiments 35 to 50, further comprising the step of preparing a complex for detecting disease-related biomarkers or patterns of one or more biomarkers in a protein corona.

[0215] Embodiment 52: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to Embodiment 51, including the method described above.

[0216] Embodiment 53: The method according to any one of Embodiments 35 to 52, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

[0217] Embodiment 54: The method according to Embodiment 53, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

[0218] Embodiment 55: The method according to Embodiment 53, wherein the disease is a neurological disease selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0219] Embodiment 56: The method according to any one of Embodiments 35 to 55, wherein one or more small molecule compounds interact with one or more unique / different proteins in a biological sample.

[0220] Embodiment 57: The method according to any one of Embodiments 35 to 56, wherein one or more low molecular weight compounds improve the detection of low-abundance proteins.

[0221] Embodiment 58: The method according to Embodiment 45, wherein phosphatidylcholine binds to albumin.

[0222] Embodiment 59: The method according to any one of Embodiments 35 to 58, wherein a protein binder in an amount of about 1 pg / ml to about 1 g / ml is added to the biological sample.

[0223] Embodiment 60: A method for diagnosing a disease in a subject, comprising the following steps: A step of adding one or more low-molecular-weight compounds to a biological sample derived from the target; A step of adding one or more protein binders having a surface capable of binding proteins to a biological sample, forming a protein corona on the surface of one or more protein binders, and generating a complex containing the protein corona and one or more protein binders; and, A process for detecting one or more disease-related biomarkers, or patterns of one or more biomarkers, in a protein coronavirus using antibody-based or proteomics technology.

[0224] Embodiment 61: The method according to Embodiment 60, wherein the step of detecting one or more biomarkers, or a pattern of one or more biomarkers, in a protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

[0225] Embodiment 62: The method according to Embodiment 60 or 61, wherein one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0226] Embodiment 63: The method according to any one of Embodiments 60 to 62, wherein one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0227] Embodiment 64: The method according to any one of Embodiments 60 to 63, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0228] Embodiment 65: The method according to any one of Embodiments 60 to 64, wherein one or more protein binders are of the same type.

[0229] Embodiment 19: The method according to any one of Embodiments 13 to 18, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0230] Embodiment 66: The method according to any one of Embodiments 60 to 65, wherein one or more low molecular weight compounds include a biomolecule.

[0231] Embodiment 67: The method according to any one of Embodiments 60 to 66, wherein one or more low molecular weight compounds include metabolites, lipids, nutrients, plant-derived molecules, or a combination thereof.

[0232] Embodiment 68: One or more low molecular weight compounds: Triacylglycerols and / or derivatives thereof; Diacylglycerols such as 1,2-diacylglycerol and 1,3-diacylglycerol, and / or derivatives thereof; Glycerophospholipids and / or derivatives thereof; Triacylglycerols and / or their derivatives, diacylglycerols and / or their derivatives, and combinations of glycerophospholipids and / or their derivatives; Glucose and / or its derivatives; Inosine 5'-monophosphate and / or derivatives thereof; Vitamin B complex and / or derivatives thereof; Glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; Triacylglycerols and / or derivatives, diacylglycerols and / or derivatives; glycerophospholipids and / or derivatives, glucose and / or derivatives, inosine 5'-monophosphate and / or derivatives, and combinations of vitamin B complex and / or derivatives; Phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives; Phosphatidylserine and / or its derivatives; Phosphatidic acid and / or its derivatives; Phosphatidylinositol and / or its derivatives; Phosphatidylglycerol and / or its derivatives; Cardiolipin and / or its derivatives; L-α-phosphatidylinositol and / or derivatives thereof; Glucose and / or its derivatives, triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; and combinations of phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; or, Those combinations, The method according to embodiment 67, including the method described in embodiment 67.

[0233] Embodiment 69: The method according to any one of Embodiments 60 to 68, wherein one or more low molecular weight compounds are phosphatidylcholine.

[0234] Embodiment 70: The method according to Embodiment 69, wherein a biological sample is given approximately 1 pg / ml to approximately 1 g / ml of phosphatidylcholine, for example, approximately 1000 μg / ml.

[0235] Embodiment 71: The method according to any one of Embodiments 60 to 70, wherein a biological sample is passed through a resin-based removal column or spin column.

[0236] Embodiment 72: Compared to a biological sample that does not contain one or more protein binders and one or more low molecular weight compounds, an increase in the number of biomarkers or biomarker patterns is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders and without one or more small molecule compounds, the increase in the number of biomarkers or biomarker patterns is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 60 to 71.

[0237] Embodiment 73: The method according to any one of Embodiments 60 to 72, wherein one or more low molecular weight compounds are added to a biological sample at varying concentrations.

[0238] Embodiment 74: The method according to Embodiment 73, wherein the various concentrations range from approximately 1 pg / ml to approximately 1 g / ml.

[0239] Embodiment 75: The method according to any one of Embodiments 60 to 74, further comprising the step of preparing a complex for detecting one or more disease-related biomarkers or patterns of one or more biomarkers in a protein corona.

[0240] Embodiment 76: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to embodiment 75, including the method described in embodiment 75.

[0241] Embodiment 77: The method according to any one of Embodiments 60 to 76, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

[0242] Embodiment 78: The method according to Embodiment 77, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

[0243] Embodiment 79: The method according to Embodiment 77, wherein the disease is a neurological disease selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0244] Embodiment 80: The method according to any one of Embodiments 60-79, wherein one or more small molecule compounds interact with one or more unique / different proteins in a biological sample.

[0245] Embodiment 81: The method according to any one of Embodiments 60 to 80, wherein one or more low molecular weight compounds improve the detection of low-abundance proteins.

[0246] Embodiment 82: The method according to Embodiment 69, wherein phosphatidylcholine binds to albumin.

[0247] Embodiment 83: The method according to any one of Embodiments 60 to 82, wherein a protein binder at a concentration of about 1 pg / ml to about 1 g / ml is added to a biological sample.

[0248] Embodiment 84: A method for detecting a protein and / or its proteoform in a biological sample, the method comprising the following steps: adding means for binding to one or more highly abundant proteins and / or their proteoforms in the biological sample; adding to the biological sample one or more protein binders having a surface capable of binding proteins, forming a protein corona on the surface of the one or more protein binders, and generating a complex comprising the protein corona and another protein binder; and detecting the number of proteins and / or their proteoforms in the protein corona by an antibody-based technique or a proteomics technique.

[0249] Embodiment 85: The method according to Embodiment 84, wherein the highly abundant protein is albumin.

[0250] Embodiment 86: The method according to Embodiment 84 or 85, wherein the step of detecting the highly abundant protein and / or its proteoform in the protein corona is performed by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

[0251] Embodiment 87: The method according to any one of Embodiments 84 to 86, wherein the one or more protein binders comprise an inorganic agent, a metal-based agent, a metal oxide-based agent, a polymer-based agent, a lipid-based agent, a carbon-based agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0252] Embodiment 88: The method according to any one of Embodiments 84 to 87, wherein one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0253] Embodiment 89: The method according to any one of Embodiments 84 to 88, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0254] Embodiment 90: The method according to any one of Embodiments 84 to 89, wherein when one or more protein binders are present, the protein binders are of the same type.

[0255] Embodiment 91: The method according to any one of Embodiments 84 to 90, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0256] Embodiment 92: The method according to any one of Embodiments 84 to 91, wherein a biological sample is passed through a resin-based removal column or spin column.

[0257] Embodiment 93: Compared to a biological sample that does not contain one or more protein binders and means for binding one or more high-abundance proteins and / or their proteoforms, an increase in the number of proteins and / or their proteoforms is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders without the addition of means for binding one or more high-abundance proteins and / or their proteoforms, the increase in the number of proteins and / or their proteoforms is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 84 to 92.

[0258] Embodiment 94: The method according to any one of Embodiments 84 to 93, further comprising the step of preparing a complex for detecting proteins and / or their proteoforms in a protein corona.

[0259] Embodiment 95: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to Embodiment 94, including the method described in Embodiment 94.

[0260] Embodiment 96: The method according to any one of Embodiments 84 to 30, wherein a protein binder is added to a biological sample in an amount of approximately 1 pg / ml to approximately 1 g / ml.

[0261] Embodiment 97: A method for detecting one or more disease-related biomarkers, or a pattern of one or more biomarkers, comprising the following steps: A step of adding means to bind one or more high-abundance proteins and / or their proteoforms to each of at least two biological samples derived from different subjects diagnosed with a certain disease; A step of adding one or more protein binders having a surface capable of binding proteins to a biological sample, forming a protein corona on the surface of one or more protein binders, and generating a complex containing the protein corona and one or more protein binders; and, A process for detecting one or more biomarkers, or patterns of one or more biomarkers, in a protein corona using antibody-based technology or proteomics technology.

[0262] Embodiment 98: The method according to embodiment 97, wherein the high-abundance protein is albumin.

[0263] Embodiment 99: The method according to embodiment 97 or 98, wherein the step of detecting one or more biomarkers in the protein corona, or the pattern of one or more biomarkers, is performed by proteomic techniques including top-down, middle-down, or bottom-up LC-MS / MS.

[0264] Embodiment 100: The method according to any one of embodiments 97 to 99, wherein the one or more protein binders include inorganic agents, metal-based agents, metal oxide-based agents, polymer-based agents, lipid-based agents, carbon-based agents, core-shell agents, composite agents, mesoporous agents, or combinations thereof.

[0265] Embodiment 101: The method according to any one of embodiments 97 to 100, wherein the one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodisks, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or combinations thereof.

[0266] Embodiment 102: The method according to any one of embodiments 97 to 202, wherein the one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0267] Embodiment 103: The method according to any one of embodiments 97 to 102, wherein when the one or more protein binders are present, the protein binders are of the same type.

[0268] Embodiment 104: The method according to any one of embodiments 97 to 103, wherein the biological sample is whole blood in systemic circulation, plasma, serum, bronchoalveolar lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0269] Embodiment 105: The method according to any one of Embodiments 97 to 104, wherein a biological sample is passed through a resin-based removal column or spin column.

[0270] Embodiment 106: Compared to a biological sample that does not contain one or more protein binders and means for binding one or more high-abundance proteins and / or their proteoforms, an increase in the number of biomarkers or biomarker patterns is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders without the addition of means for binding to one or more high-abundance proteins and / or their proteoforms, the increase in the number of biomarkers or biomarker patterns is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 97 to 105.

[0271] Embodiment 107: The method according to any one of Embodiments 97 to 106, further comprising the step of preparing a complex for detecting disease-related biomarkers or patterns of one or more biomarkers in a protein corona.

[0272] Embodiment 108: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to Embodiment 107, including the method described above.

[0273] Embodiment 109: The method according to any one of Embodiments 97 to 108, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

[0274] Embodiment 110: The method according to Embodiment 109, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

[0275] Embodiment 111: The method according to Embodiment 109, wherein the disease is a neurological disease selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0276] Embodiment 112: The method according to any one of Embodiments 97 to 111, wherein a protein binder is added to the biological sample in an amount of approximately 1 pg / ml to approximately 1 g / ml.

[0277] Embodiment 113: A method for diagnosing a disease in a subject, comprising the following steps: A step of adding means to bind one or more high-abundance proteins and / or their proteoforms to a biological sample derived from the target; A step of adding one or more protein binders having a surface capable of binding proteins to a biological sample, forming a protein corona on the surface of one or more protein binders, and generating a complex containing the protein corona and one or more protein binders; and, A step of detecting one or more biomarkers or patterns of one or more biomarkers associated with the disease in a protein corona by antibody-based technology or proteomics technology.

[0278] Embodiment 114: The method according to Embodiment 113, wherein the high-abundance protein is albumin.

[0279] Embodiment 115: The method according to Embodiment 113 or 114, wherein the step of detecting one or more biomarkers, or a pattern of one or more biomarkers, in a protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

[0280] Embodiment 116: The method according to any one of Embodiments 113 to 115, wherein one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

[0281] Embodiment 117: The method according to any one of Embodiments 113 to 116, wherein one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

[0282] Embodiment 118: The method according to any one of Embodiments 113 to 117, wherein one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.3.

[0283] Embodiment 119: The method according to any one of Embodiments 113 to 118, wherein when one or more protein binders are present, the protein binders are of the same type.

[0284] Embodiment 120: The method according to any one of Embodiments 113 to 118, wherein the biological sample is systemic circulating (whole) blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

[0285] Embodiment 121: The method according to any one of Embodiments 113 to 120, wherein a biological sample is passed through a resin-based removal column or spin column.

[0286] Embodiment 122: Compared to a biological sample that does not contain one or more protein binders and means for binding one or more high-abundance proteins and / or their proteoforms, an increase in the number of biomarkers or biomarker patterns is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders without the addition of means for binding to one or more high-abundance proteins and / or their proteoforms, the increase in the number of biomarkers or biomarker patterns is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of embodiments 113 to 121.

[0287] Embodiment 123: The method according to any one of Embodiments 113 to 122, further comprising the step of preparing a complex for detecting one or more disease-related biomarkers or patterns of one or more biomarkers in a protein corona.

[0288] Embodiment 124: The process of isolating the complex from the rest of the biological sample; A step to wash the complex; A step to resuspend the complex; A process to reduce proteins derived from the complex; A step of alkylating the protein derived from the complex; and, The process of digesting proteins derived from the complex, The method according to Embodiment 123, including the method described in Embodiment 123.

[0289] Embodiment 125: The method according to any one of Embodiments 113 to 124, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

[0290] Embodiment 126: The method according to Embodiment 125, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, gastrointestinal cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

[0291] Embodiment 127: The method according to Embodiment 125, wherein the disease is a neurological disease selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

[0292] Embodiment 128: The method according to any one of Embodiments 113 to 127, wherein a protein binder is added to the biological sample in an amount of approximately 1 pg / ml to approximately 1 g / ml. [Brief explanation of the drawing]

[0293] [Figure 1] Figure 1 is an exemplary overview of the experimental process described herein. [Figure 2] Figure 2 includes SDS-PAGE images analyzing protein corona-coated NPs in the presence of eight individual small molecule compounds and combinations of two small molecule compounds. [Figure 3A]Figure 3A is a bar graph showing the analysis of dynamic light scattering (DLS) (Figure 3A) and zeta potential (Figure 3B) for uncoated NPs and untreated protein corona-coated NPs. [Figure 3B] Figure 3B is a bar graph showing the analysis of dynamic light scattering (DLS) (Figure 3A) and zeta potential (Figure 3B) for uncoated NPs and untreated protein corona-coated NPs. [Figure 4] Figures 4A to 4C are transmission electron microscope (TEM) images of NPs. Figures 4A and 4B are TEM images of uncoated polystyrene NPs, and Figure 4C is a TEM image of a protein corona-coated NP. [Figure 5] Figure 5 is a bar graph showing the number of quantified proteins in plasma, untreated protein corona, and protein corona in the presence of small molecule compounds and combinations of small molecule compounds (mean ± standard deviation of 3 technical replicates). [Figure 6] Figure 6 is a box plot showing the distribution of normalized intensities of quantified proteins in plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules. [Figure 7] Figure 7 is a heatmap showing the hierarchical clustering of all proteins (1793 proteins in total) quantified in all samples (plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules). [Figure 8] Figure 8 is a heatmap showing the clustering of 117 proteins common to all samples (plasma, untreated protein corona, and protein corona in the presence of small molecules and combinations of small molecules). [Figure 9] Figure 9 is a heatmap showing the correlation of plasma proteome profiles using Pearson correlation coefficients for 117 proteins common to all samples (plasma, untreated protein corona, and protein corona in the presence of low molecular weight compounds and combinations of low molecular weight compounds) at concentrations from 10 to 1000 μg / ml. [Figure 10] Figure 10 includes two charts showing that different combinations of small molecules can enrich or deplete specific proteins. [Figure 11A] Figure 11A shows the enriched and depleted proteins in combinations 1 (Figures 11A and 11B) and 2 (Figures 11C and 11D) of small molecule compounds compared to untreated protein corona. [Figure 11B] Figure 11B shows the enriched and depleted proteins in combinations 1 (Figures 11A and 11B) and 2 (Figures 11C and 11D) of small molecule compounds compared to untreated protein corona. [Figure 11C] Figure 11C shows the enriched and depleted proteins in combinations 1 (Figures 11A and 11B) and 2 (Figures 11C and 11D) of small molecule compounds compared to untreated protein corona. [Figure 11D] Figure 11D shows the enriched and depleted proteins in combinations 1 (Figures 11A and 11B) and 2 (Figures 11C and 11D) of small molecule compounds compared to untreated protein corona. [Figure 12] Figure 12 includes a scatter plot showing the enrichment and decrease of specific proteins (only those that are shared) by spike addition of small molecule compounds to NP protein coronas, compared to the protein abundance in untreated NP protein coronas. [Figure 13A] Figure 13A includes bar graphs showing the KEGG process and pathway enrichment in biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13B] Figure 13B includes bar graphs showing the KEGG process and pathway enrichment in biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13C]Figure 13C includes bar graphs showing the pathway enrichment in the KEGG process and biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13D] Figure 13D includes bar graphs showing the KEGG process and pathway enrichment in biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13E] Figure 13E includes bar graphs showing the pathway enrichment in the KEGG process and biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13F] Figure 13F includes bar graphs showing the KEGG process and pathway enrichment in biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 13G] Figure 13G includes bar graphs showing the KEGG process and pathway enrichment in biological processes for all proteins that were significantly enriched or depleted, with all concentrations cumulative for each small molecule compound. [Figure 14A] Figure 14A includes integrated enrichment plots in KEGG and biological processes for all small molecule compounds and combinations of small molecule compounds, with all concentrations cumulative and compared to untreated protein corona. [Figure 14B] Figure 14B includes integrated enrichment plots in KEGG and biological processes for all small molecule compounds and combinations of small molecule compounds, with all concentrations cumulative and compared to untreated protein corona. [Figure 15A] Figure 15A includes a bar graph classifying quantified protein coronas of various small molecules and combinations of small molecules according to their physiological function. [Figure 15B]Figure 15B includes a bar graph classifying quantified protein coronas of various small molecules and combinations of small molecules according to their physiological function. [Figure 16] Figure 16 is a graph showing the effect of adding spikes of small molecules or combinations of small molecules on the dynamic range of the proteome. [Figure 17A] Figure 17A is a graph comparing the abundance and ranking of protein (albumin) between untreated plasma and the protein corona profile. [Figure 17B] Figure 17B is a graph comparing the abundance and ranking of protein (serotransferrin) between untreated plasma and the protein corona profile. [Figure 17C] Figure 17C is a graph comparing the abundance and ranking of proteins (haptoglobin) between untreated plasma and the protein corona profile. [Figure 18A] Figure 18A includes a chart showing protein abundances after incubation with NP and phosphatidylcholine (PtdCho). Figure 18A shows a stream plot or alluvial plot (including only proteins shared with plasma) showing a significant decrease in abundant plasma proteins, particularly albumin, after incubation of plasma with NP and PtdCho. [Figure 18B] Figure 18B includes a chart showing the protein abundance after incubation with NP and phosphatidylcholine (PdtCho). Figure 18B shows the total number of proteins identified in plasma samples incubated with NP, comparing treatment with and without the addition of various concentrations of PtdCho (mean ± standard deviation of 3 technical replicates). [Figure 18C]Figure 18C includes a chart showing protein abundances after incubation with NP and phosphatidylcholine (PdtCho). Figure 18C is a stream plot showing the decrease in abundant plasma proteins, particularly albumin, in response to NP addition, indicating that this decrease is intensified with increasing PtdCho concentration (including only proteins shared with plasma).

Claims

1. A method for detecting proteins and / or their proteoforms in a biological sample, comprising the following steps: A step of adding one or more low molecular weight compounds to the biological sample; A step of adding one or more protein binders having a surface capable of binding proteins to the biological sample, forming a protein corona on the surface of the one or more protein binders, and generating a complex containing the protein corona and the one or more protein binders; and A step of detecting the number of proteins and / or their proteoforms in the protein corona by antibody-based technology or proteomics technology.

2. The method according to claim 1, wherein the step of detecting the protein and / or its proteoform in the protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

3. The method according to claim 1 or 2, wherein the one or more protein binders include an inorganic agent, a metal agent, a metal oxide agent, a polymer agent, a lipid agent, a carbon agent, a core-shell agent, a composite agent, a mesoporous agent, or a combination thereof.

4. The method according to any one of claims 1 to 3, wherein the one or more protein binders include one or more nanoscale or microscale materials, such as nanoparticles, nanorods, nanospheres, nanodiscs, nanoclusters, nanofibers, nanotubes, microparticles, microrods, microspheres, microbeads, or a combination thereof.

5. The method according to any one of claims 1 to 4, wherein the one or more protein binders have a polydispersity index (PDI) of about 0.01 to about 0.7, preferably about 0.

3.

6. The method according to any one of claims 1 to 5, wherein one or more protein binders are of the same type.

7. The method according to any one of claims 1 to 6, wherein the biological sample is blood, plasma, serum, lung lavage fluid, cell lysate, menstrual blood, urine, tissue sample, amniotic fluid, cerebrospinal fluid, tear fluid, liquid biopsy, saliva, or semen, preferably a plasma sample.

8. The method according to any one of claims 1 to 7, wherein the one or more low molecular weight compounds include a biomolecule.

9. The method according to any one of claims 1 to 8, wherein the one or more low molecular weight compounds include metabolites, lipids, nutrients, plant-derived molecules, or a combination thereof.

10. The one or more of the aforementioned low molecular weight compounds: Triacylglycerols and / or derivatives thereof; Diacylglycerols such as 1,2-diacylglycerol and 1,3-diacylglycerol, and / or derivatives thereof; Glycerophospholipids and / or their derivatives; Triacylglycerols and / or their derivatives, diacylglycerols and / or their derivatives, and combinations of glycerophospholipids and / or their derivatives; glucose and / or its derivatives; Inosine 5'-monophosphate and / or derivatives thereof; Vitamin B complex and / or derivatives thereof; Glucose and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; Triacylglycerols and / or derivatives thereof, diacylglycerols and / or derivatives thereof; glycerophospholipids and / or derivatives thereof, glucose and / or derivatives thereof, inosine 5'-monophosphate and / or derivatives thereof, and combinations of vitamin B complex and / or derivatives thereof; Phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives; Phosphatidylserine and / or its derivatives; Phosphatidic acid and / or its derivatives; Phosphatidylinositol and / or its derivatives; Phosphatidylglycerol and / or its derivatives; Cardiolipin and / or its derivatives; L-α-phosphatidylinositol and / or its derivatives; Glucose and / or its derivatives, triacylglycerol and / or its derivatives, diacylglycerol and / or its derivatives; and combinations of phosphatidylcholine and / or its derivatives; Phosphatidylethanolamine and / or its derivatives, L-α-phosphatidylinositol and / or its derivatives, inosine 5'-monophosphate and / or its derivatives, and combinations of vitamin B complex and / or its derivatives; or, Those combinations, The method according to claim 9, including the method described in claim 9.

11. The method according to any one of claims 1 to 10, wherein the one or more low molecular weight compounds are phosphatidylcholine.

12. The method according to claim 11, wherein approximately 1 pg / ml to approximately 1 g / ml, for example, approximately 1000 μg / ml of phosphatidylcholine is added to the biological sample.

13. The method according to any one of claims 1 to 12, wherein the biological sample is passed through a resin-based removal column or spin column.

14. Compared to a biological sample that does not contain the one or more protein binders and the one or more low molecular weight compounds, an increase in the number of proteins and / or their proteoforms is observed to be at least 0.1 times, 0.5 times, 1 time, or more; and / or, Compared to a biological sample containing one or more protein binders but without the addition of the one or more low molecular weight compounds, the increase in the number of proteins and / or their proteoforms is at least 0.1 times, 0.5 times, 1 time, or more. The method according to any one of claims 1 to 13.

15. The method according to any one of claims 1 to 14, wherein one or more low molecular weight compounds are added to the biological sample at various concentrations.

16. The method according to claim 15, wherein the various concentrations are approximately 1 pg / ml to approximately 1 g / ml.

17. The method according to any one of claims 1 to 16, further comprising the step of preparing a complex for detecting the protein and / or its proteoform in the protein corona.

18. A step of isolating the complex from the remainder of the biological sample; A step of washing the composite; A step of resuspending the composite; A step of reducing the protein derived from the complex; A step of alkylating the protein derived from the complex; and, A step of digesting the protein derived from the complex, The method according to claim 17, including the method described in claim 17.

19. A method for detecting one or more biomarkers associated with a disease, or a pattern of one or more biomarkers, comprising the following steps: A step of adding one or more low molecular weight compounds to each of at least two biological samples derived from different subjects diagnosed with the aforementioned disease; A step of adding one or more protein binders having a surface capable of binding proteins to the biological sample, forming a protein corona on the surface of the one or more protein binders, and generating a complex containing the protein corona and the one or more protein binders; and A step of detecting one or more biomarkers or a pattern of one or more biomarkers in the protein corona using antibody-based technology or proteomics technology.

20. The method according to claim 19, wherein the step of detecting one or more biomarkers or a pattern of one or more biomarkers in the protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

21. The method according to claim 19 or 20, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

22. The method according to claim 21, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, digestive tract cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

23. A method for diagnosing a disease in a subject, comprising the following steps: A step of adding one or more low-molecular-weight compounds to a biological sample derived from the subject; A step of adding one or more protein binders having a surface capable of binding proteins to the biological sample, forming a protein corona on the surface of the one or more protein binders, and generating a complex containing the protein corona and the one or more protein binders; and A step of detecting one or more biomarkers or patterns of one or more biomarkers in the protein corona that are associated with the disease, using antibody-based technology or proteomics technology.

24. The method according to claim 23, wherein the step of detecting one or more biomarkers or a pattern of one or more biomarkers in the protein corona is carried out by a proteomics technique including top-down, middle-down, or bottom-up LC-MS / MS.

25. The method according to claim 23 or 24, wherein the disease is a neoplastic disease, a cardiovascular disease, a metabolic disease, an infectious disease, an inflammatory disease, a congenital and genetic disease, a degenerative disease, a neurological disease, or a combination thereof.

26. The method according to claim 25, wherein the disease is a neoplastic disease selected from lung cancer, pancreatic cancer, myeloma, myeloid leukemia, meningioma, glioblastoma, breast cancer, esophageal squamous cell carcinoma, digestive tract cancer, liver cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, neuroendocrine cancer, and combinations thereof.

27. The method according to claim 25, wherein the disease is a neurological disease selected from Alzheimer's disease, brain tumor, epilepsy, Parkinson's disease, ALS, arteriovenous malformation, cerebrovascular disease, cerebral aneurysm, epilepsy, multiple sclerosis, peripheral neuropathy, postherpetic neuralgia, stroke, frontotemporal dementia, demyelinating disease, multiple sclerosis, Devic's disease, central pontine myelinolysis, progressive multifocal leukoencephalopathy, leukodystrophy, Guillain-Barré syndrome, progressive inflammatory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, anti-MAG antibody-associated peripheral neuropathy, and combinations thereof.

Images