Functionalized magnetic bead and preparation and use method thereof

Abstract

Disclosed herein are various aspects of magnetic beads, including functional groups suitable for binding biomolecules, such as peptides, polypeptides, proteins, RNA, and / or DNA. The magnetic beads may include a vinyl azalactone-based cross-linked polymer and a magnetic material dispersed in the magnetic beads. In certain aspects, the magnetic beads may be used to separate biomolecules, such as from lysates, or to purify biomolecules suitable for analysis, such as mass spectrometry.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 517,010, filed August 1, 2023, and U.S. Provisional Patent Application No. 63 / 604,531, filed November 30, 2023, both of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to magnetic beads containing functional groups suitable for binding biomolecules, including polypeptides, proteins, RNA and / or DNA, and also to methods for preparing such magnetic beads and for using these magnetic beads to separate and / or purify biomolecules for analysis (such as by mass spectrometry). Background Technology

[0004] Mass spectrometry (MS)-based proteomics has become the most comprehensive method for protein identification, quantification, interaction analysis, modification, and structural characterization. However, it is also a challenging subject requiring expertise in sample preparation, as obtaining high-quality samples through robust and reproducible preparation is crucial for success.

[0005] Proteomics sample preparation is extremely complex and involves a variety of methods, with many different operational protocols. For example, bottom-up proteomics sample preparation requires multiple steps, including protein extraction, cysteine ​​reduction and alkylation, protein digestion into peptides, purification, and peptide concentration for LC-MS analysis. The lack of standardization in proteomics sample preparation makes it difficult to accurately compare results from different laboratories and using different protocols. Summary of the Invention

[0006] This document discloses several aspects of magnetic beads suitable for biological sample preparation protocols, such as sample preparation for proteomics analysis (e.g., by MS). Methods for preparing and using magnetic beads, as well as kits comprising these magnetic beads, are also disclosed. In some aspects, the magnetic beads comprise a cross-linked polymer and a magnetic material (e.g., an iron source). The cross-linked polymer may comprise one or more azalactone moieties. or one or more functional groups derived from the azalactone moiety (optionally derived from the reaction of the azalactone moiety with a nucleophilic reactive group). In some aspects, the crosslinking polymer is a crosslinked azalactone polymer, and the magnetic beads are azalactone beads. In some aspects, the magnetic material is or comprises iron oxide particles, such as Fe3O4 and / or Fe2O3 particles.

[0007] This document also discloses several aspects of a method for using the disclosed magnetic beads. The method may include: forming a first suspension comprising a plurality of magnetic beads and a first liquid containing biomolecules; removing the magnetic beads from the first suspension and optionally washing the magnetic beads with a second liquid; forming a second suspension comprising the magnetic beads and an elution solution; and removing the magnetic beads from the second suspension, leaving a third liquid containing biomolecules. In some aspects, each magnetic bead comprises a crosslinked polymer and a magnetic material, wherein the crosslinked polymer comprises one or more azalactone moieties. , or one or more functional groups derived from the azalactone moiety (optionally derived from the reaction of the azalactone moiety with a nucleophilic reactive group).

[0008] The foregoing and other objects, features and advantages of this disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0009] Figure 1 is a flowchart illustrating one aspect of the standard workflow using the disclosed magnetic beads.

[0010] Figure 2 is a flowchart illustrating the SP3 workflow, which includes the PAC workflow and the SP2 workflow.

[0011] Figure 3 is a digital image showing one aspect of the disclosed magnetic bead.

[0012] Figure 4 shows the relationship between differential volume and particle size, illustrating the average particle size of the disclosed magnetic beads, and providing a table with the average particle size and standard deviation.

[0013] Figure 5 is a digital image showing one aspect of the disclosed magnetic beads synthesized using iron oxide clusters as the magnetic material.

[0014] Figure 6 is a digital image showing one aspect of the disclosed magnetic beads synthesized using iron oxide particles as the magnetic material.

[0015] Figure 7 illustrates the use of Dynabeads TM Digital images of the disclosed magnetic beads as one aspect of magnetic material synthesis.

[0016] Figure 8A Figure 8A and Figure 8B show digital images of the microplate at each stage of the SP2 workflow and the results table (Figure 8B), illustrating the surface adsorption loss for different types of magnetic beads.

[0017] Figure 9 shows the relationship between peptide yield and magnetic bead type, illustrating the peptide yield from different magnetic bead types as identified using a quantitative colorimetric peptide assay.

[0018] Figure 10 shows the relationship between peptide number and magnetic bead type, illustrating the number of unique peptides separated using different magnetic bead types and identified using nanoLC-MS analysis (75 inner diameter column, 150 min gradient, QE plus mass spectrometer, PD 2.5 data processing).

[0019] Figure 11 shows the relationship between proteome and magnetic bead type, illustrating the number of different proteomes separated using different magnetic bead types and identified using nanoLC-MS analysis (75 inner diameter column, 150 min gradient, QE plus mass spectrometer, PD 2.5 data processing).

[0020] Figure 12 shows the relationship between the amount of bound antibody and the magnetic beads, illustrating the binding ability of the disclosed magnetic beads coupled with base-stable protein A (asPA) to rabbit IgG.

[0021] Figure 13 shows gel images comparing the anion exchange properties of magnetic beads with three different ligand chemical structures. Detailed Implementation

[0022] I. Terminology

[0023] The following terms and methods are provided to better describe this disclosure and to guide those skilled in the art in implementing it. Unless otherwise expressly stated, the singular forms “an,” “a,” and “the / described” mean one or more. Unless the context clearly indicates otherwise, the term “or” means a single element or a combination of two or more elements among the said alternative elements. As used herein, “comprising” means “including.” Therefore, “comprising A or B” means “including A, B, or A and B,” without excluding additional elements. Unless otherwise specified, all references cited herein (including patents and patent applications) are incorporated herein by reference in their entirety.

[0024] Unless otherwise stated, all figures representing component quantities, molecular weights, percentages, temperatures, times, etc., as used in this specification or claims should be understood to be modified by the term "about". Therefore, unless otherwise implied or express, the numerical parameters are approximate values ​​that may depend on the desired properties sought and / or the detection limits of standard test conditions / methods. When directly and clearly distinguishing the embodiments from the prior art discussed, the figures for the embodiments are not approximate values ​​unless explicitly stated with the word "about".

[0025] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While similar or equivalent methods and materials may be used in practice or testing of this disclosure, suitable methods and materials are described below. These materials, methods, and examples are illustrative only and not intended to be limiting.

[0026] "alkyl" refers to a compound having 1 to 25 carbon atoms. 1-25 ) or more carbon atoms, more typically having 1 to 10 (C 1-10 Carbon atoms (such as 1 to 6 C atoms) 1-6 ) carbon atoms or 1 to 4 (C 1-4 A saturated aliphatic hydrocarbon group consisting of carbon atoms.

[0027] "Carboxyl group" refers to the -CO2H functional group.

[0028] "Ester group" refers to the -CO2R functional group, where R is an alkyl group, such as a C1-6 alkyl group.

[0029] "Nucleic acid" refers to a polynucleotide molecule. The polynucleotide can be naturally occurring or synthetic. Nucleic acid can be DNA, RNA, or a mixture of DNA and RNA nucleotides. Typically, nucleic acids contain 20 to 10,000 or more nucleotides, such as 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 nucleotides up to 10,000 nucleotides.

[0030] A peptide is a compound that contains amino acid residues linked together by peptide bonds. Typically, peptide compounds have 2 to 50 amino acid residues.

[0031] A polypeptide is a compound comprising amino acid residues linked by peptide bonds. In some respects, a polypeptide has from about 50 amino acid residues to 2,000 or more amino acid residues. The amino acids may include α-amino acids and may include L-optic isomers, D-optic isomers, or combinations thereof.

[0032] "Protein" refers to a molecule or complex containing one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are typically stabilized using non-covalent bonds (such as hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions) and / or covalent bonds (e.g., disulfide bonds, such as between thiol groups of cysteine ​​residues).

[0033] II. Magnetic beads

[0034] This document discloses several aspects of magnetic beads comprising a magnetic material dispersed in polymer beads. In some aspects, the polymer beads comprise a crosslinked polymer comprising one or more azalactone moieties. , and / or one or more functional groups derived from the azalactone moiety. The functional groups derived from the azalactone moiety may include portions derived from the reaction between the azalactone moiety and a nucleophilic reactive group (such as, but not limited to, amine-containing compounds, thiol-containing groups, alcohols, or water).

[0035] Hydrolysis of azalactones or the reaction of azalactone groups with amine-containing compounds can increase the functionality of magnetic beads and / or alter their surface properties. For example, hydrolysis of the azalactone ring can produce magnetic beads with multiple carboxylic acid functional groups on their surface. Alternatively, reacting the azalactone groups with amine-containing compounds can increase or decrease the hydrophobicity or hydrophilicity of the magnetic beads.

[0036] In some respects, the azalactone beads are reacted with an amine-containing compound comprising a primary, secondary, or tertiary amine group and optionally further comprising one or more additional functional groups. In other respects, the azalactone beads are reacted with a thiol- or alcohol-containing compound to yield thioamide and ester-functionalized beads.

[0037] In other respects, azalactone beads are reacted with amine-containing compounds to provide linking arms that provide one or more additional functional groups for further reaction with ligands or biomolecules. For example, amine-containing compounds containing additional amine reactive groups can be reacted with azalactone beads, wherein the reactive amine groups can be converted to epoxy, maleimide, or iodoacetyl groups using standard methods known in the art.

[0038] In other respects, the azalactone beads are reacted with compounds to increase the number of functional groups attached to the beads. Such methods can be effective for improving the binding ability of the beads to specific target molecules. For example, the beads can be reacted with amine compounds (e.g., polymers or dendritic macromolecules) containing multiple reactive groups.

[0039] In other respects, the amine-containing compound is reacted with the azalactone groups on the beads to provide a spacer group to overcome steric hindrance during ligand linkage, or to provide a linker arm for subsequent reactions with complementary reactive groups using the compound and methods. For example, the amine-containing compound may include reactive functional groups such as azide, alkynyl (e.g., cyclic alkynyl, such as dibenzocyclooctyne (DBCO)), maleimide, amino, hydroxyl, carboxylic acid, sulfonic acid, or chelating agents (such as NTA or IDA).

[0040] Amine-containing compounds also include biomolecules (e.g., proteins, peptides, nucleic acids, nucleotides, and oligosaccharides / polysaccharides), organic ligands, and macromolecular synthetic compounds (such as polymers, e.g., PEG or polyethyleneimine (PEI)) and dendritic macromolecules. Representative amine-containing organic ligands include, but are not limited to: alkylamines (e.g., straight-chain or branched C4-C...). 12 Alkylamines, such as 1,5-diaminopentane, diethylamine, butylamine, and octylamine; arylamines (e.g., benzylamine and 4-aminobenzoic acid); aminohexanoic acid; ethanolamine; 5-amino-2-methylbenzenesulfonic acid; aminoethyltrimethylammonium chloride (AETMA); taurine; tris(hydroxymethyl)aminomethane (Tris); and the like. Other examples of amine-containing compounds are positively or negatively charged ligands that can be used for applications such as ion exchange (cation and anion). Representative examples of ligands that can be used for anion exchange include, but are not limited to: alkylamines (e.g., straight-chain or branched C4-C12 alkylamines, such as 1,5-diaminopentane (PDA) and butylamine); arylamines (e.g., benzylamine and naphthylamine); and polymeric amines (e.g., PEG, or polyethyleneimine (PEI) such as PEI-25K, PEI-800K, etc.). In other embodiments, representative examples of ligands that can be used for cation exchange include, but are not limited to, any alkyl or aryl carboxylic acids, sulfonic acids, and phosphonic acids.

[0041] In some respects, the amine-containing moiety is a protein, such as an antibody, antibody-binding proteins (e.g., protein A, protein A / G, protein G, and protein L), streptavidin, neutral avidin, glutathione, enzymes, or other amine-containing biomolecules. In one respect, azalactone beads are conjugated to an amine-containing biomolecule (such as alkali-resistant protein A (asPA)), and the final conjugate can then be used to purify IgG. When the biomolecule is an enzyme, it is desirable that it retains its enzyme selectivity after conjugation with the beads. Examples of enzymes include proteases (e.g., trypsin) and other types of enzymes known in the art. Figure 12 shows that azalactone beads (asPA-magUL) have similar properties to magnetic agarose beads (asPA-magAg). This assay was performed using replicate samples of each magnetic bead.

[0042] In some respects, the amine-containing compound is or includes a metal-charged chelate (e.g., nickel-charged hypoazolidinyltriacetic acid (Ni-NTA)). In one respect, azalactone beads are coupled to Ni-NTA, and the final conjugate is used to purify multihistidine-labeled proteins from soluble protein extracts.

[0043] In one aspect, azalactone beads are coupled to metal-loaded chelates (such as Fe-NTA, Ti-NTA, Zr-NTA, or Ga-NTA), and the final conjugate is used to purify phosphorylated peptides or proteins from biological samples (e.g., cell lysates).

[0044] In some respects, the amine-containing compound is or includes a positively charged ligand. For example, the beads can be coupled with a positively charged ligand (such as 1,5-diaminopentane). Positively charged bead conjugates can be used to purify negatively charged substances (e.g., nucleic acids, extracellular vesicles, viral particles, etc.) from biological samples (e.g., plasma, blood, cell culture supernatant, urine, etc.).

[0045] In some aspects, the polymer beads are azalactone beads formed from vinylazalactone and a crosslinking agent. The vinylazalactone may be 4,4-dimethyl-2-vinyloxazol-5(4H)-one.

[0046] 4,4-Dimethyl-2-vinyloxazol-5(4H)-one

[0047] The crosslinking agent can be any crosslinking agent suitable for promoting polymerization and forming beads. In some aspects, the crosslinking agent is bisacrylamide, agarose, or vinyl ether. In other aspects, the crosslinking agent is methylenebisacrylamide.

[0048] In certain aspects, the vinylazanolide is 4,4-dimethyl-2-vinyloxazol-5(4H)-one, and the crosslinking agent is bisacrylamide (such as methylenebisacrylamide).

[0049] In some respects, the bead has a plurality of functional groups on its surface. The functional groups may be selected from... , , , , , , , or In some respects, the functional groups are selected from... , , , , , , or In some respects, the functional group is or comprises: .

[0050] In any respect, the magnetic material in the magnetic beads can be in any suitable form, such as, but not limited to, particles, powder, flakes, clusters, beads, or combinations thereof. In some embodiments, the magnetic material is or comprises magnetic particles.

[0051] In any respect, the magnetic material in the magnetic beads may contain an iron source. The iron source can be any suitable iron source, such as an iron source added during the polymer bead formation reaction. The iron source can be in the form of iron-containing particles and / or can be iron oxide, such as Fe3O4, Fe2O3, or combinations thereof. Alternatively or additionally, the beads may include magnetic particles as an iron source, such as Dynabeads. TM (Available from Thermo Fisher Scientific) and / or Sera-Mag TM Speedbeads (available from Speedbeads Biotech) and / or other magnetic beads or magnetic particles. In some aspects, the magnetic material is or comprises Fe3O4 particles. In some aspects, the magnetic material is an activated iron oxide source, such as Fe3O4-NHS, to provide an additional source of reactivity during particle synthesis.

[0052] In some aspects, the magnetic material is an activated magnetic material and may contain one or more functional groups (such as, for example, carboxyl groups or ester groups). In other aspects, the magnetic material is an activated iron source containing additional functional groups (such as, but not limited to, carboxyl or ester groups).

[0053] In some aspects, the magnetic material is contained within agarose beads, and the agarose beads are contained within a crosslinked polymer to form magnetic beads.

[0054] The magnetic particles may have sizes suitable for use in the disclosed beads, such as 5 nm to 1,000 nm or larger, such as 5 nm to 200 nm, or 100 nm to 800 nm. In some aspects, the magnetic particles have a size of 15 nm to 100 nm (such as 15 nm to 50 nm, or 50 nm to 100 nm). In some other aspects, the magnetic particles have a size of 100 nm to 800 nm; for example, iron clusters may have an average particle size of 100 nm to 800 nm (such as 100 nm to 600 nm, or about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, or 600 nm).

[0055] The average particle size of the magnetic particles can be determined using a particle size analyzer. In some respects, the magnetic particles are substantially spherical, but in others, they are not. In some respects, the magnetic particles are substantially spherical iron clusters. In some other respects, the magnetic particles are neither iron clusters nor substantially spherical. However, such magnetic particles can be coated with another material that provides a substantially spherical shape.

[0056] The magnetic beads have an average size suitable for use in separation techniques. The average particle size is determined by a particle analyzer (e.g., Beckman Coulter). TM The magnetic beads are determined by means of an LS13320XR or by scanning electron microscopy (SEM). In some aspects, the magnetic beads are substantially spherical, and the average particle size is the average diameter of the beads. In some aspects, the magnetic beads have an average particle size of 20 μm to 100 μm or larger (such as 20 μm to 80 μm, 30 μm to 60 μm, 30 μm to 50 μm, or 40 μm to 80 μm).

[0057] The magnetic beads can be porous or non-porous. In some aspects, the magnetic beads have a porous structure with pore sizes ranging from 100 angstroms to 200 angstroms. In other aspects, the pores extend from the surface of the particle into its interior, but do not completely penetrate the particle. In some aspects, at least a portion (and potentially substantially all) of the magnetic material is located within the pores.

[0058] The magnetic beads can be in a hydrated or dehydrated state. Typically, the disclosed magnetic beads are the size in the hydrated state, and the size of the hydrated beads (hydrated particle size) is larger than that of the dehydrated beads. These magnetic beads exhibit swelling properties, and their size can increase by 3 to 10 times or more, or 5 to 8 times, or 5 to 6 times. In contrast, non-magnetic magnetic beads typically swell to about 8 times their size when hydrated compared to their dehydrated state. Furthermore, to the inventors' knowledge, other magnetic beads do not expand to a measurable extent. The expansion of the disclosed magnetic beads can make functional portions (such as azalactone portions) on the bead surface more accessible, and / or reduce steric hindrance caused by their size due to the increased spacing between functional groups caused by the expansion, making it easier for large biomolecules to bind to the functional groups.

[0059] In some respects, the disclosed magnetic beads exhibit lower bead loss during use in automated processes (e.g., Kingfisher equipment) compared to other magnetic beads. Bead loss can be attributed at least in part to the adsorption of the beads onto equipment surfaces (e.g., onto the suction head plate and sample plate). In some respects, the bead loss of the disclosed bead group is less than about 5% by weight when used in automated processes. In comparative experiments, the loss rate of the disclosed magnetic beads was 4.7% (by weight), while that of the Cytiva Sera-Mag... TM The loss rate of the magnetic beads is 6.0%, Invitrogen. TM DynaGreen TM The loss rate of silica magnetic beads is 11.5%, and Dynabeads TM MyOne TM The loss rate of silica magnetic beads is 6.8%.

[0060] III. Synthesis of Magnetic Beads

[0061] A first solution of monomer and optionally polymer stabilizer is prepared in a suitable solvent (such as an alkane solvent, e.g., heptane or hexane, or a chlorinated solvent, e.g., carbon tetrachloride, chloroform, or dichloromethane). The monomer may be a azalide monomer, such as vinylazalide, e.g., 4,4-dimethyl-2-vinyloxazol-5(4H)-one. The polymer stabilizer may be any polymer stabilizer suitable for use in this polymerization reaction. For example, polymer stabilizers include, but are not limited to, isooctyl acrylate / acrylic acid copolymers, isobutyl methacrylate / acrylic acid copolymers, or hexyl acrylate / sodium acrylate copolymers. After stirring, a magnetic material in a suitable form (such as, but not limited to, granules, powders, flakes, clusters, beads, or combinations thereof) is added. In some aspects, the magnetic material comprises an iron source, such as iron oxide. In some aspects, iron oxide particles are used. Additionally, in some aspects, the first solution is prepared and maintained in a temperature range of 35°C to 50°C, such as 40°C to 45°C.

[0062] A second solution is prepared, comprising a crosslinking agent in a suitable solvent. The crosslinking agent is any crosslinking agent suitable for promoting the formation of magnetic beads. Exemplary crosslinking agents include, but are not limited to, acrylamide, bisacrylamide, agarose, or vinyl ether. In some aspects, the crosslinking agent is methylenebisacrylamide. The solvent can be any solvent suitable for promoting the polymerization reaction, such as alcohols (e.g., methanol, ethanol, 2-propanol, 1-propanol, or combinations thereof), water, or combinations thereof. In some aspects, a free radical initiator (e.g., sodium persulfate) is added to the second solution. Additionally, in some aspects, the second solution is prepared and maintained at a temperature range of 25°C to 40°C, such as 30°C to 35°C.

[0063] The first solution is mixed with the second solution, and the mixture is stirred (e.g., by stirring, shaking, vortexing, etc.) at a temperature suitable for promoting polymerization and magnetic bead formation, followed by the addition of a polymerization initiator such as tetramethylethylenediamine (TEMED). In some aspects, the temperature is 30°C to 50°C.

[0064] After cooling to room temperature, the magnetic beads are filtered and washed with a suitable solvent (such as acetone). The magnetic beads are then resuspended in a suitable solvent (such as acetone) and agitated (e.g., by shaking, stirring, vortexing or sonication) under cooling conditions (such as in an ice bath).

[0065] The magnetic beads are then sieved to select those within the target particle size range. Magnetic bead sieving may involve using one or more sieves (such as two sieves) to obtain magnetic beads with a target particle size or range. In some cases, a 63 μm sieve and a 25 μm sieve are used to select a population of magnetic beads with a particle size range of 63–25 μm.

[0066] The magnetic beads are then placed in a magnetic separation system to remove any non-magnetic residues. After filtration, the magnetic beads are dried, for example, under vacuum and / or by heating at 30°C to 60°C.

[0067] Before use, magnetic beads typically need to be swollen in an aqueous liquid (such as water, buffer solution, or aqueous / organic solvent mixture).

[0068] For more information on the synthesis of crosslinked beads, see U.S. Patent Nos. 5,403,902 and 5,292,840, the entire contents of which are incorporated herein by reference.

[0069] IV. Reagent kit containing the magnetic beads

[0070] This document also discloses several aspects of kits comprising the disclosed magnetic beads. The composition of the kits may vary depending on the intended use. In some aspects, in addition to the disclosed magnetic beads, the kits may also comprise a lysis solution, a universal nuclease, a reduction solution, an alkylation solution, an enzyme reconstitution solution, a trypsin / Lys-C protease mixture, a magnetic bead washing / binding solution, a washing solution, an elution solution, or any combination thereof.

[0071] In some respects, the kit comprises the disclosed magnetic beads, lysis solution, universal nuclease, reduction solution, alkylation solution, enzyme reconstitution solution, and trypsin / Lys-C protease mixture. In other respects, the kit comprises the disclosed magnetic beads, magnetic bead washing / binding solution, washing solution, and elution solution.

[0072] V. Biomolecules and reagents

[0073] Successful peptide mass spectrometry analysis requires high-quality input samples. Sample preparation prior to mass spectrometry (MS) analysis often introduces chemicals incompatible with downstream MS analysis, and these chemicals must be removed from the sample before LC-MS analysis. However, completely removing salts, detergents, and other chemical / non-chemical contaminants while retaining all peptides and achieving reasonable recoveries is very difficult, typically requiring specialized equipment, highly complex processes, and / or a significant time investment.

[0074] The disclosed magnetic beads can be used to simplify and accelerate sample purification, while improving sample quality in terms of cleanliness and enhancing its adaptability to automated operations. These magnetic beads are also compatible with plastics and solvents commonly used in bioassays. These characteristics make the magnetic beads suitable for downstream applications prior to MS, and also suitable for other applications such as chemical labeling, enrichment, and fractionation.

[0075] The disclosed magnetic beads are suitable for use with a variety of biomolecules, including proteins, polypeptides, peptides, DNA, RNA, and combinations thereof. Additionally, the disclosed magnetic beads are suitable for use with isotopically labeled biomolecules. Isotopic labeling may include labeling with deuterium, carbon-13, nitrogen-15, oxygen-18, or combinations thereof. Alternatively or alternatively, the biomolecules may be labeled with isotopically enriched mass tags, such as, but not limited to, tandem mass tagging (TMT and TMTpro) reagents (available from Thermo Fisher Scientific). TM Stable isotope-labeled peptides and / or proteins prepared using the SILAC metabolic labeling system (available from Thermo Fisher Scientific). TM Rare earth metals (e.g., lanthanides); fluorescent labels (e.g., dyes); tissues and / or cells cultured with heavy amino acids; or combinations thereof.

[0076] In some respects, the disclosed magnetic beads, due to their unique binding properties to different types of biomolecules, can be used in multianalyte analysis applications. For example, the disclosed magnetic beads typically bind DNA in low-concentration organic solvents (such as about 30% organic matter) and are unaffected by the presence of guanidine salts. In contrast, the disclosed magnetic beads bind RNA in higher-concentration organic solvents (such as about 90% organic matter). Furthermore, in the presence of guanidine hydrochloride, the disclosed magnetic beads can bind proteins in solvents with up to 45% organic matter; or in the absence of guanidine salts, they can bind in solvents with about 80% organic matter. These combinations of guanidine salts and organic matter can be uniquely used in the disclosed magnetic beads for the sequential extraction of DNA, protein, and RNA from a single sample, with the potential to significantly reduce cross-contamination by other analytes. In addition, the disclosed magnetic beads are compatible with a variety of organic solvents, including but not limited to: alcohols such as methanol, ethanol, and isopropanol; acetonitrile; esters such as ethyl acetate; and acids such as trifluoroacetic acid and formic acid.

[0077] VI. Application

[0078] In some aspects, a general method for using the magnetic beads disclosed herein includes forming a first suspension comprising a plurality of the disclosed magnetic beads and a first liquid containing the target biomolecule. After the biomolecule binds to the magnetic beads, the magnetic beads are removed from the first suspension and optionally washed to remove any unwanted or unbound material. A second suspension is then formed comprising the magnetic beads bound to the biomolecule and an elution solution. The magnetic beads are then removed from the second suspension to obtain a solution containing the target biomolecule.

[0079] The biomolecule can be any biomolecule suitable for binding to the disclosed magnetic beads. In some aspects, the biomolecule is a protein, peptide, polypeptide, DNA, or RNA molecule. In other aspects, the biomolecule is a complex molecule comprising a combination of protein, peptide, polypeptide, DNA, and / or RNA portions.

[0080] By appropriately selecting ligands, the magnetic beads disclosed herein can be applied to anion and cation exchange purification methods. A general ion exchange method using the magnetic beads disclosed herein includes forming a first suspension comprising a plurality of the disclosed magnetic beads and a first liquid containing a target substance carrying a negative or positive charge. The target substance can bind to ligands on the magnetic beads and can then be separated from other components of the suspension. In some embodiments, the target substance can be released from the magnetic beads.

[0081] In any respect, the biomolecules may be labeled. In some respects, the label may be any label suitable for use in analytical techniques such as mass spectrometry. Exemplary labels include, but are not limited to, stable isotopes (such as deuterium, carbon-13, nitrogen-15, oxygen-18); isotope-enriched mass tags, such as, but not limited to, tandem mass tagging (TMT and TMTpro) reagents (available from Thermo Fisher Scientific). TM Stable isotope-labeled peptides and / or proteins prepared using the SILAC metabolic labeling system (available from Thermo Fisher Scientific). TM Rare earth metals (e.g., lanthanides); fluorescent labels (e.g., dyes); tissues and / or cells cultured with heavy amino acids; or any combination thereof.

[0082] Figure 2 provides a flowchart of the SP3 workflow using the disclosed magnetic beads, which includes the PAC process and the SP2 process, which are discussed in more detail below.

[0083] A. Purification of peptides and / or polypeptides

[0084] In some aspects, the disclosed magnetic beads are used to purify peptides and / or polypeptides. In some aspects, a mixture is formed comprising the disclosed magnetic beads and a sample containing the peptides and / or polypeptides to be purified. The sample may be a protein digestion sample. Optionally, the magnetic beads may be washed, for example, to remove storage buffer before being mixed with the sample.

[0085] The mixture may further comprise a magnetic bead washing / binding solution. In some aspects, the magnetic bead washing / binding solution is 100% acetonitrile containing 0.1% formic acid. The magnetic bead washing / binding solution may be added to the magnetic beads to form a magnetic bead slurry before adding the magnetic beads to the sample. The amount of magnetic bead slurry added to the sample may be adjusted according to the amount of protein in the sample. In some aspects, when the amount of protein input is 15 to 30 µg, 30 to 60 µg, or 60 to 150 µg, the total volume of the magnetic bead slurry should be 250 µL, 500 µL, or 1000 µL, respectively. In some aspects, the magnetic bead slurry has a magnetic bead concentration of 25%. In some aspects, approximately 20 μL of the 25% magnetic bead slurry is used, providing 5 mg of magnetic beads per sample. In some cases, 3 mg to 10 mg of magnetic beads are used for 10 to 25 μg of protein, such as using about 5 mg of magnetic beads per 10 to 25 μg of protein.

[0086] After mixing the disclosed magnetic beads with the sample, the resulting mixture can be incubated at a temperature suitable for promoting protein binding (such as 20°C to 30°C, or approximately room temperature). The incubation time is suitable for promoting protein binding, such as 15 minutes to 180 minutes or longer, or 30 minutes to 60 minutes. During incubation, the mixture can be agitated, such as by shaking, vortexing, stirring, or sonication.

[0087] After incubation, the magnetic beads are removed from the mixture along with any unbound peptides or polypeptides using a suitable technique (such as the use of a magnetic rack). The magnetic beads can be washed to remove any remaining unbound material, solvent, etc. In some cases, the washing solution is a 90% to 99% aqueous solution of organic matter containing 0.1% to 1% organic acid, and may be approximately 85% acetonitrile, 10% ethanol, 0.5% formic acid, and 4.5% water.

[0088] The magnetic beads are then treated with an elution solution to remove bound peptides and / or polypeptides from the beads. In some aspects, the elution solution is a 1% to 5% aqueous solution of an organic compound containing 0.05% to 0.25% acid, such as a 2% acetonitrile / 0.1% formic acid aqueous solution. The magnetic beads are incubated with the elution solution at a suitable temperature for a suitable time to promote the elution of peptides / polypeptides from the magnetic beads. Incubation can be carried out at a temperature of 20°C to 30°C or at approximately room temperature, and / or for 1 minute to 10 minutes or longer, such as 2 minutes to 5 minutes, or about 3 minutes.

[0089] After incubation, the magnetic beads are removed from the eluted peptide and / or polypeptide solution using a suitable technique (such as a magnetic rack). The eluted peptide and / or polypeptide solution can then be transferred to a suitable container for analysis, such as by mass spectrometry.

[0090] In one aspect, a method for obtaining peptides or polypeptides for LC-MS analysis includes: A first suspension is formed, the first suspension comprising a plurality of disclosed magnetic beads and a first liquid, the first liquid containing a protein digestion sample comprising a target peptide or polypeptide; Stir the first suspension at room temperature for 30 to 60 minutes; Remove the magnetic beads from the first suspension and wash them with the second liquid; A second suspension comprising magnetic beads and an elution solution is formed, and the second suspension is stirred at room temperature for 1 to 5 minutes; and The magnetic beads are separated from the second suspension to leave a third liquid containing the target peptide or polypeptide.

[0091] In some aspects, each of the plurality of magnetic beads comprises iron oxide particles distributed throughout the azalactone bead, and a plurality of [missing information - likely referring to a number of particles] located on the surface of the magnetic bead. part.

[0092] B. Protein Aggregation Capture (PAC)

[0093] Several aspects of the disclosed magnetic beads can be used in protein aggregation capture (PAC) applications. In some aspects, a mixture comprising the disclosed magnetic beads and a sample containing protein is formed. Optionally, the magnetic beads can be washed, for example, to remove storage buffer, prior to mixing with the sample. In some aspects, the washing solution may contain 70% to 95% by volume an organic solvent, such as 75% to 85% by volume an organic solvent (e.g., alcohols, such as 2-propanol, or acetonitrile), and may also contain an organic acid, such as trifluoroacetic acid (TFA), in an amount greater than 0% to 1% by volume, such as 0.25% to 1% by volume an organic acid (such as TFA).

[0094] In some respects, the washed magnetic beads are redissolved in a solution containing 30% to 95% organic solvent, such as 75% to 85% organic solvent (e.g., alcohol, such as 2-propanol, or acetonitrile), and an organic acid, such as trifluoroacetic acid (TFA), in an amount greater than 0% to 1%, such as 0.25% to 0.75% organic acid.

[0095] In some cases, approximately 80% of 2-propanol containing 0.5% TFA will be used in washing solutions, reconstitution solutions, or both.

[0096] Organic solvents and optional organic acids can be added to the protein sample to achieve a final organic concentration of 70% to 95% or higher, such as 75% to 85% organic solvent, and optionally the organic acid concentration can be adjusted to 0% to 1%, such as 0.25% to 0.57% or about 0.5% organic acid concentration.

[0097] In any aspect of the PAC method, unless otherwise stated, the organic solvent may be any suitable organic solvent, such as alcohols (e.g., 2-propanol, ethanol, methanol, 1-propanol), ester solvents (e.g., ethyl acetate), or acetonitrile, or combinations thereof.

[0098] In any aspect of the PAC method, unless otherwise stated, the organic acid may be any suitable organic acid, such as trifluoroacetic acid (TFA) or formic acid (FA).

[0099] In other respects, organic acids can be replaced by organic bases such as trialkylamines (e.g., triethylamine).

[0100] The disclosed magnetic beads are then added to the protein sample. In some cases, the magnetic beads are added at a weight ratio of 5:1 to 15:1 (such as 8:1 to 12:1, or about 10:1) to the protein. The magnetic beads can be added to a buffer solution having an organic concentration of 70% to 95% (such as 75% to 85%). And / or the protein concentration can be greater than 0 to 5 mg / mL, such as 0.5 mg / mL to 3 mg / mL, or 1 mg / mL to 2 mg / mL.

[0101] After mixing the disclosed magnetic beads with the sample, the resulting mixture can be incubated at a temperature suitable for promoting protein binding (such as 20°C to 30°C, or approximately room temperature). The incubation time is suitable for promoting protein binding, such as 15 minutes to 180 minutes or longer, or 30 minutes to 60 minutes. During incubation, the mixture can be agitated, such as by shaking, vortexing, stirring, or sonication.

[0102] After incubation, the magnetic beads are separated from the mixture. The separated magnetic beads are washed in a solution containing 65% to 100% organic solvent. The magnetic beads may be washed once or more. In some aspects, the magnetic beads are washed with 100% organic solvent, followed by washing with 65% to 95% organic solvent (such as 70% to 80% organic solvent). The organic solvent can be any suitable organic solvent for use in the PAC process. In some aspects, the organic solvent can be acetonitrile or alcohol. In a particular aspect, the magnetic beads are washed with 100% acetonitrile, followed by washing with 70% alcohol (such as ethanol).

[0103] After washing, expose the magnetic beads to the digestion mixture. In some cases, for protein samples of 1 to 2 mg / mL, add 10 μL to 25 μL of the digestion mixture.

[0104] The digestion mixture can be any suitable digestion mixture. In some aspects, the digestion mixture comprises a buffer (such as a trialkylammonium buffer, e.g., triethylammonium bicarbonate (TEAB)), an inorganic salt (such as calcium chloride), and one or more enzymes (e.g., trypsin and / or LysC). The amount of enzyme or enzyme mixture added is from 1:7 to 1:30 (e.g., 1:10 to 1:25) by weight of enzyme to protein.

[0105] The magnetic beads are incubated in the digestion mixture for an appropriate time to promote digestion, such as 1 to 5 hours or longer, or 2.4 to 3.5 hours. During incubation, the mixture may be agitated, such as by shaking, vortexing, stirring, or sonication. Incubation can be carried out at temperatures suitable for promoting digestion, such as 25°C to 50°C, 30°C to 40°C, or 35°C to 40°C.

[0106] After incubation, an organic solvent is added to the mixture to make the concentration of organic matter in the mixture greater than 90%, such as greater than 92% or greater than 92.5%.

[0107] Magnetic beads can be separated and washed using a solution with a high concentration of organic matter (such as greater than 90%, greater than 92%, or at least 95%). In some aspects, the washing solution comprises acetonitrile, an alcohol (such as ethanol), and water, and may further contain an organic acid such as formic acid. In one example, the washing solution comprises about 85% acetonitrile, about 10% ethanol, and about 5% water, and may further contain 0.5% formic acid.

[0108] After washing, the magnetic beads are eluted with an aqueous solution containing 1% to 5% organic solvent (such as 2% to 4% organic solvent) and 0.1% to 0.5% organic acid (such as 0.2% to 0.3% organic acid). In some aspects, the organic solvent is acetonitrile, and / or the organic acid is formic acid.

[0109] After elution, the magnetic beads are removed from the mixture.

[0110] In one aspect, a method for obtaining proteins includes: A first suspension is formed, the first suspension comprising a plurality of magnetic beads and a first liquid containing protein, wherein the first liquid is an aqueous solution comprising 0.5% trifluoroacetic acid and 80% of an organic solvent selected from 2-propanol and acetonitrile; Stir the first suspension at room temperature for 30 to 60 minutes; Remove the magnetic beads from the first suspension and wash them with a second liquid containing an organic solvent; Incubate the magnetic beads with a digestion mixture containing trypsin and LysC for 1 to 4 hours; The magnetic beads were washed with a washing solution containing water, organic solvents and formic acid to remove the digestion mixture; A second suspension comprising magnetic beads and an elution solution is formed, and the second suspension is stirred at room temperature for 1 to 5 minutes; and The magnetic beads are separated from the second suspension to leave a third liquid containing proteins.

[0111] Additionally, in some aspects of the method, each magnetic bead comprises iron oxide particles distributed throughout the azalactone bead, and a plurality of [unclear text - possibly related to surface markings or markings] located on the surface of the magnetic bead. part.

[0112] C. Multi-omics sequential aggregation capture

[0113] Several aspects of the disclosed magnetic beads can be used for the sequential aggregation and capture of samples containing combinations of nucleic acids and proteins. In some aspects, a mixture comprising the disclosed magnetic beads and a sample containing the target molecule is formed. The sample can be cell lysate. In some aspects, each 1 x 10 6 Each cell lysing agent uses 10 mg to 20 mg of the disclosed magnetic beads, such as per 1 x 102 6 Each cell uses approximately 15 mg of the disclosed magnetic beads. Optionally, the magnetic beads can be washed, for example, to remove storage buffer, before mixing with the sample. Several aspects of a method for isolating DNA, protein, and RNA from a mixed sample are described below. Those skilled in the art will understand that these method steps can be used or omitted in any order, depending on the nature of the sample and target molecule provided.

[0114] a. DNA binding

[0115] After the magnetic beads are added to the sample, an organic solvent is added to bring the organic concentration in the sample to 10% to 40%, such as 20% to 35%, 25% to 35%, or about 30%. The organic solvent can be any organic solvent suitable for promoting DNA binding. Unless otherwise specified, the organic solvent can be any suitable organic solvent, such as alcohols (e.g., 2-propanol, ethanol, methanol, 1-propanol) and ester solvents (e.g., acetate esters, such as ethyl acetate) or acetonitrile, or combinations thereof.

[0116] After combining the disclosed magnetic beads with the sample, the resulting mixture can be incubated at a temperature suitable for promoting DNA binding (such as 20°C to 30°C, or approximately room temperature). The incubation time is suitable for promoting DNA binding, such as 15 minutes to 180 minutes or longer, or 30 minutes to 60 minutes. During incubation, the mixture can be agitated, such as by shaking, vortexing, stirring, or sonication.

[0117] After incubation, the DNA-bound magnetic beads are separated from the mixture using a suitable technique, such as a magnetic rack. Remaining lysates may be processed with additional magnetic beads as needed to remove RNA and / or proteins, as disclosed herein.

[0118] DNA-bound magnetic beads are placed in an aqueous solvent containing approximately 20% to 40% organic solvent (such as 25% to 35% organic solvent) and treated with guanidine hydrochloride, a buffer solution, and a chelating agent. The organic solvent can be an alcohol, such as 2-propanol, ethanol, methanol, or 1-propanol. The resulting mixture is incubated with stirring for 5 to 30 minutes. After incubation, the magnetic beads are removed from the liquid using a suitable technique (such as a magnetic rack) and then treated with a second guanidine hydrochloride solution containing a buffer solution, a surfactant, and a chelating agent. The buffer solution can be Tris HCl, PBS, HEPES, triethylammonium bicarbonate (TEAB), or a combination thereof. The surfactant can be Tween, Triton X-100, SDS, or a combination thereof. And / or the chelating agent can be ethylenediaminetetraacetic acid (EDTA).

[0119] After incubation, remove the supernatant, and if necessary, add proteinase K and / or RNase to the supernatant. Incubate the mixture at 40°C to 65°C (such as 50°C to 60°C) for 5 to 30 minutes, while adding water to the magnetic beads to prevent drying. After incubation, remove the water and remix the supernatant with the magnetic beads.

[0120] The mixture was treated with another buffer solution of guanidine hydrochloride containing EDTA and about 60% organic solvent, and the resulting mixture was incubated with stirring at room temperature for 30 to 60 minutes.

[0121] After washing the magnetic beads with a solution containing buffer (such as Tris hydrochloride buffer) and approximately 60% to 80% organic solvent, the DNA is eluted with an aqueous solution containing buffer (such as Tris-HCl) and a chelating agent (such as EDTA). The magnetic beads are then removed using appropriate techniques, and the resulting DNA can be used directly or stored for future applications.

[0122] b. Protein isolation

[0123] Treat lysate samples (such as those following the DNA binding step) with fresh magnetic beads as disclosed herein, along with an organic solvent sufficient to produce a total organic matter concentration of approximately 45%. Organic acids, such as TFA or formic acid, may also be added. Incubate the resulting mixture with agitation for 30 to 60 minutes or longer to promote protein binding. After incubation, remove the magnetic beads using a suitable technique, such as a magnetic rack. The RNA-containing supernatant can be used in the RNA binding protocol described herein.

[0124] The protein-bound magnetic beads are washed once or multiple times (e.g., 1, 2, 3 or more times) with a 70% to 100% organic solvent (e.g., 70% ethanol and / or 100% acetonitrile). After washing, the magnetic beads can be selectively reduced and / or alkylated, such as by treatment with tris(2-carboxyethyl)phosphine (TCEP) and / or 2-chloroacetamide.

[0125] The magnetic beads were then treated with a universal nuclease, followed by trypsin / LysC. After digestion, proteins were eluted as described herein in relation to the PAC method.

[0126] c. RNA binding

[0127] Guanidine hydrochloride is added to a liquid sample containing RNA (such as the RNA-containing supernatant described herein), and the mixture is incubated at 30°C to 40°C for 1 to 20 minutes. A clean sample containing the disclosed magnetic beads is added along with a sufficient amount of organic solvent, such that the sample is at least 85% organic, such as 90% organic solvent. Suitable organic solvents include, but are not limited to, acetonitrile or alcohols (such as 2-propanol, 1-propanol, ethanol, methanol) or combinations thereof.

[0128] The mixture is incubated at room temperature for a sufficient time to promote RNA binding to the magnetic beads, such as 30 to 60 minutes. After incubation, the magnetic beads are separated from the supernatant using a suitable technique, such as a magnetic rack.

[0129] The magnetic beads are treated with a mixture of buffer solution, guanidine hydrochloride, surfactant, and chelating agent. The buffer solution may be PBS, Tris HCl, or a combination thereof. And / or the surfactant may be 10% Tween, Triton X-100, or a combination thereof. And / or the chelating agent may be EDTA.

[0130] After incubation, remove the supernatant, and if necessary, add proteinase K and / or RNase to the supernatant. Incubate the mixture at 40°C to 65°C (such as 50°C to 60°C) for 5 to 30 minutes, while adding water to the magnetic beads to prevent drying. After incubation, remove the water and remix the supernatant with the magnetic beads.

[0131] An organic solvent is added to the magnetic bead / sample mixture to achieve a final organic concentration of 90% in the sample. Suitable organic solvents include, but are not limited to, acetonitrile or alcohols (such as 2-propanol, 1-propanol, ethanol, methanol) or combinations thereof. The mixture is incubated at room temperature with stirring for a time sufficient to promote binding, such as 30 to 60 minutes. The magnetic beads are then washed with a mixture of 60% to 80% organic solvent (such as 65% to 75% organic solvent), a chelating agent, and a buffer. The chelating agent may be EDTA, and / or the buffer may be Tris HCl, PBS, or combinations thereof. Suitable organic solvents include, but are not limited to, acetonitrile or alcohols (such as 2-propanol, 1-propanol, ethanol, methanol) or combinations thereof.

[0132] The RNA was then eluted with water and a chelating agent (such as EDTA), and the magnetic beads were separated from the RNA-containing mixture.

[0133] D. Ion exchange applications

[0134] The disclosed magnetic beads can be used in applications involving the capture of charged substances from biological samples. In an exemplary ion exchange application, a mixture is formed comprising magnetic beads carrying suitable ligands as disclosed herein and a sample containing the target charged substance. For example, in one method, each 1 x 10 6 Cell lysates were prepared using approximately 10 mg to approximately 20 mg of the disclosed magnetic beads, such as per 1 x 103 cells. 6 Each cell uses approximately 15 mg of the disclosed magnetic beads. The magnetic beads may be selectively washed before mixing with the sample to remove storage buffer solutions or other contaminants. In some embodiments, the target charged substance may be released from the magnetic beads for potential use in downstream processes. This document describes several aspects of methods for isolating DNA, proteins, and RNA from a mixed sample, and these method steps may be used or omitted in any order, depending on the nature of the sample and target molecule provided.

[0135] VII. Examples

[0136] Example 1

[0137] Preparation of magnetic beads

[0138] Toluene (100 to 150 mL) was added to a 1-liter Morton round-bottom flask (flask 1) equipped with a top stirrer, nitrogen inlet, and thermocouple, and heated to 40°C with stirring at 200 rpm. After adding the polymer stabilizer (1 g / mL, 282 μL), the temperature was raised to 45°C. Heptane (400 mL) was slowly added to the reaction mixture, ensuring the temperature remained above 40°C throughout. Simultaneously, methylenebisacrylamide (10.5 g, 68 mmol), isopropanol (72.5 mL), and product water (40 mL) were added to a 250 mL round-bottom flask (flask 2) equipped with a magnetic stirrer, nitrogen inlet, and thermocouple, and heated to 30°C.

[0139] After adding heptane to flask 1, vinyl dimethylazanolide (VDM or VDMA) (1.0 g, 7.2 mmol) was added to flask 1. The remaining steps were carried out over 15 minutes. The stirring rate was increased, and then ferric oxide (3 g, 13 mmol) (Fe3O4) was added. Sodium persulfate (0.435 g, 1.8 mmol) dissolved in 8 mL of deionized water (purged with nitrogen for 1 hour before use) was added to flask 2, and the mixture was stirred for 1 to 2 minutes. The magnetic stir bar was removed, and the contents of flask 2 were transferred to flask 1. The final mixture was stirred at 35°C for 5 to 10 minutes, and then pure TEMED (0.435 mL, 3 mmol) was added to initiate polymerization. An exothermic reaction of approximately 5 to 8°C was observed within one minute, followed by the formation of gray / black magnetic beads. The reaction mixture was stirred at 40°C for 2 hours.

[0140] Once the reaction solution has cooled to room temperature, filter the magnetic beads and wash with acetone. Resuspend the beads in acetone and sonicate in an ice bath for 2 to 3 hours. Then sieve the beads using a 63 μm top sieve and a 25 μm bottom sieve, with most beads collected above the 25 μm sieve. Separate the 63–25 μm bead population, transfer it to a 1 L Pyrex bottle, and place it on a magnet (such as one from Sepmag). TM The magnetic beads are then filtered through a magnetic separation system (or equivalent) to remove any non-magnetic residue. The beads are then filtered using a Buchner funnel and transferred to glass vials for drying overnight in a vacuum oven at 35 to 40°C.

[0141] After the magnetic beads swelled in water, they were observed and analyzed under a microscope. Figure 3 shows that this process produced spherical magnetic beads with iron oxide distributed throughout the beads. Particle size analysis was performed using an LS13-320 XRSW particle size analyzer from Beckman Coulter (Indianapolis, IN). Figure 4 shows that the average particle size was approximately 48 μm.

[0142] Figure 5 Figures 7 through 7 provide digital images showing magnetic beads prepared using different magnetic material sources, including iron oxide clusters (Figure 5), iron oxide particles (Figure 6), and Dynabeads. TM (Figure 7). Figures 5 to 7 show that, despite using different magnetic materials, these magnetic beads are all basically spherical and have an average size of 30 μm to 60 μm.

[0143] Example 2

[0144] Preparation of magnetic beads

[0145] Toluene (160 mL) was added to a 1-liter Morton round-bottom flask (flask 1) equipped with a top stirrer, nitrogen inlet, and thermocouple, and heated to 40°C with stirring at 200 rpm. After adding the polymer stabilizer (1 g / mL, 282 μL), the temperature was raised to 45°C. Heptane (400 mL) was slowly added to the reaction mixture, ensuring the temperature remained above 40°C. In parallel, methylenebisacrylamide (20 g), isopropanol (72.5 mL), and product water (40 mL) were added to a 250 mL round-bottom flask (flask 2) equipped with a magnetic stirrer, nitrogen inlet, and thermocouple, and heated to 30°C.

[0146] After adding heptane to flask 1, vinyl dimethylazanolide (VDM or VDMA) (1.0 g, 7.2 mmol) was added to flask 1. The stirring rate was increased to 500 rpm. The iron source and sodium sulfate were added to flask 2 containing MBA and then to flask 1. The final mixture was stirred for 5 to 10 minutes, and then pure TEMED was added to initiate polymerization. An exothermic reaction of approximately 5 to 8°C was observed within one minute, followed by the formation of gray / black magnetic beads. The reaction mixture was stirred at 600 rpm and 40°C for 2 hours.

[0147] Once the reaction solution has cooled to room temperature, the magnetic beads are filtered and washed with acetone. Using the procedure described in Example 1, the magnetic beads are sonicated, sieved, and tested for microscopic analysis and particle size analysis.

[0148] Example 3

[0149] Preparation of magnetic beads

[0150] Toluene (160 mL) was added to a 1-liter Morton round-bottom flask (flask 1) equipped with a top stirrer, nitrogen inlet, and thermocouple, and heated to 40°C with stirring at 400 rpm. After adding the polymer stabilizer (1 g / mL, 282 μL), the temperature was raised to 45°C. Heptane (200 mL) was slowly added to the reaction mixture, ensuring the temperature remained above 40°C throughout. Simultaneously, methylenebisacrylamide (20 g), isopropanol (72.5 mL), and product water (40 mL) were added to a 250 mL round-bottom flask (flask 2) equipped with a magnetic stirrer, nitrogen inlet, and thermocouple, and heated to 30°C.

[0151] After adding heptane to flask 1, add vinyldimethylazanolide (VDM or VDMA) (1.0 g, 7.2 mmol) to flask 1. Increase the stirring speed to 500 rpm, then add Dynabeads... TM (1 g) and sodium sulfate were added to flask 2 containing MBA. The mixture was stirred for 2 minutes and then added to flask 1. The final mixture was stirred for 5 to 10 minutes, and then pure TEMED was added to initiate polymerization. An exothermic reaction of about 5 to 8°C was observed within one minute, followed by the formation of gray / black magnetic beads. The reaction mixture was stirred at 850 rpm and 40°C for 2 hours.

[0152] Once the reaction solution has cooled to room temperature, the magnetic beads are filtered and washed with acetone. Using the procedure described in Example 1, the magnetic beads are sonicated, sieved, and tested for microscopic analysis and particle size analysis.

[0153] Example 4

[0154] Hydrolysis of magnetic beads

[0155] The magnetic beads were hydrolyzed according to the following procedure: 0.50 g of magnetic beads were added to a 15 mL conical tube containing 10 mL of water and incubated overnight at room temperature. The next day, the magnetic beads were washed with water using a suitable magnetic rack. The hydrolyzed magnetic beads were then adjusted to a 25% slurry in water.

[0156] Unless otherwise stated, the solutions used in Examples 5 through 8 are provided in Table 1.

[0157] Table 1. List of Solutions

[0158] Example 5

[0159] Example program (SP2) using the disclosed magnetic beads.

[0160] Use 15 to 150 µg of protein per sample preparation. Wash cultured cells or tissues 2 to 3 times with 1 × PBS solution to remove cell culture medium or excess blood, respectively. Resuspend the protein, cells, or tissue in lysis solution without the need for additional buffer.

[0161] A. Protein extraction, reduction, and alkylation

[0162] 1. For cultured cells, add at least 1 × 10⁻⁶ 6 Add 50 µL of lysis buffer and 1 µL of universal nuclease to each cell. Use a P200 pipette tip to pipette up and down 10–15 times until the sample viscosity decreases.

[0163] Note: Lyse cells in an appropriate volume of lysis buffer to achieve a final protein concentration of 2–5 µg / µL, with final volumes of 7.5 µL, 15 µL, or 30 µL, corresponding to input protein amounts of 15–30 µg, 30–60 µg, or 60–150 µg, respectively. Centrifugation is generally not required after pipetting the cell lysis buffer.

[0164] 2. For tissue samples, add 50 µL of lysis buffer containing 1 µL of universal nuclease per 5 mg of tissue, and homogenize using a tissue homogenizer until the sample is completely homogenized. Homogenize the tissue lysis buffer at 16,000 × 10⁻⁶. g Centrifuge for 10 minutes.

[0165] 3. For purified protein, plasma and serum samples with or without high-abundance protein removal, dilute the samples directly with lysis buffer to 2–5 µg / µL, and adjust the final volume to 7.5 µL, 15 µL or 30 µL, respectively, corresponding to input protein amounts of 10–25 µg, 25–50 µg or 50–100 µg.

[0166] Note: The protein concentration in plasma is typically around 60–70 µg / µL. No universal nuclease needs to be added for purified protein and plasma samples.

[0167] 4. Determine the protein concentration of the supernatant using standard methods, such as the Pierce™ BCA Protein Assay Kit (product number 23227) or the Pierce™ Rapid Gold BCA Protein Assay Kit (product number A53226).

[0168] Note: For high-concentration samples, dilution with lysis buffer may be necessary before BCA determination.

[0169] 5. Transfer 15–150 µg of protein sample to a 1.5-mL low-protein-adsorption centrifuge tube or a 96-well plate, and adjust the final volume to 7.5 µL, 15 µL, or 30 µL using lysis buffer, corresponding to input protein amounts of 15–30 µg, 30–60 µg, or 60–150 µg, respectively.

[0170] 6. Add 3.75 µL, 7.5 µL or 15 µL of reducing solution to the sample, corresponding to protein input amounts of 15–30 µg, 30–60 µg or 60–150 µg respectively, and mix gently.

[0171] 7. Add 3.75 µL, 7.5 µL, or 15 µL of alkylation solution to the sample, corresponding to an input protein amount of 15–30 µg, 30–60 µg, or 60–150 µg, respectively, and mix gently.

[0172] 8. Place the sample in the heating module and incubate at 95°C for 10 minutes, or at 50°C for 20–30 minutes, to complete the reduction and alkylation of the protein sample.

[0173] 9. After incubation, cool the sample to room temperature.

[0174] 10. Briefly centrifuge the centrifuge tubes or 96-well plates before digestion.

[0175] B. Digesting proteins

[0176] 1. Add 150 µL of enzyme reconstitution solution to one bottle of Trypsin / Lys-C protease mixture to prepare an enzyme mixture with a concentration of 0.67 µg / µL.

[0177] 2. Add 3.75 µL, 7.5 µL, or 15 µL of reconstituted enzyme solution to the reduced and alkylated protein sample solution, corresponding to an input protein amount of 15–30 µg, 30–60 µg, or 60–150 µg, respectively.

[0178] 3. Incubate at 37°C with shaking for 1–2 hours to digest the protein sample.

[0179] Note: TMT reagents can be selectively used for labeling before peptide purification in this step.

[0180] C. Purification of peptides

[0181] 1. Transfer 20 µL of magnetic bead slurry to a microcentrifuge tube or 96-well plate and remove the storage buffer using a magnetic rack.

[0182] 2. Add 200 µL of magnetic bead washing / binding buffer to the resin for washing, briefly vortex to mix, and then remove the buffer using a magnetic rack.

[0183] 3. Add the magnetic bead washing / binding solution to the resin and transfer the magnetic bead slurry to the protein digestion sample. For samples with input protein amounts of 15–30 µg, 30–60 µg, or 60–150 µg, the total volume after dilution should be 250 µL, 500 µL, or 1000 µL, respectively.

[0184] 4. Incubate the sample at room temperature for 30–60 minutes.

[0185] 5. Use a magnetic rack to collect the magnetic beads and remove unbound peptide solutions.

[0186] 6. Add 500 µL of washing solution to the resin for washing, briefly vortex to mix, and then remove the buffer solution using a magnetic rack.

[0187] 7. Add 30–100 µL of elution buffer, briefly vortex to mix, and then incubate at room temperature for 3 minutes.

[0188] 8. Use a magnetic rack to transfer the eluted peptide solution to a new container for LC-MS analysis.

[0189] Example 6

[0190] Example program (PAC or multianalyte aggregation capture, protein processing using SP3)

[0191] Note: As long as the ratio of magnetic beads to protein is adjusted proportionally, any amount of protein can be used.

[0192] Wash cultured cells or tissues 2–3 times with 1 × PBS to remove cell culture medium or excess blood. Resuspend proteins, cells, or tissues in lysis buffer, such as the lysis buffer described herein. For protein extraction, various buffer components can be used, including various detergents, salts, and denaturants. For DNA extraction, denaturants should be avoided. For RNA extraction, guanidine hydrochloride should be added to the lysis buffer.

[0193] Pyrolysis and Reduction / Alkylation – General Considerations and Getting Started: 1. For cultured cells, add at least 1 × 10⁻⁶ 6 Add lysis buffer to each cell. Use sufficient lysis buffer to achieve the desired protein concentration of 2–12 mg / mL. Incubate on ice for 15 minutes.

[0194] Note: For 1 x 10 6 Each cell, presumably containing 450 μg protein, 5 μg DNA and 10 μg RNA, was used with 15 mg of the magnetic beads for each sample type.

[0195] Note: For tissue samples, add lysis buffer to the tissue and homogenize using a tissue homogenizer until the sample is completely homogenized.

[0196] Note: For protein extraction only, add 1% (v / v) of universal nuclease to the lysis buffer. If you plan to perform multi-omics sequential extraction of any combination of RNA, DNA, or protein, do not add nuclease to the sample or sonicate it. For this purpose, please proceed directly to the “Binding DNA to Magnetic Beads” section described in Example 5.

[0197] 2. Centrifuge the lysate at 16,000 × g at 4°C for 15 minutes.

[0198] 3. Determine the protein concentration of the supernatant using standard methods, such as the Pierce™ BCA Protein Assay Kit (product number 23227) or the Pierce™ Rapid Gold BCA Protein Assay Kit (product number A53226).

[0199] 4. If necessary, perform reduction and alkylation to achieve a final concentration of 10 mM TCEP and 20 mM 2-chloroacetamide (or an equivalent scheme using dithiothreitol and iodoacetamide). TCEP and 2-chloroacetamide can be used simultaneously, but DTT and iodoacetamide must be used sequentially and quenched with DTT after incubation.

[0200] Note: Reduction and alkylation can be performed before or after the protein-binding magnetic beads. If reduction and alkylation are performed after the protein-binding magnetic beads, heat at 50°C for 45 minutes. If reduction and alkylation are performed before the protein-binding magnetic beads, heat at 95°C for 10 minutes. After incubation, cool the sample to room temperature before adding the magnetic beads or enzyme.

[0201] Proteins are bound to magnetic beads (for SP3 protein treatment only).

[0202] Note: This protocol assumes that the protein is reduced and alkylated at 95°C for 10 minutes before being bound to the magnetic beads, and cooled for 5 minutes before the addition of the magnetic beads.

[0203] Note: For applications that only process proteins, 2-propanol and acetonitrile can be used interchangeably.

[0204] Note: Upstream of the PAC workflow, the chemical steps of cracking, reduction, and alkylation are compatible.

[0205] 1. Vortex the magnetic beads to ensure complete suspension.

[0206] 2. Remove the supernatant from the magnetic beads and wash in 500 µL of 80% 2-propanol and 0.5% TFA. Remove the washing solution.

[0207] 3. Resuspend the magnetic beads in 80% 2-propanol and 0.5% TFA to [50 μg / μL].

[0208] 4. Add 99.5% 2-propanol and 0.5% TFA to the protein solution to achieve a final organic concentration of 80% 2-propanol and 0.5% TFA.

[0209] 5. Add magnetic beads (magnetic beads: protein = 10:1, by weight) to the protein sample in the buffer solution to achieve an organic concentration of 80% and a protein concentration of 1–2 mg / mL.

[0210] 6. Incubate at room temperature for 45 minutes, mixing thoroughly to keep the magnetic beads suspended, and then remove the effluent.

[0211] 7. Add 100% MeCN, equivalent to 4 times the volume of the magnetic beads, and vortex thoroughly to mix. Remove the washing liquid.

[0212] 8. Add 70% EtOH in a volume equivalent to four times the volume of the magnetic beads and vortex thoroughly to mix. Remove the washing solution.

[0213] 9. Remove the centrifuge tube from the magnetic rack and add 10–25 µL of digestion mixture to the magnetic beads. This mixture consists of 50 mM TEAB, 10 mM CaCl2, and trypsin and LysC in a 1:10–1:25 (enzyme:protein, weight ratio) ratio.

[0214] 10. Briefly vortex mix and incubate for 3 hours in a thermostatic mixer at 37°C and 1200 rpm.

[0215] 11. After incubation, briefly centrifuge to collect any evaporated liquid.

[0216] 12. Add 100% acetonitrile to the sample and the magnetic bead eluent to bring the final organic concentration to at least 92.5%.

[0217] Note: Starting from this step, SP2 is also compatible.

[0218] 13. Vortex the sample for 30 seconds. The total peptide concentration should be at least 0.2 mg / mL.

[0219] 14. Incubate the sample at room temperature for 10 minutes.

[0220] 15. Collect the magnetic beads and discard the supernatant.

[0221] 16. Wash the sample twice with a solution of 85% acetonitrile, 10% ethanol, 5% water, and 0.5% formic acid, which is equivalent to four times the volume of the magnetic beads.

[0222] 17. Collect the magnetic beads and discard the supernatant.

[0223] 18. Elute the sample with an aqueous solution of 2–4% MeCN and 0.2% FA, which is twice the volume of the magnetic beads, and vortex for 2 minutes (per 15 mg of magnetic beads).

[0224] 19. Place the centrifuge tube on a magnetic rack for 2 minutes and transfer the supernatant to a clean centrifuge tube. Centrifuge the sample at 10,000 × g for 30 seconds, and then transfer the sample to a new centrifuge tube without disturbing the precipitate.

[0225] 20. Quantify using a fluorescence or colorimetric peptide quantification kit or UV-Vis and / or proceed to downstream applications.

[0226] Example 7

[0227] Multi-omics sequential aggregation capture

[0228] DNA is bound to magnetic beads

[0229] Note: This protocol is for sequential elution of DNA-protein-RNA. Other sequences can also be achieved using these magnetic beads. For protein-DNA extraction only, first use 2% SDS, 100 mM TEAB, 1 mM EDTA, followed by reduction and alkylation with TCEP (10 mM) and 2-chloroacetamide (20 mM), and then bind the protein in 80% 2-propanol and 0.5% TFA. Collect the eluent and dilute with water to 30–40% 2-propanol to bind the DNA. The presence of CH6ClN3 (guanidine hydrochloride) significantly alters the binding properties of each analyte on these magnetic beads; therefore, the organic solvent concentration will vary depending on the timing and concentration of CH6ClN3 addition.

[0230] 1. Dilute the sample 1:1 with 0.2% SDS and 100 mM TEAB. Incubate on ice for 15 minutes.

[0231] 2. Wash 15 mg of the magnetic beads (corresponding to each 1 x 10⁻⁶) with 100% 2-propanol. 6 (cell lysate).

[0232] 3. Add the required amount of magnetic beads to the lysis solution.

[0233] 4. Add sufficient 2-propanol to the sample to bring the total organic concentration to 30%.

[0234] 5. Combine at room temperature for 45 minutes and mix thoroughly to keep the magnetic beads suspended.

[0235] 6. Place the centrifuge tube on the magnetic rack and transfer the lysate, now containing RNA and protein, to the “binding protein” section of this protocol.

[0236] Continue with the following DNA processing steps using only magnetic beads.

[0237] 7. For every 15 mg of magnetic beads, add approximately 250 μL of 30% 2-propanol, 4.2 M CH6ClN3, 150 mM Tris-HCl, and 20 mM EDTA. Remove from the magnetic rack and incubate at room temperature for 10 minutes, mixing thoroughly to keep the magnetic beads suspended.

[0238] 8. Place it on the magnetic rack and discard the supernatant.

[0239] 9. Add the following formulation to 100 μL for every 15 mg of magnetic beads: 0.5× PBS, 28% CH6ClN3, 10% Tween, 0.5% Tris-HCl, 0.5% EDTA, 0.5% Triton X-100. Invert several times to mix thoroughly with the magnetic beads, then place on a magnetic rack.

[0240] 10. Transfer the supernatant to a new centrifuge tube. If necessary, add proteinase K and RNase, invert several times to mix, and incubate at 55 °C and 400 rpm for 10 minutes.

[0241] Note: Add water to the magnetic beads during incubation to prevent them from drying out.

[0242] 11. After incubation, remove the moisture from the magnetic beads and add the sample back into the magnetic beads.

[0243] 12. Add equal volumes of 60% 2-propanol, 2.4 M CH6ClN3, 150 mM Tris-HCl, and 20 mM EDTA to the magnetic bead / sample mixture and bind for 45 minutes at room temperature, while mixing thoroughly to keep the magnetic beads suspended.

[0244] 13. Wash the DNA-bound magnetic beads twice with 200 μL 70% EtOH, 1 mM EDTA, and 25 mM Tris-HCl (per 15 mg of magnetic beads).

[0245] 14. Elution was performed using 50 μL of 25 mM Tris-HCl and 0.1 mM EDTA (per 15 mg magnetic beads).

[0246] 15. Place on a magnetic rack and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0247] 16. Centrifuge at 12,000 × g at 4°C for 2 minutes.

[0248] 17. Transfer the sample to a new centrifuge tube without magnetic beads.

[0249] 18. Add EDTA to 1 mM and store at 4°C until downstream application.

[0250] Protein binding to magnetic beads (for sequential methods only)

[0251] 1. Retrieve the effluent from "DNA binding to magnetic beads" or "lysis and reduction / alkylation".

[0252] 2. Wash the new 30 μL (15 mg) magnetic beads with 200 μL of 99.5% 2-propanol and 0.5% trifluoroacetic acid, and then remove the washing solution.

[0253] 3. Transfer the sample to 15 mg of the magnetic beads (corresponding to each 1 × 10⁻⁶ mg / mL). 6 (cells).

[0254] 4. Add sufficient 99% 2-propanol and 1% TFA to bring the total organic concentration to 45%.

[0255] 5. Combine at room temperature for 45 minutes and mix thoroughly to keep the magnetic beads suspended.

[0256] 6. Place on a magnetic rack and transfer the supernatant containing RNA to a new 1.5 mL centrifuge tube.

[0257] a. Add 0.1 mM EDTA to the supernatant containing RNA and store it at 4°C for a short period of time.

[0258] 7. Wash with 200 μL of 70% EtOH and briefly vortex to mix (per 15 mg of magnetic beads).

[0259] 8. Wash with 200 μL of 100% MeCN and briefly vortex to mix (per 15 mg of magnetic beads).

[0260] 9. Resuspend the protein bound to the magnetic beads in 40 μL of 100 mM TEAB and 10 mM CaCl2.

[0261] a. If not performed before binding with magnetic beads, reduce and alkylate using a total of 10 μL of TCEP and 2-chloroacetamide. The final concentration is 20 mM 2-chloroacetamide and 10 mM TCEP. Vortex the sample and incubate at 50°C for 45 minutes.

[0262] b. If the sample has been pre-reduced and alkylated, add 50 μL of 100 mM TEAB and 10 mM CaCl2 to the magnetic beads instead of 40 μL.

[0263] 10. Add 0.6 μL of universal nuclease. Mix thoroughly by repeatedly pipetting up and down.

[0264] 11. Remove one bottle of Trypsin / LysC from -20°C and thaw for 5 minutes.

[0265] a. Redissolve the reagent vial in 50 μL of 0.1% CH3COOH to a concentration of 2.0 mg / mL. Mix gently and let stand for 5 minutes.

[0266] b. Add enzyme: protein = 1:10–1:25 (by weight).

[0267] c. Incubate for 2 hours in a thermostatic mixer at 37°C and 1200 rpm.

[0268] Continue with the following steps to complete the SP3 protein preparation process. Alternatively, the SP2 preparation method is also compatible and can be used as an alternative to this procedure, and can still be used as SP3 (adjusted according to the amount of 15 mg magnetic beads).

[0269] 12. Add sufficient 2-propanol to achieve an organic concentration of >92.75% and begin peptide purification.

[0270] a. Incubate the sample at room temperature and 1200 rpm for 10 minutes.

[0271] b. Collect the magnetic beads and discard the supernatant.

[0272] c. Wash the sample twice with 200 μL of 100% HPLC grade MeCN (or use 85% MeCN, 10% EtOH, 5% water, and 0.5% formic acid to improve salt removal, for example, for TMT or TMTpro labeled samples).

[0273] d. Collect the magnetic beads and discard the supernatant.

[0274] e. Elute the sample with 50 μL of 2–4% MeCN and 0.2% FA aqueous solution for 2 minutes, and vortex intermittently to mix.

[0275] f. Place on a magnetic rack for 2 minutes, and transfer the supernatant to a clean centrifuge tube.

[0276] g. The sample was centrifuged at 10,000 × g for 30 seconds.

[0277] h. Transfer the sample to a new centrifuge tube without disturbing the precipitate.

[0278] 13. Quantify using a fluorescence or colorimetric peptide quantification kit or UV-Vis, and / or proceed to downstream applications.

[0279] RNA is bound to magnetic beads

[0280] 1. Retrieve the RNA effluent (containing 45% 2-propanol and RNA) from storage conditions at 4°C.

[0281] 2. Add 50 mg of dried CH6ClN3 to 200 μL of sample to achieve 2.5 M CH6ClN3, and incubate at 37°C and 1200 rpm for 10 minutes.

[0282] 3. Wash 30 μL of the fresh magnetic beads described herein with 200 μL of 2-propanol.

[0283] 4. Transfer 200 μL of sample to 15 mg of dry magnetic beads.

[0284] 5. Add approximately 1200 μL of 2-propanol to bring the final organic concentration of the sample to 90%.

[0285] 6. Combine at room temperature for 45 minutes and mix thoroughly to keep the magnetic beads in suspension.

[0286] 7. Place on a magnetic rack and discard the supernatant.

[0287] 8. Add the following formulation to 100 μL for every 15 mg of magnetic beads: 0.5× PBS, 28% CH6ClN3, 10% Tween, 0.5% Tris-HCl, 0.5% EDTA, 0.5% Triton X-100. Invert several times to mix thoroughly with the magnetic beads, then place on a magnetic rack.

[0288] 9. Remove the supernatant containing RNA from the magnetic beads and transfer it to a new 1.5 mL centrifuge tube.

[0289] a. Resuspend the magnetic beads in 200 μL of water.

[0290] 10. Transfer the supernatant containing RNA to a new centrifuge tube. If necessary, add proteinase K and RNase, invert several times to mix, and incubate at 55 °C and 400 rpm for 10 minutes.

[0291] Note: Add water to the magnetic beads during incubation to prevent them from drying out.

[0292] 11. After incubation, remove the moisture from the magnetic beads and add the sample back into the magnetic beads.

[0293] 12. Add 2-propanol to the magnetic bead / sample mixture to bring the final organic concentration of the sample to 90%, and bind at room temperature for 45 minutes while mixing thoroughly to keep the magnetic beads suspended.

[0294] 13. Wash the RNA-bound magnetic beads twice with 400 μL of 70% EtOH, 1 mM EDTA, and 25 mM Tris-HCl (per 15 mg of magnetic beads) (do not vortex!).

[0295] 14. Elute with 50 μL water and 0.1 mM EDTA (per 15 mg magnetic beads).

[0296] 15. Extract and purify the RNA from the magnetic beads, place it in a new 1.5 mL centrifuge tube, and proceed to downstream applications.

[0297] Example 8

[0298] Comparative experiment

[0299] To compare the magnetic beads with other commercially available magnetic beads, Thermo Fisher Scientific was used. TM King Fisher TM A comparative procedure was developed for the sample purification system. Table 2 lists the magnetic beads used in the comparative experiments.

[0300] Table 2. Magnetic beads used in the comparative experiment

[0301] Each magnetic bead was used with 25 μg of HeLa cell lysis buffer sample. The comparison workflow is as follows: A. Protein digestion (non-KingFisher step) Reduction / alkylation: Heat at 95°C for 10 minutes, cool to room temperature, and centrifuge for a few seconds.

[0302] Digestion: Shake at 37°C and 1,000 rpm for 1 hour.

[0303] Trypsin / Lys-C: 0.67 μg / μL (100 μg trypsin / Lys-C was reconstituted in 150 μL of 0.1% acetic acid / 10 mM CaCl2 for use, with a sample to trypsin ratio of 10:1).

[0304] Table 3. Sample / Reagent Volume for Protein Digestion

[0305] B. Peptide purification (performed on KingFisher) Magnetic bead pretreatment: Add 20 μL (5.0 mg) of magnetic beads to 80 μL of magnetic bead washing / binding buffer. Wash the magnetic beads with 200 μL of magnetic bead washing / binding buffer.

[0306] Dilution: Transfer the magnetic beads to the diluted sample. The total volume is 500 μL (organic phase >9% after dilution).

[0307] Combination: Mix the sample / magnetic beads at a medium speed for 45 minutes at room temperature.

[0308] Washing: Transfer the sample / magnetic beads to 500 μL of washing buffer for peptide washing. Mix the plate at medium speed for 2 minutes at room temperature.

[0309] Elution: Transfer the sample / magnetic beads to 100 μL of elution buffer for peptide elution. Mix the plate at medium speed for 5 minutes at room temperature. Collect the eluted peptide sample for LC-MS analysis.

[0310] Table 4. Peptide purification volume

[0311] The experimental results are shown in Figures 8A, 8B, and 9–11. Figures 8A and 8B show that, in the SP2 workflow, the surface adsorption loss of the magnetic beads is lower than that of Sera-Mag. TM Carboxyl-modified particles, MyOne TM Or silicone magnetic beads. SeraMag TM MyOne TM Both silica gel magnetic beads and the magnetic beads exhibited significant magnetic bead loss on the comb-shaped pick plate and sample plate surface (magnetic bead transfer / mixing and bonding steps). In contrast, the magnetic beads did not exhibit significant magnetic bead loss. The data for the magnetic beads in Figures 8A and 8B refer to hydrolyzed magnetic beads, i.e., magnetic beads whose surface azalactone groups have been hydrolyzed. However, similar results were obtained using the magnetic beads with azalactone groups treated with aminobenzoic acid or aminoethyltrimethylammonium chloride (AETMA) (data not shown).

[0312] Figure 9 shows that, using a quantitative colorimetric peptide assay, the peptide yield of the magnetic beads in the KingFisher SP2 workflow was significantly higher than that of the Sera-Mag. TM MyOne TM Or silicone magnetic beads. Using silicone magnetic beads did not recover any peptides.

[0313] Figure 10 shows that the number of unique peptides identified using the magnetic beads via nano-LCMS analysis was significantly greater than that of other types of magnetic beads. Figure 11 shows that the number of proteomes identified using the magnetic beads was significantly greater.

[0314] Example 9

[0315] An experiment was designed to demonstrate that the magnetic beads, when coupled with alkali-resistant protein A (asPA), exhibit superior binding performance compared to commercially available magnetic beads containing asPA conjugates.

[0316] Couple asPA to the magnetic bead

[0317] The alkali-resistant protein A is coupled to the azalactone magnetic beads according to the following steps: 1. Weigh 77 mg of the azalactone magnetic beads.

[0318] 2. Dilute asPA (20 mg / mL, 250 mM sodium acetate, pH 5.5, 0.5 M NaCl, 0.05% Tween-20) at a 1:3 (v / v) ratio in coupling buffer (0.1 M sodium carbonate, 1.2 M sodium citrate, pH 9); the final component concentration is 5 mg / mL asPA, 0.075 M sodium carbonate, 0.9 M sodium citrate, pH 9.

[0319] 3. Add 3.6 mL of asPA dissolved in coupling buffer to the azalactone magnetic beads.

[0320] 4. Couple asPA with magnetic beads overnight at room temperature.

[0321] 5. Wash the asPA-magnetic beads with 2-3 mL of water.

[0322] 6. Block the asPA-magnetic beads with 2–3 mL of 3 M ethanolamine for 2 hours.

[0323] 7. Wash the asPA-magnetic beads with 2-3 mL of water.

[0324] 8. Wash the asPA-magnetic beads twice with 2-3 mL of 0.1M glycine pH 2 (Pierce IgG elution buffer, #21028).

[0325] 9. Wash the asPA magnetic beads three times with 2-3 mL of water.

[0326] 10. Use 20% ethanol to slurry asPA-magnetic beads to 25%.

[0327] The binding capacity of asPA-azalactone beads was measured by comparing them with asPA-magnetic agarose beads.

[0328] The procedure for measuring the binding capacity of asPA-azalactone beads by comparing them with asPA-magnetic agarose beads (Pierce High Capacity Protein A MagBeads, alkali stable, #A53036, 25% slurry) is as follows: 1. Prepare 3 mg / mL rabbit IgG (RIgG) in PBS.

[0329] 2. Add 2 x 50uL of 25% magnetic bead slurry (asPA-azalactone beads and asPA-magnetic agarose beads) to a 1.5mL microcentrifuge tube.

[0330] 3. Wash the magnetic beads twice with 500uL PBS.

[0331] 4. Add 500 μL of 3 mg / mL (RIgG) to the magnetic beads and incubate at room temperature for 1 hour with shaking / mixing.

[0332] 5. Collect and preserve the effluent for analysis.

[0333] 6. A standard curve for RIgG ranging from 0 to 3 mg / mL was created by measuring the absorbance at 280 nm.

[0334] 7. Measure the absorbance of the RIgG starting solution and the effluent from each magnetic bead sample.

[0335] 8. Use the standard curve to calculate the concentration of the initial solution and the concentration of the effluent.

[0336] 9. Calculate the RIgG bound to the magnetic beads by subtracting the RIgG in each effluent from the initial RIgG. Extrapolate to the amount of bound RIgG per mL of settled magnetic beads.

[0337] result

[0338] The results clearly show that asPA coupled with the disclosed magnetic beads has an RIgG binding capacity that is approximately 15% higher than that of asPA magnetic agarose beads (Figure 12). Without being bound by specific theories, this may be at least partly due to the fact that the disclosed azalactone magnetic beads can couple a greater amount of asPA than the same amount of magnetic agarose beads. Furthermore, in some respects, the amount of asPA bound with the disclosed azalactone magnetic beads is approximately twice as high as the amount bound with the same amount of magnetic agarose beads.

[0339] Example 10

[0340] Preparation of magnetic beads for anion exchange

[0341] Anion exchange magnetic beads were prepared according to the following procedure: 0.50 g of magnetic beads and 0.50 g of 1,5-pentanediamine were added to a 50 mL conical tube containing citrate / carbonate buffer (pH 8-12, 30 mL) and incubated overnight at room temperature. The next day, the beads were washed three times with water using a suitable magnetic rack. After washing, the beads were adjusted to a slurry concentration of 25% in water. Other types of anion exchange magnetic beads can be prepared using different types of diamines. Cation exchange magnetic beads can be prepared similarly using different ligands (such as sulfonic acids).

[0342] Example 11

[0343] Preparation of magnetic beads for enriching phosphorylated peptides

[0344] Magnetic NTA beads were prepared according to the following procedure. 0.50 g of magnetic beads and 1.0 g of amino-linker-NTA or amino-linker-IDA (e.g., 6-amino-carboxyhexyliminodiacetic acid) were added to a 50 ml conical tube containing a suitable buffer (pH 8–12) and incubated overnight at room temperature. The next day, the magnetic beads were washed three times with water using a suitable magnetic rack. The magnetic NTA beads could then be loaded with a suitable metal (e.g., Fe, Ti, Zr, or Ga) for enriching phosphorylated peptides from simple or complex mixtures using immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC).

[0345] Magnetic NTA beads are loaded with FeCl2 or FeCl3 to produce magnetic Fe-NTA beads. Using a binding buffer, the magnetic Fe-NTA beads can bind to phosphorylated peptides in protein digests (such as cells, tissues, cerebrospinal fluid, peptide mixtures, or other sources), and the phosphorylated peptides are eluted using an elution buffer that uses buffers known to those skilled in the art. The sample can then be analyzed using mass spectrometry, and phosphorylation modifications can be searched using Proteome Discover software to measure phosphorylated peptides and phosphorylation specificity in a given sample.

[0346] Magnetic NTA beads can also be immobilized with appropriate metals (such as nickel (Ni) or cobalt (Co)) for the purification of His-tagged proteins and analysis using the procedures described above.

[0347] Example 12

[0348] Extracellular vesicle capture

[0349] Isolation of extracellular vesicles (EVs) from cell culture supernatant

[0350] 1. Add 2 mL of equilibration buffer (EquilB, 50 mM HEPES, 100 mM NaCl, pH 8) to a 5 mL tube.

[0351] 2. Add 100-200 μL of 25% anion exchange magnetic beads to EquilB. Vortex mix for 5 seconds, collect the beads on a magnetic rack, and remove EquilB.

[0352] 3. Add 2 mL of EquilB to the magnetic beads and incubate at room temperature for 2 minutes. Collect the magnetic beads and remove the EquilB.

[0353] 4. Add 3 mL of clear cell culture supernatant (Expi293F) to the magnetic beads and incubate for 1 hour, while mixing end to end on a tube rotator at 25 rpm.

[0354] 5. Collect the magnetic beads and remove the effluent.

[0355] 6. Wash the magnetic beads once with 3 mL of washing buffer (WB, 50 mM HEPES, 250 mM NaCl, pH).

[0356] 7. Gently flip and mix end to end 3-5 times until the magnetic beads are completely suspended.

[0357] 8. Collect the magnetic beads and remove the WB. Transfer the magnetic beads to a 1.5 mL microcentrifuge tube using 2 x 0.3 mL WB.

[0358] 9. Collect the magnetic beads and remove the WB.

[0359] 10. Add 0.3 mL of elution buffer (EB, 50 mM HEPES, 1 M NaCl, pH 8) to the magnetic beads. Mix at 1200 rpm for 10 minutes at room temperature in a thermostat.

[0360] 11. Collect the magnetic beads and preserve the eluent for analysis.

[0361] Identification of EV markers (CD81) and contaminants (calcitonin) in isolated EVs using Western blotting. white)

[0362] 1. Prepare gel samples by mixing the eluent with 5X SDS loading buffer.

[0363] 2. After volume normalization, separate the eluent on a 4-20% SDS-PAGE gel.

[0364] 3. Using an Invitrogen Power Blotter, electroblot at a constant 5A and 25V for 10 minutes to transfer the gel to a nitrocellulose membrane.

[0365] 4. Cut the membrane in half and use each half to detect different targets.

[0366] 5. Use a solution of 10% BSA blocking solution and TBS containing 0.05% Tween-20 mixed at a ratio of 1:1, and seal the membrane at room temperature for 1 hour.

[0367] 6. Incubate the membrane with a primary antibody (anti-calcinin or anti-CD81) overnight at 4°C.

[0368] 7. Wash the membrane with TBST 5 times, 5 minutes each time.

[0369] 8. Dilute the horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse (H+L) secondary antibodies (Thermo Fisher Scientific, #32460 and #32430, respectively) at a ratio of 1:50,000 in blocking buffer: TBST (1:4). Incubate the membrane in the secondary antibody at room temperature for 30 minutes.

[0370] 9. Wash the membrane with TBST 5 times, 5 minutes each time.

[0371] 10. Incubate the membrane for 5 minutes at room temperature with SuperSignal West Dura persistent substrate (Thermo Fisher Scientific, #34076).

[0372] 11. Scan the membrane on an Invitrogen iBright FL1500 imaging system (Thermo Fisher Scientific, #A44115) and perform chemiluminescence detection simultaneously.

[0373] Total protein in isolated EVs was identified by gel electrophoresis.

[0374] 1. Prepare gel samples by mixing the eluent with 5X loading buffer.

[0375] 2. After volume normalization, separate the eluent on a 4-20% SDS-PAGE gel.

[0376] 3. Stain the gel with GelCode Blue Safe protein staining solution (Thermo Fisher Scientific, #24594) for 1 hour.

[0377] 4. Decolorize the gel in water and scan it.

[0378] result

[0379] The above protocol was used to compare magnetic beads coupled with three ligands (1,5-pentanediamine (PDA), PEI-800, and PEI-25K) at two different pH levels (pH 7 and pH 9) as described in Example 10. The results showed that multiple ligands (including but not limited to 1,5-pentanediamine (PDA), PEI-800, and PEI-25K) could be successfully used for EV separation. As seen in the gel image (Figure 13), anion exchange magnetic beads with three different ligand chemical structures were successfully used to separate EVs from Expi293 cell culture supernatant. It was found that the type of buffer and pH used to prepare the magnetic bead-ligand conjugates affected the yield of separated EVs. Compared to PBS (pH 7), using sodium citrate / sodium carbonate (pH 9) buffer for PDA-magnetic bead coupling yielded a higher EV marker CD81 yield. Conversely, using PBS with a neutral pH (approximately pH 7) to couple PEI ligands to magnetic beads yielded higher CD81 yields. Unintentionally, the higher EV capture yield of the magnetic beads can be attributed to the higher ligand density on the beads. While all the ligands tested in this experiment effectively separated EVs, smaller organic ligands (PDAs) were found to have some advantages over the polymeric ligands PEI-800 or PEI-25K. Overall, PDA beads yielded the highest EV yield with the lowest background. Calcinin (a contaminant) was found to be present in PEI ligands but negligible in PDA ligands. Furthermore, numerous protein bands were visible in PEI samples stained with Coomassie Brilliant Blue gels, while only a few bands were visible in PDA samples.

[0380] Given the many embodiments to which the principles of this disclosure may be applied, it should be understood that the illustrated embodiments are merely preferred embodiments of this disclosure and should not be construed as limiting the scope of this disclosure. Rather, the scope of this disclosure is defined by the following claims. We therefore assert that all content falling within the scope and spirit of these claims is within the protection scope of this disclosure.

Claims

1. A method comprising: forming a first suspension comprising a plurality of magnetic beads and a first liquid containing biomolecules; removing the magnetic beads from the first suspension and optionally washing the magnetic beads with a second liquid; forming a second suspension comprising the magnetic beads and an elution liquid; and removing the magnetic beads from the second suspension to leave a third liquid comprising the biomolecules; 2. The method of claim 1, wherein the magnetic material comprises an iron source. wherein each magnetic bead comprises a cross-linked polymer and a magnetic material, wherein the cross-linked polymer comprises one or more azlactone groups or one or more functional groups derived from the azlactone group, optionally from the reaction of the azlactone group with a nucleophilic reactive group.

3. The method of claim 2, wherein the iron source comprises iron oxide.

4. The method of any one of claims 1 to 3, wherein the magnetic material is contained within agarose beads and the agarose beads are contained within a cross-linked polymer.

5. The method of any one of claims 1 to 4, wherein the magnetic material is in the form of particles, powder, flakes, or clusters.

6. The method of any one of claims 1 to 5, wherein the cross-linked polymer is a cross-linked azlactone polymer and the magnetic beads are azlactone beads.

7. The method of any one of claims 1 to 6, wherein the magnetic beads comprise one or more functional groups derived from the reaction of an azlactone group with an amine-containing compound selected from a biomolecule, an organic ligand, a polymer, a dendrimer, or a combination thereof.

8. The method of claim 6, comprising: iron oxide particles distributed throughout the azlactone beads; and a plurality of functional groups located on the surface of the magnetic beads.

10. The method of any one of claims 1 to 9, wherein the magnetic material comprises Fe3C>4, Fe2C>3, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein the azlactone beads are formed from a vinyl azlactone and a cross-linking agent selected from bisacrylamide, agarose, and vinyl ether. wherein the functional group is selected from , , , , , , , .

9. The magnetic bead of claim 8, wherein the functional group comprises .

12. The method of claim 11, wherein the vinyl azlactone is 4,4-dimethyl-2-vinyl oxazol-5(4H)-one.

13. The method of claim 11 or 12, wherein the cross-linking agent is methylene bisacrylamide.

14. The method of any one of claims 1 to 13, wherein the biomolecule is a protein, a peptide, a polypeptide, a DNA, or an RNA molecule.

15. The method of claim 14, wherein the biomolecule is labeled with a stable isotope, an isotope-enriched mass tag, a stable isotope-labeled peptide and / or protein, a rare earth metal, a fluorescent label, a tissue and / or cell cultured with a heavy amino acid, or a combination thereof.

16. The method of claim 15, wherein the biomolecule is labeled with a stable isotope selected from deuterium, carbon-13, nitrogen-15, oxygen-18, or a combination thereof.

17. The method of any one of claims 1 to 16, wherein the biomolecule is a protein, a polypeptide, or a peptide and the first liquid has an organic concentration of 80% or greater. ​ ​ 18. The method of claim 17, wherein the first liquid comprises 80% or more 2-propanol, acetonitrile, or a combination thereof.

19. The method of claim 17 or 18, wherein the elution liquid comprises greater than zero to 0.5% formic acid in water.

20. The method of any one of claims 17 to 19, wherein the first liquid is a protein sample.

21. The method of claim 20, wherein after washing the magnetic beads with a second liquid and before adding an elution liquid, the method further comprises: incubating the magnetic beads with a digestion mixture; and after incubation, washing the magnetic beads to remove the digestion mixture.

22. The method of any one of claims 17-19, wherein the first liquid is a protein digest sample.

23. The method of any one of claims 1 to 16, wherein the biomolecule is DNA.

24. The method of claim 23, wherein the second liquid comprises from 25% to 35% of an organic solvent and a chelating agent, guanidine hydrochloride, a buffer, or a combination thereof.

25. The method of claim 24, wherein before forming the second suspension, the method further comprises: separating the magnetic beads from the second liquid; adding to the magnetic beads a surfactant mixture comprising a buffer, guanidine hydrochloride, a chelating agent, and a surfactant and agitating; separating the magnetic beads from the surfactant mixture and adding to the surfactant mixture proteinase K and / or RNase, agitating and remixing with the magnetic beads; adding yet another mixture comprising from 50% to 70% of an organic solvent in water, a chelating agent, guanidine hydrochloride, and a buffer; and washing the magnetic beads with a mixture comprising from 65% to 80% of an organic solvent, a chelating agent, and a buffer.

26. The method of any one of claims 23 to 25, wherein the elution solvent comprises a buffer and a chelating agent.

27. The method of any one of claims 24 to 26, wherein the chelating agent is EDTA.

28. The method of any one of claims 24 to 27, wherein the buffer is a tris HC1 buffer, PBS, or a combination thereof.

29. The method of any one of claims 24 to 28, wherein the organic solvent is 2-propanol, ethanol, or a combination thereof.

30. The method of any one of claims 24 to 29, wherein the surfactant is Tween, Triton X-100, or a combination thereof.

31. The method of any one of claims 1 to 16, wherein the biomolecule is RNA.

32. The method of claim 31, wherein the first liquid has an organic concentration of 85% or more.

33. The method of claim 31 or 32, wherein the eluent comprises a buffer, guanidine hydrochloride, a chelating agent, and a surfactant.

34. The method of claim 33, wherein: the buffer is PBS, Tris HC1, or a combination thereof; the chelating agent is EDTA; the surfactant is Tween, Triton X-100, or a combination thereof; or a combination thereof.

35. The method of claim 33 or 34, further comprising adding proteinase K and / or RNase to the third liquid.

36. The method of claim 35, further comprising remixing the third liquid with the magnetic beads to form a third suspension and adjusting the organic concentration of the third suspension to at least 85%.

37. The method of claim 36, further comprising washing the magnetic beads with a solution comprising an organic solvent, a chelating agent, and a buffer.

38. The method of claim 37, wherein the organic solvent is ethanol, the chelating agent is EDTA, the buffer is Tris HC1, or a combination thereof.

39. The method of claim 37 or claim 38, further comprising eluting the RNA from the magnetic beads with an aqueous EDTA solution.

40. The method of any one of claims 1 to 39, wherein the method further comprises analyzing the biomolecule by mass spectrometry.

41. A method of obtaining a peptide or polypeptide for LC-MS analysis, the method comprising: forming a first suspension comprising a plurality of magnetic beads and a first liquid, the first liquid containing a protein digestion sample comprising a peptide or polypeptide; stirring the first suspension at room temperature for 30 to 60 minutes; removing the magnetic beads from the first suspension and washing the magnetic beads with a second liquid; forming a second suspension comprising the magnetic beads and an eluent and stirring the second suspension at room temperature for 1 to 5 minutes; and separating the magnetic beads from the second suspension to leave a third liquid comprising the peptide or polypeptide; wherein each magnetic bead comprises iron oxide particles distributed throughout the azlactone bead and a plurality of groups located on the surface of the magnetic bead.

42. A method of obtaining a protein, the method comprising: forming a first suspension comprising a plurality of magnetic beads and a first liquid containing a protein, wherein the first liquid is an aqueous solution containing 0.5% trifluoroacetic acid and 80% of an organic solvent selected from 2-propanol and acetonitrile; stirring the first suspension at room temperature for 30 to 60 minutes; removing the magnetic beads from the first suspension and washing the magnetic beads with a second liquid comprising an organic solvent; incubating the magnetic beads with a digestion mixture comprising trypsin and LysC for 1 to 4 hours; washing the magnetic beads with a wash solution consisting of water, an organic solvent, and formic acid to remove the digestion mixture; forming a second suspension comprising the magnetic beads and an eluent and stirring the second suspension at room temperature for 1 to 5 minutes; and separating the magnetic beads from the second suspension to leave a third liquid comprising the protein; wherein each magnetic bead comprises iron oxide particles distributed throughout the azlactone bead and a plurality of groups located on the surface of the magnetic bead.

43. A magnetic bead comprising a cross-linked polymer and a magnetic material, wherein the cross-linked polymer comprises one or more azlactone groups or one or more functional groups derived from an azlactone group, optionally from the reaction of an azlactone group with a nucleophilic reactive group.

44. The magnetic bead of claim 43, wherein the magnetic material comprises a source of iron.

45. The magnetic bead of claim 44, wherein the iron source comprises iron oxide.

46. The magnetic bead of any one of claims 43-45, wherein the magnetic material is contained within an agarose bead, and the agarose bead is contained within a cross-linked polymer.

47. The magnetic bead of any one of claims 43-46, wherein the magnetic material is in the form of a particle, a powder, a flake, or a cluster.

48. The magnetic bead of any one of claims 43-47, wherein the cross-linked polymer is a cross-linked azlactone polymer, and the magnetic bead is an azlactone bead.

49. The magnetic bead of any one of claims 43-48, wherein the magnetic bead comprises one or more functional groups derived from reaction of an azlactone group with an amine-containing compound selected from a biomolecule, an organic ligand, a polymer, a dendrimer, or a combination thereof.

50. The magnetic bead of claim 48, comprising: iron oxide particles distributed throughout the azlactone bead; and a plurality of functional groups located on the surface of the magnetic bead. wherein the functional group is selected from , , , , , , , or .

51. The magnetic bead of claim 43, wherein the functional group comprises .

52. The magnetic bead of claim 43 or 51, wherein the azlactone bead is formed from a vinyl azlactone and a cross-linking agent selected from a bisacrylamide, an agarose, and a vinyl ether.

53. The magnetic bead of any one of claims 43 to 52, wherein: the vinyl azlactone is 4,4-dimethyl-2-vinyl oxazol-5(4H)-one; the cross-linking agent is bisacrylamide; or the vinyl azlactone is 4,4-dimethyl-2-vinyl oxazol-5(4H)-one and the cross-linking agent is bisacrylamide.

54. The magnetic bead of claim 52 or 53, wherein the cross-linking agent is methylene bisacrylamide.

55. The magnetic bead of any one of claims 43 to 54, wherein the magnetic material is Fe3O4 or Fe2O3 or a combination thereof.

56. The magnetic bead of any one of claims 43 to 55, wherein the magnetic bead has a bead diameter of 20 to 80 microns.

57. The magnetic bead of claim 56, wherein the magnetic bead has a bead diameter of 30 to 60 microns.

58. The magnetic bead of any one of claims 56-57, wherein the magnetic bead size is the size in a non-hydrated state, and the hydrated size of the magnetic bead is 6 to 8 times larger than its size in the non-hydrated state.

59. The magnetic bead of any one of claims 43 to 58, wherein the magnetic bead is a porous magnetic bead.

60. A population of magnetic beads of any one of claims 43 to 59, wherein the population has a magnetic bead loss of less than about 5% by weight when used in an automated process.

61. A kit comprising a plurality of magnetic beads according to any one of claims 43 to 59 or claims 64 to 70.

62. A method of removing contaminants from a sample, comprising: providing a sample comprising a liquid matrix and a protein fragment or polypeptide, wherein the liquid matrix comprises a contaminant; combining the sample with a population of magnetic beads according to any one of claims 36 to 45 in the presence of a binding solution, wherein the protein fragment or polypeptide binds to the magnetic beads; magnetically separating the population of magnetic beads from the liquid matrix; optionally washing the separated population of magnetic beads with a wash solution to remove any remaining contaminants from the population of magnetic beads; eluting the protein fragment or polypeptide from the separated magnetic beads with an elution solution to form a solution comprising the protein fragment or polypeptide; and magnetically separating the population of magnetic beads from the solution comprising the protein fragment or polypeptide.

63. A method of capturing protein aggregates from a sample, the method comprising: providing a sample comprising a liquid matrix and a plurality of proteins; combining the sample with a population of magnetic beads according to any one of claims 36 to 45 in the presence of a binding solution, wherein at least a portion of the proteins bind to the magnetic beads to form bound proteins on the magnetic beads; magnetically separating the population of magnetic beads from the liquid matrix; optionally exposing the population of magnetic beads with the bound proteins to a reducing agent and / or an alkylating agent, and subsequently separating the population of magnetic beads from the reducing agent and / or the alkylating agent; optionally washing the separated population of magnetic beads; eluting the bound proteins from the population of magnetic beads with an elution solution to form a solution containing unbound proteins; and optionally digesting the unbound proteins with a digestion reagent.

64. The magnetic bead of claim 49, wherein the amine-containing compound further comprises a positively charged group or a negatively charged group.

65. The magnetic bead of claim 49, wherein the amine-containing compound is an alkyl amine group or an aryl amine group.

66. The magnetic bead of claim 65, wherein the amine-containing compound is pentanediamine, PEI-25K, or PEI-800.

67. The magnetic bead of claim 49, wherein the amine-containing compound is selected from the group consisting of carboxylate salts, sulfonate salts, and phosphonate salts.

68. The magnetic bead of claim 49, wherein the amine-containing compound comprises an azido group or an alkyne group.

69. The magnetic bead of claim 49, wherein the amine-containing compound comprises a metal-charged chelator selected from the group consisting of nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), and ethylenediaminetetraacetic acid (EDTA).

70. The magnetic bead of claim 69, wherein the metal is selected from the group consisting of metals such as nickel, iron, cobalt, aluminum, titanium, zirconium, gallium, and the like, and any combination thereof.

71. A method of isolating a negatively charged substance from a biological sample, the method comprising: providing a magnetic bead, wherein each magnetic bead comprises iron oxide particles distributed throughout the aza-lactone magnetic bead and a positively charged group located on the surface of the magnetic bead; combining the magnetic bead with a biological sample comprising a negatively charged substance to form a magnetic bead conjugate, wherein the conjugate comprises the negatively charged substance bound to the positively charged group on the magnetic bead; and separating the magnetic bead conjugate from the biological sample.

72. The method of claim 71, wherein the negatively charged species is an extracellular vesicle, a nucleic acid, or a virion.

73. A method of isolating a positively charged species from a biological sample, the method comprising: providing a population of magnetic beads, wherein each magnetic bead comprises iron oxide particles distributed throughout an azlactone magnetic bead and a negatively charged group on the surface of the magnetic bead; combining the magnetic beads with a biological sample comprising a positively charged species to form a magnetic bead conjugate, wherein the conjugate comprises the positively charged species bound to the negatively charged group on the magnetic bead; and isolating the magnetic bead conjugate from the biological sample.

74. A method of removing contaminants from a sample, comprising: providing a sample comprising a liquid matrix and a protein, protein fragment, or polypeptide, wherein the liquid matrix comprises a contaminant; combining the sample with a population of magnetic beads according to claim 69 or claim 70 in the presence of a binding solution, wherein the protein, protein fragment, or polypeptide binds to the magnetic beads; magnetically separating the population of magnetic beads from the liquid matrix; optionally washing the separated population of magnetic beads with a wash solution to remove any remaining contaminants or non-specifically bound analytes from the population of magnetic beads; eluting the protein, protein fragment, or polypeptide from the separated magnetic beads with an elution solution to form a solution comprising the protein, protein fragment, or polypeptide; magnetically separating the population of magnetic beads from the solution comprising the protein, protein fragment, or polypeptide.

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