Protein and Peptide Purification Methods

Abstract

Provided herein are methods, reagents and kits for separating polypeptides, such as proteomes. Also provided herein are modified trypsin polypeptides that are resistant to autolysis and can be selectively separated from biological samples once digestion is complete.

Description

[0001] Statement on Federal Funding

[0002] This invention was made with government support under Grant No. IIP-1700833 from the National Science Foundation. The government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 763,298, filed on June 8, 2018, and U.S. Provisional Patent Application No. 62 / 919,469, filed on March 14, 2019, each of which is hereby incorporated by reference in its entirety.

[0005] Proteomics studies all expressed proteins in cells, tissues, and organisms, where cells express thousands of different proteins at concentrations ranging from hundreds to tens of millions of copies per cell. The main obstacle to proteomic research and diagnostics is sample preparation. Protein samples come from a variety of sources, each with its own preparation problems. Some samples are high in salts, some are high in DNA and RNA, some are very dilute, and some have large amounts of lipids and small organic compounds. There is no universal method for protein sample preparation.

[0006] A significant stumbling block for proteomic research and diagnostics is the preparation of peptide samples. A common step in peptide production is the proteolytic digestion of proteins. Trypsin is the most common proteolytic enzyme used for proteomic peptide analysis. Once the protein is digested, trypsin must be removed prior to mass spectrometry analysis. In addition, trypsin is prone to autodigestion, which can contaminate peptide samples with peptides generated by trypsin autolysis. These trypsin autolytic peptides can strongly interfere with the analysis of proteomic samples.

[0007] There is a need for improved methods of preparing protein samples for proteomics. SUMMARY OF THE INVENTION

[0009] According to one aspect or embodiment of the present invention, there is provided a compound comprising a member of a bioorthogonal coupling pair linked to a dicarboxylic anhydride moiety.

[0010] According to another aspect of the present invention, a modified trypsin polypeptide is provided. The modified trypsin polypeptide comprises: a trypsin polypeptide; a biotin moiety connected to the trypsin polypeptide; and a group comprising a member of a bioorthogonal coupling pair connected to the trypsin polypeptide, wherein the ratio of the group comprising a member of the bioorthogonal coupling pair on the trypsin polypeptide to the biotin moiety is 1:4 to 1:6. A method for preparing a trypsin polypeptide fragment is also provided, comprising digesting a polypeptide with the modified trypsin polypeptide.

[0011] In another aspect or embodiment of the present invention, a protein or peptide isolation kit is provided, comprising a compound of any one of claims 1-13 in a container, or a modified trypsin polypeptide of claim 14 or 15 in a packaged container.

[0012] According to another aspect or embodiment of the present invention, there is provided a method for purifying a polypeptide, comprising:

[0013] mixing a basic pH sample comprising the polypeptide with an amount of a coupling compound comprising a moiety comprising a first member of a bioorthogonal coupling pair connected to a maleic anhydride moiety via a linker;

[0014] coupling the polypeptide in the sample to a second member of a bioorthogonal coupling pair attached to a substrate;

[0015] optionally washing the matrix-bound polypeptide to remove any unbound material from the matrix-bound polypeptide; and

[0016] The peptides are eluted from the matrix in an elution solution at an acidic pH.

[0017] In another aspect or embodiment, a method of making a modified trypsin polypeptide is provided, comprising conjugating a trypsin polypeptide with a first compound comprising an amine-reactive moiety or a sulfhydryl-reactive moiety attached to biotin and a second compound comprising an amine-reactive moiety or a sulfhydryl-reactive moiety attached to a first member of a bioorthogonal coupling pair in a molar ratio of 4:1 to 6:1.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A and 1B Non-limiting examples of methods for purifying proteins or polypeptides described herein are schematically shown.

[0020] Figure 2A and 2B The procedure for protein / peptide purification using mTet-CDM and TCO-beads is illustrated. Figure 2A The first half of the reaction is shown, where mTet-CDM is coupled to any protein or peptide and then mixed with the TCO-beads. Figure 2B The second half of the process is shown, where the mTet-CDM-conjugated protein / peptide is coupled to the TCO beads, contaminants are washed away, and finally the attachment of the CDM to the protein / peptide is reversed at a mildly acidic pH.

[0021] Figure 3 An exemplary sequence of a trypsin polypeptide is provided (SEQ ID NO: 1).

[0022] Figure 4 is a schematic diagram of an exemplary cartridge.

[0023] Figure 5 An exemplary protein purification workflow as described in Example 1 is illustrated.

[0024] Figure 6 The synthetic scheme of mTet-PEG4-CDM is shown.

[0025] Figure 7 An illustrative synthesis scheme for TCO beads is provided.

[0026] Figure 8 Labeling of primary amines with mTet-CDM was more efficient in 100 mM HEPES and reached equilibrium at ∼80% labeling of carbonic anhydrase. Carbonic anhydrase protein was labeled with mTet-CDM in the presence of 10 mM HEPES pH 8.0 (shown in light blue) or 100 mM HEPES pH 8.0 (shown in dark blue). Although labeling in 10 mM HEPES pH 8.0 reached equilibrium at ∼30%, labeling in 100 mM HEPES pH 8.0 reached equilibrium at ∼80%, indicating that higher HEPES concentrations are important for extensive labeling.

[0027] Fig. 9 A buffer containing 0.3 M NaCl and 1% SDS performed best in preventing nonspecific binding of proteins to TCO beads.

[0028] Fig.10 .The binding of mTet-CA to TCO beads reached 94% within 30 min (minutes). In order to find the optimal incubation time for the binding of mTet-tagged proteins to TCO beads, a 1 hour time course was performed and the binding of mTet-CA to TCO beads was measured. After 30 min, 94% of mTet-CA was bound to TCO beads and equilibrium was reached at this time point. For all future binding events, an incubation time of 30 min was used.

[0029] Fig.11 .mTet-CA was eluted from TCO beads in three washes with incubation times as short as 15 min. With increasing incubation time, mTet-CA bound to TCO beads was eluted in four washes. The total elution yield for each incubation time (A) and the elution yield for each individual wash were calculated and compared (15 min incubation elution was used as a representative figure B). All elution incubation times produced yields above 80%, with 30 min producing the highest yield of 94%, with 89% of the eluted protein found in the first two elution washes.

[0030] Fig.12 Capture, wash and release of whole yeast lysate resulted in 81% yield. A sample containing 100 μg of yeast proteome was labeled with mTet-CDM and cleaned up using our capture, wash and release sample preparation workflow. After elution, more than 80% of the proteome was recovered.

[0031] Fig.13 .2D-DIGE of eluted proteins showed similar proteomic profiles, with some spots being more enriched in the load sample or the eluted sample. Yeast proteomic samples that were not subjected to a proteomic sample preparation workflow (A) and yeast proteomic samples that were subjected to a proteomic sample preparation workflow (B) were compared using 2D-DIGE.

[0032] Fig.14 .Evaluation of the entire protein cleanup workflow. Band 1: Load, Band 2: Unbound, Band 3: Elution 1, Band 4: Elution 2, Band 5: Mock Elution. Note that the urea in the elution fraction reduces the Coomassie Blue staining of the protein sample. Band 5 shows the same amount of protein in the load sample, but in elution buffer. This indicates that almost 100% of the loaded protein was eluted.

[0033] Fig.15 .2D electrophoresis gel of Cy3 Burkholderia lysate after purification.

[0034] Fig.16 The graph provided shows the recovery of the peptide of Example 5 relative to the amount of mTet-CDM conjugated to the peptide. DETAILED DESCRIPTION OF THE INVENTION

[0036] Unless otherwise expressly stated, the use of numerical values ​​in the various ranges specified in this application is expressed as approximate values, as if the minimum and maximum values ​​in the range are all preceded by the word "about". In this way, slight changes above and below the range can be used to obtain results substantially the same as the values ​​in the range. Similarly, unless otherwise stated, the disclosure of the range is intended to be a continuous range of each value between the minimum and maximum values. As used herein, "a" and "an" refer to one or more. Patients are humans or non-human animals.

[0037] The term "comprising" as used herein is open ended and can be synonymous with "including", "containing" or "characterized by..." As used herein, embodiments "comprising" one or more of the elements or steps also include, but are not limited to, embodiments "consisting essentially of" and "consisting of" these elements or steps.

[0038] "Moiety" ("moieties") refers to a portion of a compound, and includes groups such as functional groups. Thus, a nucleobase moiety refers to a nucleobase that has been modified by attachment to another compound moiety, such as a polymer monomer, e.g., a nucleic acid or nucleic acid analog monomer described herein, or a polymer, e.g., a nucleic acid or nucleic acid analog described herein.

[0039] "Alkyl" refers to a straight chain, branched chain or cyclic hydrocarbon group containing 1 to about 20 carbon atoms, such as but not limited to C 1-3 , C 1-6 , C 1-10 "Alkyl" refers to an alkyl radical substituted at one or more positions, such as 1, 1, 2, 3, 4, 5 or even 6 positions, and the substituent is connected to any available atom to produce a stable compound through substitution as described herein. "Optionally substituted alkyl" refers to an alkyl radical or a substituted alkyl radical. "Halogen", "halide" and "halo" refer to -F, -Cl, -Br and / or -I. "Alkylene" and "substituted alkylene" refer to divalent alkyl and divalent substituted alkyl radicals, respectively, including but not limited to vinyl (-CH2-CH2-). "Optionally substituted alkylene" refers to an alkyl radical or a substituted alkyl radical.

[0040] "Alkenyl" refers to a straight chain, branched chain or cyclic hydrocarbon group containing 2 to about 20 carbon atoms, such as but not limited to C 1-3 , C 1-6 , C 1-10A "substituted alkene" refers to an alkene substituted at one or more, such as 1, 2, 3, 4 or 5, positions, wherein the substituents are attached to any available atom to produce a stable compound by substitution as described herein. An "optionally substituted alkene" refers to an alkene or a substituted alkene. Similarly, an "alkenylene" refers to a divalent alkene. Examples of alkenylene include, but are not limited to, vinylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof. A "substituted alkenylene" refers to a divalent substituted alkene. An "optionally substituted alkenylene" refers to an alkenylene or a substituted alkenylene.

[0041] "Cycloalkyl" refers to a monocyclic, bicyclic, tricyclic or polycyclic 3-14-membered saturated, unsaturated or aromatic ring system. "Cycloalkenyl" refers to a monocyclic, bicyclic, tricyclic or polycyclic 3-14-membered ring system, and contains at least one carbon-carbon double bond in the ring system. Cycloalkyl can be connected by any atom. Cycloalkyl also considers fused rings, wherein the cycloalkyl is fused to an aryl ring or a heteroaryl ring. Representative examples of cycloalkyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Cycloalkyl can be unsubstituted or optionally substituted by one or more substituents described below. "Cycloalkylene" refers to a divalent cycloalkyl. The term "optionally substituted cycloalkylene" refers to a cycloalkylene substituted by 1, 2 or 3 substituents connected to any available atom to produce a stable compound.

[0042] As used herein, "polypeptide" includes proteins and oligopeptides as classes, and generally refers to polypeptides comprising two or more amino acid residues, although longer amino acid chains are often referred to.

[0043] A "sample" comprising a polypeptide can be blood, plasma, serum, urine, cerebrospinal fluid, cell lysate, cell culture medium, or any other biological composition comprising a polypeptide. In one embodiment, the sample is a cell lysate from a patient tissue sample. In another embodiment, the sample is prepared by lysing a pelleted cell from a cell culture, for example to produce a recombinant protein or to analyze the proteome of a cell, tissue or organ culture.

[0044] A joint is a part that makes one part connected to another part in a compound. An "inert" joint is a part that makes one part in a compound covalently connected to another part, and optionally makes one part in a compound spaced apart from another part, and has no substantial negative impact on the activity of the entire compound, such as in the context of the present invention, a reactive group (such as a tetrazine group, a succinyl diimide group or a dicarboxylic anhydride group such as maleic anhydride) reacts with its expected target and forms and maintains the ability of a key according to the methods described herein. In addition to being used to covalently connect two parts, a joint can also have a beneficial effect, such as in the physical separation of the part connected thereto, for example, optimizing the interval to avoid steric effects. The joint can also play some other functions, such as changing the hydrophobicity / hydrophilicity of the entire molecule, to provide additional sites, such as amines protected by protecting groups, for making additional parts connected to the compound, or making the entire molecule rigid. The linker is attached to the remainder of the compound by any suitable linking moiety ("link"), for example, by a carbon-carbon bond, an ester, a thioester, an amine, an ether, an amide, a carbonate or a carbamate. The linker may be a hydrocarbon group containing only carbon and hydrogen, or optionally containing one or more heteroatoms such as N, O and / or S. In the context of the present invention, in one embodiment, a suitable linker is a PEG group -(O-CH2-CH2) n - a divalent moiety, wherein n is 2-100, such as 2-10 (PEG 2-10 ) or 2-5(PEG 2-5 ), such as 2, 3, 4 (PEG4), 5, 6, 7, 8, 9 or 10. In addition to suitable linkages to attach the PEG group to tetrazine and dicarboxylic anhydride moieties such as maleic anhydride moieties, the PEG linker may also contain one or more methylene groups at either end.

[0045] In one aspect or embodiment of the invention, there is provided a compound comprising a first member of a bioorthogonal coupling pair, such as a tetrazine moiety, linked to a dicarboxylic anhydride moiety. Dicarboxylic anhydrides, such as maleic anhydride, such as citraconic anhydride, have been used for protein conjugation (see, e.g., Klapper ML and IM Klotz,

[46] Acylation with Dicarboxylic Acid Anhydrides Methods Enzymol. 1972; 25:531-6; Atassi MZ and AF Habeeb,

[49] Reaction of proteins with citraconic anhydride Methods Enzymol. 1972; 25:546-53; Butler PJ, Hartley BS

[14] Maleylation of amino groups Methods Enzymol. 1972; 25:191-9; AJ Kirby and PW Lancaster, Structure and efficiency in intramolecular and enzymic catalysis. Catalysis of amide hydrolysis by the carboxy-group of substituted maleamic acids Journal of the Chemical Society Perkin Transactions 2 2(9) January 1972). The first member of the bioorthogonal coupling pair, such as a tetrazine moiety, is linked to a dicarboxylic acid anhydride to generate a compound that can be used to purify the polypeptide.

[0046] In use, after the compound is linked to the polypeptide by forming an amide bond between the dicarboxylic anhydride of the compound and the primary amine of the polypeptide, the first member of the bioorthogonal coupling pair is reacted with the second member of the bioorthogonal coupling pair attached to a matrix, such as a bead, a porous surface, or a solid surface, thereby producing a polypeptide (e.g., a protein or peptide) bound to the matrix. The reaction of the dicarboxylic anhydride moiety with the primary amine of the polypeptide forms an acid-labile amide bond. A weak acid (e.g., pH in the range of >2 to 6) cleaves the amide bond of the conjugate, thereby allowing the polypeptide to be eluted. In this way, a washing solution that is used to wash any cellular components from the polypeptide and does not cleave the amide bond between the matrix and the bound polypeptide can be suitable for the purpose of washing the polypeptide.

[0047] It should be recognized that some non-protein compounds present in the sample may also contain primary amines and can be purified together with the polypeptide. However, for most proteomic assays, such non-protein contamination is not expected to have a significant impact on the assay. Therefore, purification refers to enriching one or more components in a sample by removing other components from the sample. It should be understood that, in an embodiment, the purification process of the polypeptide or protein is by washing the polypeptide bound to the matrix under conditions where the polypeptide is not eluted from the column, for example, with a suitable washing solution such as water, saline, TRIS-EDTA, PBS, etc., to effectively separate the polypeptide from other cellular materials such as nucleic acids, carbohydrates and lipids.

[0048] In one aspect, the polypeptide is bound to a chromatography column matrix, such as a porous matrix or beads, so that washing and elution can be performed in a single chromatography column (see, e.g., Figure 1A ). A chromatographic column is any container capable of retaining a matrix and allowing a solution to pass through the matrix without loss of the matrix. A chromatographic column is usually a tube with a fixed mesh or screen.

[0049] In another aspect of the present invention, a purification method for protein or polypeptide is provided. The method is universal because it only targets the primary amine and lysine residues present in all polypeptide N-terminals. Any biological sample can be used, such as a biological sample obtained from a patient, or a cell, tissue or organ culture, a biopsy, a clumping cell from any biological fluid sample such as urine, blood, saliva, mucus, cerebrospinal fluid, semen, aspirate, etc., for producing tissue culture of recombinant protein, etc. In the method, a cell lysate is prepared, such as by homogenizing, optionally in the presence of any suitable salt, buffer, surfactant, emulsifier, chaotropic agent, chelating agent, etc., such as in urea, SDS or RIPA buffer. On the one hand, a cell lysate is prepared in a weakly alkaline buffer or saline solution, such as to produce a pH range of>7 to 10, such as a lysate of 8-9.5. The sample may be cell-free and may not require lysis, such as in the case of analysis of the secretome, when producing recombinant proteins that are secreted into the culture medium, or when analyzing cell-free preparations of biological fluids (e.g., supernatants from centrifugation).

[0050] If a cell lysate is to be analyzed, once the cells are lysed, the pH of the lysate is adjusted to a pH range of >7-10, such as 8-9.5, using, for example, a salt or buffer solution, if necessary. The lysate is mixed with a compound comprising a first member of a bioorthogonal coupling pair connected to a dicarboxylic acid anhydride portion, the dicarboxylic acid anhydride portion being, for example, but not limited to, mTET-PEG4-CDM, the first member being, for example, but not limited to, a tetrazine portion. The sample is then reacted with a matrix to which a second member of a bioorthogonal coupling pair, such as TCO, is connected. The second member of the bioorthogonal coupling pair can be connected to the matrix by any suitable chemical method, such as coupling it to the primary amines of the matrix by NHS. The matrix can be a bead, such as, but not limited to, a magnetic bead, an agarose bead, a plastic surface or a glass surface (e.g., a hole of a multi-well plate such as a 96-well plate) or a porous matrix. The matrix can include any suitable matrix material that does not interfere with the reactions and processes described herein to any substantial extent. When coupling, washing and eluting the polypeptide in a manner well known in the biological and chemical fields, the matrix can be contained in a chromatographic column, a spin column, or any other physical form that retains the matrix. The magnetic beads are treated in any manner recognized in the art.

[0051] Then use for example pH to be 6.0 or any suitable washing solution of less non-acidic washing of the matrix of binding polypeptide, to prevent premature hydrolysis of the amide bond that makes polypeptide connected to matrix.Suitable washing solution includes but is not limited to water, saline, PBS, Tris-EDTA or other saline solution or buffered saline solution that can not hydrolyze the amide bond that makes polypeptide connected to matrix.For any matrix selected, matrix can be washed once or repeatedly in the manner recognized in the art.Two or more washing steps can be applied, and use the same or different washing solutions.

[0052] In the above and Figure 1A In a variation of the method shown, peptides are purified rather than the entire protein. This is schematically depicted in Figure 1B In brief, a protease digestion step is added after cell or tissue lysis. In one embodiment, the protease is trypsin, in another embodiment, the protease is a modified trypsin as described below.

[0053] Once the matrix to which the polypeptide is bound is washed, the polypeptide is eluted from the matrix using a weakly acidic eluent. The composition of the elution solution may vary, depending on the desired end use of the eluted polypeptide. The pH of the elution solution may range from 2 to <7 or 2.5-6. By using a carboxylic anhydride coupled to the amines of the polypeptide, the primary amines of the polypeptide are restored once the amide bond coupling the polypeptide to the matrix is ​​cleaved.

[0054] "Click Chemistry" describes reactions that are high yield, wide range, produce only byproducts that can be removed without chromatography, are stereospecific, easy to implement, and can be performed in easily removable or benign solvents. In the context of the present disclosure, click chemistry reactions are biorthogonal, meaning that they are selective enough that they can be reliably performed even in complex biological environments. These reactions must be performed efficiently in the presence of numerous functional groups present in living systems, such as nucleophiles, electrophiles, reducing agents, oxidants, and water. At the same time, the reactions themselves should have minimal impact on biology.

[0055] In the context of bioorthogonal reactions such as bioorthogonal click chemistry reactions, the formation of bonds between molecules or moieties not found in natural compounds, referred to herein as bioorthogonal coupling pairs, is relied upon. Such reactions are preferably selective for other potential reactive functional groups present on biomolecules, carried out in aqueous media close to physiological pH, and have a fast reaction rate using low reactant concentrations at room temperature (or up to 37 ° C), all of which ensure high modification efficiency (Lopes Bernardes, G., Oliveira, B., & Guo, Z. (2017). Inverse electron demand Diels–Alder reactions in chemical biology. Chemical Society Reviews https: / / doi.org / 10.17863 / CAM.10698). Bioorthogonal reaction reagents (bioorthogonal coupling pairs) do not react with natural cell products such as proteins or nucleic acids. Coupling occurs under very broad aqueous conditions and is stable once formed. In one embodiment, the bioorthogonal reaction is an inverse electron demand Diels-Alder reaction (IEDDA, ​​e.g., inverse electron demand [4+2] Diels-Alder cycloaddition), in which an electron-rich dienophile (e.g., a strained olefin) is reacted with an electron-rich dienophile (e.g., a strained olefin) to form an electron-rich dienophile. alkene) with an electron-poor diene (e.g., a tetrazine, such as 1,2,4,5-tetrazine or 4-(1,2,4,5-tetrazine)phenyl moiety, such as 4-(1,2,4,5-tetrazine-3-yl)phenyl, 6-alkyl-1,2,4,5-tetrazine, 6-pyridin-2-yl-1,2,4,5-tetrazine, 6-pyrimidin-2-yl-1,2,4,5-tetrazine, 4-(6-alkyl-1,2,4,5-tetrazine-3-yl)phenyl, 4-(6-pyridin-2-yl-1,2,4,5-tetrazine-3-yl)phenyl or 4-(6-pyrimidin-2-yl-1,2,4,5-tetrazine-3-yl)phenyl, wherein the alkyl group can be C 1-4alkyl) reaction (see, e.g., Karver, MR, et al., Synthesis and Evaluation of a Series of 1,2,4,5-tetrazines for Bio-orthogonal Conjugation. Bioconjug Chem. 2011 November 16; 22(11): 2263-2270), which is in contrast to the standard electron-demanding Diels-Alder reaction in which an electron-rich diene reacts with an electron-poor dienophile (for further details and examples providing other bioorthogonal reactions, see, e.g., Lopes Bernardes, G., Oliveira, B., & Guo, Z. (2017). Chemical Society Reviews https: / / doi.org / 10.17863 / CAM.10698). Note that "1,2,4,5-tetrazine" refers to an exact 1,2,4,5-tetrazine compound or 1,2,4,5-tetrazine moiety, while "a 1,2,4,5-tetrazine" refers to a compound or moiety comprising a 1,2,4,5-tetrazine moiety. In another example, the bioorthogonal coupling pair is an alkyne-azide coupling pair, such as the well-known propargyl moiety and an azido moiety.

[0056] In the IEDDA reaction, an electron-poor diene, such as 1,2,4,5-tetrazine, is reacted with an electron-rich dienophile, such as a strained dienophile, and the reaction kinetics are tailored using the selection of fine-tuning the electron-poor diene and the electron-rich dienophile ("IEDDA coupling pair") (supra). Non-limiting examples of suitable electron-poor dienes for the IEDDA reaction include tetrazines, such as 1,2,4,5-tetrazines, such as methyl tetrazines and triazines (see, e.g., Devaraj, NK, et al., Fast and Sensitive Pretargeted Labeling of Cancer Cells via Tetrazine / Trans-Cyclooctene Cycloaddition Agnew Chem Int Ed Engl. 2009; 48(38): 7013-7016 and Karver, MR, et al., Bioconjug Chem. 2011 November 16; 22(11): 2263-2270). A non-limiting example of an electron-rich dienophile for IEDDA reactions is trans-cyclooctene. A non-limiting example of bioorthogonal reactions and reaction pairs is the reaction of methyl tetrazine (mTet) and trans-cyclooctene (TCO). The IEDDA pair is used because the reaction kinetics are fast and the reaction does not require a catalyst. In the following examples, mTet-CDM protein / mTet-CDM-peptide / free mTet-CDM is deeply diffused into the polymer shell around the beads using NHS esters of these reagents each with a PEG spacer (linker). Other suitable bioorthogonal coupling reactions include alkyne-azide reactions, such as reactions with triazides and alkynes, or alkyne-DBCO (dibenzocyclooctyne) reactions.

[0057] The method described in this article is to use click chemistry to achieve protein and peptide purification. First, a reversible protein tag mTet derivative was synthesized, which is called mTet-CDM. mTet-CDM is a very effective capture reagent, but the excess unused mTet-CDM needs to be removed. mTet is one half of a biorthogonal coupling pair. The other half of the coupling pair is TCO. Both mTet and TCO do not react with other biological compounds and are therefore considered bioorthogonal. In this way, their reactions are very specific and there are no by-products.

[0058] In an example of this method, beads with bound TCO are used to capture free mTet-CDM and mTet-CDM coupled to proteins or peptides. The composition of the beads can be non-magnetic beads or magnetic beads. Beads with bound TCO can be prepared by reacting TCO-NHS esters with amine-derivatized beads. Since the normal density of TCO molecules on commercial amine beads may be too low for the purposes described herein, commercial beads can be modified to carry 10-50 times more amino groups by growing amine-containing polymers from the surface of the beads. These amine groups are then coupled to TCO, so that the beads carry 50 times more TCO. The process of purifying proteins / peptides using mTet-CDM and TCO beads is shown in Figure 2.

[0059] Proteases such as trypsin play an important role in proteomics and biochemistry. Trypsin cleaves proteins at the carboxyl terminus of lysine and arginine residues unless an adjacent proline is present. This cleavage specificity allows researchers to identify genes encoding individual proteins based on the predicted trypsin cleavage pattern. The protein lysates, called tryptic peptides, are introduced into a mass spectrometer (MS) that can accurately determine the mass of the tryptic peptides. MS is also able to determine the amino acid sequence of the tryptic peptides to further characterize the protein under investigation.

[0060] Trypsin is a well-known and well-studied acidic protease. An exemplary trypsin protein sequence (precursor) is shown in Figure 3As shown. "Trypsin polypeptide" refers to any natural trypsin or its proteolytic active variant. A large number of trypsins and modified trypsins that are different in sequence from natural trypsin are known and / or commercially available, including sequence variants of natural trypsin. Trypsin is commonly used in proteomics research to produce tryptic peptides that can be analyzed by various methods such as mass spectrometry or Edman sequencing. In practice, trypsin autolysis, which causes the autolysis product to mask the peptides produced by the protein sample to be digested, and also causes the production of pseudotrypsin, shows a wide range of specificity, including activity similar to chymotrypsin. In order to avoid these problems, a typical trypsin digestion is performed at a low ratio of about 1:20 to 1:50 trypsin to target protein. In addition, it is desirable to remove trypsin from the protein fragment after digestion. Trypsin contains many primary amines, such as Lys residues and their N-termini. The primary amine of lysine can be used to connect groups using any useful chemical method such as NHS joints. Trypsin has 14 lysine residues and only two arginine residues, i.e., another autolysis target. NHS-biotin, such as the N-hydroxysuccinimide ester of biotin (see below), can be used to modify lysine residues and the N-terminus of trypsin. In addition, since NHS-biotin does not completely label trypsin, it does not help to completely remove trypsin from protein samples.

[0061]

[0062] Methods for removing trypsin from peptide samples are described herein. The methods described herein produce trypsin derivatives that are highly resistant to self-digestion and can be removed from solution by chemically cross-linking the trypsin derivatives to a solid support matrix using a bioorthogonal "click" chemistry. As mentioned above, trypsin contains 14 lysine residues and only two arginine residues. Therefore, most of the self-digestion sites are located on lysine residues.

[0063] In the following example, to limit self-digestion, >95% of all lysines (plus the amino terminus) are coupled to a mixture of methyl tetrazine-PEG4-NHS and biotin-NHS. Biotin-NHS alone does not reduce the reactivity of trypsin, while extensive labeling with methyl tetrazine-PEG4-NHS alone results in protein insolubility and complete loss of enzymatic activity. Combining these two labeling agents in a molar ratio of methyl tetrazine-PEG4-NHS to biotin-NHS of 1:5 allows complete retention of trypsin activity. The trypsin derivative is referred to herein as MB-trypsin. By coupling with beads containing trans-cyclooctene (TCO), the presence of methyl tetrazine on MB-trypsin allows almost complete removal of the protein from solution. Methyl tetrazine and TCO together form a bioorthogonal coupling pair in which the two moieties react at a very fast rate to form a covalent bond. Other tetrazine moieties can be used in this click chemistry pair, or other bioorthogonal click chemistry pairs can be used in the connection of trypsin to a matrix such as a bead for removal.

[0064] In use, a protein sample is digested using a modified trypsin having a biotin side group moiety and a side group comprising a first member of a bioorthogonal coupling pair, such as a tetrazine group, e.g., a methyltetrazine group moiety, and e.g., a molar ratio of biotin to the bioorthogonal coupling pair member of 4:1 to 6:1, e.g., 5:1. Following sample digestion, the digest is contacted with a second member (e.g., a click chemistry partner) of a binding surface (e.g., a bound bead) of a bioorthogonal coupling pair. For example, in the case of a tetrazine as the first member of the bioorthogonal coupling pair, trans-cyclooctene, or another strained cyclic olefin, e.g., of 8 or more carbons, can be the second member of the binding surface of the bioorthogonal coupling pair.

[0065] Likewise, according to embodiments of the present invention, trypsin is modified with biotin linked to an amine or thiol reactive moiety (e.g., NHS, maleimide, or iodoacetamide moiety) and a first member of a bioorthogonal coupling pair linked to an amine or thiol reactive moiety, such as, but not limited to, NHS-biotin and NHS-tetrazinyl compounds. In embodiments, the NHS-tetrazinyl compound comprises a tetrazinyl moiety linked to an NHS moiety, such as, for example, having the following structure:

[0066]

[0067] Where R2 is H or C 1-3 alkyl; L is an inert linker, or

[0068]

[0069] Wherein L' is an inert linker and optionally comprises an ester linker to the N-succinimidyl moiety. In another embodiment, the compound has the following structure:

[0070]

[0071] wherein n is 2-10, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0072] Referring to the structure above, when reacting with a primary amine such as a protein, the NHS-tetrazine compound will leave behind an amide-tetrazine moiety, for example:

[0073]

[0074] Likewise, NHS-biotin (structure below) will react with primary amines of a peptide, leaving behind an amide-biotin moiety.

[0075] The molar ratio of the first member of the bioorthogonal coupling pair attached to an amine- or thiol-reactive moiety to the biotin attached to the amine- or thiol-reactive moiety, e.g., the molar ratio of the NHS-tetrazine compound to the NHS-biotin reacted with trypsin, or the ratio of the first member of the bioorthogonal coupling pair attached to an amine- or thiol-reactive moiety to the biotin attached to the amine- or thiol-reactive moiety, e.g., the ratio of the NHS-tetrazine residues to the NHS-biotin residues attached to the trypsin polypeptide (that is, e.g., the molar ratio of the amino-tetrazine moiety to the amino-biotin moiety), is 1:4 to 1:6. With respect to the use of NHS-biotin and the NHS-tetrazine compound to modify trypsin, higher amounts of biotin result in the inability of the NHS-tetrazine compound to modify a large amount of the trypsin polypeptide, resulting in less efficient capture of the protein. Higher amounts of the NHS-tetrazine compound can precipitate trypsin in an aqueous environment.

[0076] In one aspect of the invention, a protein purification method and a protein purification kit are provided. Due to the huge chemical diversity of proteins in the proteome, standardized protein separation methods that capture the entire proteome have historically been challenging. Long-standing precipitation methods rely on the hydrophobicity of unfolded proteins, but some proteins are not hydrophobic enough to precipitate, while others are too hydrophobic to return to solution. Therefore, the general protein purification method and kit described below include the following features:

[0077] (1) Bead-based: Protein precipitation has several of the problems mentioned above. In addition, the need for centrifugation limits its utility in automation. Bead-based methods provide a larger surface area, thus avoiding the protein precipitation problem, and can be automated.

[0078] (2) Tolerance to cell lysis procedures: The methods and reagents described herein are suitable for many lysis methods. Many researchers prefer to keep protein samples in a native state for various purposes. Therefore, the methods and reagents described herein are capable of purifying protein samples in a native or denatured state.

[0079] (3) Identify universal protein targeting features: Protein precipitation methods and bead capture methods rely on general protein properties such as hydrophobicity and charge, while peptide isolation methods rely on hydrophobicity, charge, and size. Proteins and peptides have a very wide range of hydrophobicity, charge, and size. Therefore, targeting these properties will not select all proteins / peptides. One feature common to all proteins and peptides is aliphatic primary amines (amino termini and lysine residues). Nucleic acids have no aliphatic primary amines; primary amines are rare in carbohydrates. Therefore, targeting primary amines has the best potential to capture the entire proteome.

[0080] (4) Use of molecular tags instead of direct coupling to beads: Direct attachment of proteins to beads via amine groups requires large amounts of beads because the coupling reaction is not very efficient due to the inherent hydrolysis rate of amine-reactive coupling reagents. Such large amounts of beads necessitate the use of large amounts of buffer to completely elute the bound protein / peptide, resulting in a dilute solution that should require additional steps to concentrate the eluted sample, which presents other problems. In contrast, the methods and reagents described herein use molecular tags, where one end of the tag is coupled to a primary amine on a protein or peptide and the other end of the tag directs the binding of the tagged protein / peptide to the beads.

[0081] (5) Covalent attachment of labeled proteins / peptides to beads using click chemistry: Since the CDM half of the tag forms a reversible covalent bond, click chemistry is used to form a stable covalent bond between the tag and the beads. In particular, for the examples below, methyltetrazine (mTet) and trans-cyclooctene (TCO) click chemistry pairs were selected because of their bioorthogonal reactivity (non-reactive with biomolecules), their fast reaction kinetics, the fact that they do not require a copper catalyst like other click pairs, and their ability to couple under the relatively harsh conditions of typical cell / tissue lysis procedures and to withstand the stringent washing steps required to remove non-protein contaminants.

[0082] (6) Binding of all Click-CDM (free protein / peptide and bound protein / peptide): The protein purification method, reagents and kits are characterized to produce a suitably concentrated protein / peptide sample for further biochemical or proteomic analysis. Appropriate small volume beads are used in the method to bind free unreacted tag and labeled protein / peptide.

[0083] In another aspect of the present invention, a kit for digesting polypeptides or for purifying proteins or polypeptides is provided. The kit includes packaging and at least the components of the kit. The packaging can be any suitable container suitable for storing and / or delivering the kit components, such as a box, sleeve, tube, carton, pouch, bag, etc. A "kit" can include one or more separate containers for kit elements, although in one embodiment, all components of the kit are packaged together, or packaged in a single container. For reagents or compositions, such as reagents and compositions described herein, the kit includes one or more containers containing the reagents or compositions. The kit may include a modified trypsin of any embodiment proposed herein, a coupling compound, such as a compound comprising a member of a bioorthogonal coupling pair connected to a dicarboxylic anhydride portion, such as an electron-poor diene or electron-rich dienophile member of an IEDDA coupling pair, such as a tetrazine portion or a TCO portion, or an alkyne-azide pair. The matrix is ​​coupled to the second member of the bioorthogonal coupling pair. The matrix can be in any suitable form, such as a magnetic bead, a bead or a porous matrix or a solid surface in a chromatographic column, such as a hole in a porous plate. The kit may include other containers comprising one or more of the following: a digestion solution, a coupling solution, one or more wash solutions, an eluent, optionally in concentrated form, such as a 2X, 5X, 10X or 25X concentrated solution.

[0084] The containers of the kit may be compartments in a cartridge for use in an automated or semi-automated device or system for digesting or purifying proteins in a sample.

[0085] Figure 4 Schematically illustrated is a box 100, which includes a housing 105, four compartments 110a, 110b, 110c, 110d, an outlet 120, a closed valve 125 and an opened valve 125'. The housing 105 can have any useful configuration and is suitable for inserting into an automated device or system to control the delivery of the composition contained in the compartments 110a, 110b, 110c, 110d. Each compartment can contain different reagents or compositions, or identical reagents or compositions. Valves 125 and 125' can be controlled by any suitable machinery or electromechanical mechanism, such as by a solenoid, and can be placed in the box 100 or at any position outside the box 100, such as an outlet can be coupled to a valve fluid, and the valve is a part of the device into which the box 100 is inserted. Figure 4 The box 100 shown is exemplary only and can include any number of compartments, any shape, any fluid path, and any fluid control mechanism. A person of ordinary skill in the engineering field can configure the box for any device or system, such as an automated system. The control of the box and / or the reagents or compositions taken out of the box can be automated, such as controlled by a computer-implemented process.

[0086] Example 1 - Isolation and purification of common proteins / peptides

[0087] An exemplary universal protein / peptide isolation and cleanup kit includes any or all of the following reagents: TCO magnetic beads or TCO-non-magnetic beads, mTet-CDM, cell lysis buffer, wash buffer to remove non-protein contaminants, elution buffer to reverse CDM-protein / peptide linkage and recover the purified protein / peptide sample.

[0088] Cell lysates are prepared by a variety of methods, such as lysing with SDS, urea or RIPA buffer. If a complete protein proteome is required, the protein in the cell lysate is coupled to mTet-CDM under weakly alkaline conditions of pH 8-9.5. If peptides are required, the protein in the lysate is digested with proteolytic enzymes such as trypsin or Lys C, such as but not limited to trypsin containing biotin and tetrazine side groups, such as MB-trypsin described below. In the case of tetrazine-modified trypsin, trypsin is removed by coupling tetrazine-modified trypsin to a TCO of a binding matrix, such as a TCO of a binding bead after digestion. Other bioorthogonal reaction chemistry methods, such as IEDDA coupling pairs, can be used to modify trypsin and remove trypsin from the digested protein mixture. Then under weakly alkaline conditions, such as pH 8-9.5, the resulting peptide fragments are coupled to mTet-CDM. For complete proteins or peptides, aliquots of TCO beads sufficient to bind the total amount of mTet-CDM in the sample preparation reaction are added to the sample. The sample is then incubated with shaking (0°C–37°C) to allow the mTet:TCO click reaction to occur. This is expected to take 1–10 hours, until no free mTet is detected. The beads are then washed repeatedly (3–5 times) to remove non-protein contaminants. A variety of wash buffers can be used to ensure complete removal of non-protein material. These buffers can include SDS, urea, or salts. The protein / peptide bound to the beads is then eluted under mildly acidic conditions (e.g., pH 2.5–6) to reverse the CDM-protein / peptide linkage. The reversal buffer also contains components suitable for downstream workflows. This may include urea, SDS, detergents, salts, or organic solvents. This produces a purified protein or peptide sample that is free of contaminants. Figure 5 The workflow is shown.

[0089] Figure 6The synthesis scheme of mTet-PEG-CDM is shown. The starting materials are mTet-PEG4-amine (compound 1) and 2-carboxyethyl-3-methyl-maleic anhydride N-hydroxysuccinimide ester (CDM-NHS) (compound 2) (custom synthesis). Since CDM-NHS has two amine-reactive parts, 2 moles of mTet-PEG-amine are combined with 1 mole of CDM-NHS to obtain intermediate 3. Since the connection between CDM and mTet-PEG-amine is unstable to acid, the connection is reversed by treatment with HCl and the activated CDM anhydride of mTet-PEG-CDM 4 is formed. The final product is purified by liquid chromatography and analyzed by NMR and MS.

[0090] A non-limiting synthesis example of mTet-CDM is as follows: mTet-PEG4-NH2 hydrochloride 1 (5 mmol) was dissolved in 1M triethylammonium bicarbonate buffer (10 ml) at pH 8.5. CDM-NHS ester 2 (2.5 mmol) dissolved in anhydrous acetonitrile (ACN, 5 ml) was added dropwise under stirring at room temperature. Stirring was continued for 1 hour. The reaction mixture was concentrated to a volume of 1 ml. 2N HCl (5 ml) was added to hydrolyze 3, and the reaction mixture was stirred for 30 min. The reaction mixture was separated by MPLC chromatography on a RP-18 system using acetonitrile / water / 0.1% TFA as mobile phase using a stepwise gradient. The product was eluted with about 20% acetonitrile (monitored at 254nm). The product fractions were analyzed by UPLC (Waters), RP-18 column, acetonitrile / water / 0.1% TFA, 0-0.5min, 0% acetonitrile, 0.5min-3min 0-100% acetonitrile linear gradient, running time 5min, monitoring at 240nm and 256nm. The pure fractions were collected and concentrated to give 486mg (34%) of pink resinous product 4.

[0091] Synthesis of TCO beads

[0092] As an example of a matrix that binds proteins or protein fragments to separate proteins, TCO beads were prepared by growing a high density of polymer chains from the surface of amine beads using ATRP. The first step in the process is to couple an initiator molecule to the amine beads ( Figure 7). The initiator N-2-bromo-2-methylpropionyl-NHS ester is coupled using standard NHS ester coupling conditions. The concentration of the initiator is adjusted to achieve nearly complete coupling. A copolymer of acrylamide and aminopropylacrylamide grows from the surface of the beads to which the initiator is bound. The goal is to increase the amine content of the beads by 10-fold to 50-fold. The ratio of acrylamide to aminoethylacrylamide and the length of the copolymer are adjusted to maximize the amine content without significantly increasing the viscosity of the protein / peptide or accessibility to the interior of the polymer coating. Acrylamide is included in the copolymer to avoid overly dense packing of the aminopropyl side chains. The final step in the synthesis is to couple TCO-PEG-NHS to the aminoethyl side chains. The concentration of TCO PEG-NHS is adjusted to maximize coupling. Any uncoupled amines are blocked with acetic anhydride to prevent nonspecific protein binding. Polyacrylamide and PEG are known for low levels of nonspecific protein binding.

[0093] Example 2 - Preparation and Characterization of MB-Trypsin

[0094] Methyl tetrazine-biotin-trypsin (MB-trypsin) was prepared by mixing: 1 ml of 1 mg / ml trypsin in 25 mM HEPES-NaOH pH 8.0, 10 mM CaCl2, 3 mM benzamidine HCl, 140 μl of 100 mM biotin-NHS in anhydrous DMF, and 28 μl of 100 mM methyl tetrazine-PEG4-NHS in anhydrous DMF. The mixture was incubated at 0°C for 30 min and dialyzed overnight against 2 x 1 L of 25 mM HEPES-NaOH pH 8.0, 10 mM CaCl2. M-trypsin can be stored at 4°C for up to 60 days with <10% loss of activity, or -80°C for longer periods.

[0095] MB-trypsin can be characterized by the following assay. 1 ml of reaction buffer (46 mM HEPES-NaOH pH 8.0, 11.5 mM CaCl2, 1 mM tosyl arginine methyl ester (TAME)) is combined with 1 μl of 1 mg / ml trypsin solution. The light absorbance at 247 nm is measured and the nanomoles of TAME hydrolyzed per minute per microgram of trypsin are calculated. Labeling trypsin with methyl tetrazine and biotin does not reduce the catalytic activity of trypsin.

[0096] According to the following, the degree of primary amine labeling can be assessed using the fluorescamine assay. A standard curve is drawn by dissolving 0-10 μg trypsin or 10 μg MB-trypsin in 200 μl of 100 mM HEPES-NaOH pH 8.0. 50 μl of 3 mg / ml fluorescamine is added to acetone. Mix and transfer 200 μl of each reaction solution to a black 96-well plate for fluorescence measurement in a plate reader. Fluorescence is measured using an excitation wavelength of 390 ± 5 nm and an emission wavelength of 465 ± 10 nm. The degree of free amines in the MB-trypsin sample relative to the standard curve is calculated. Typically, > 95% of amines are labeled under the above-mentioned labeling reaction conditions.

[0097] Trypsin protein digestion protocol: Mix equal amounts of MB-trypsin and target protein [usually 100 μg MB-trypsin and 100 μg target protein in a total volume of 400 μl] in a buffer containing 100 mM HEPES-NaOH pH 8.0 and a denaturant mixture containing 1% sodium dodecyl sulfate (SDS), 1% and 0.5% sodium deoxycholate. The target protein was previously reduced with dithiothreitol and alkylated with iodoacetamide. Digestion was usually completed within 2 hours at 37°C.

[0098] Stability of MB-Trypsin: MB-Trypsin is fully active in buffers containing up to 0.1% SDS, in buffers containing up to 4 M urea, and at –80°C after up to 3 cycles of freeze-thaw.

[0099] Example 3

[0100] Labeling of proteins and peptides with mTet-CDM. Proteins or peptides were labeled with mTet-CDM under two different buffer conditions. Briefly, proteins at a concentration of 0.5 mg / mL in 100 mM HEPES pH 8.0 and 25 mM NaCl were incubated with mTet-CDM (30 mg / mL in ACN) where mTet-CDM was 1-6 times more abundant than the protein. Labeling is reported as mTet-CDM was always added in a volume less than 10% of the total reaction volume. The reaction was incubated for 1 hour at 4°C. This labeling process was also repeated in a buffer containing 10 mM HEPES pH 8.0 and 25 mM NaCl.

[0101] Determine the extent of mTet labeling using a fluorescamine assay to detect free primary amines. Make a standard curve for unlabeled samples using 0-10 μg of unlabeled protein / proteome / peptide. Dilute each standard to a final volume of 250 μL of 10 mM HEPES, pH 8.0. Dissolve fluorescamine in acetone at a concentration of 3 mg / mL and add 50 μL of this solution to each solution and mix thoroughly. Repeat this for the mTet-labeled samples. Transfer the solutions to a black polystyrene flat-bottom Greiner from MilliporeSigma (St. Louis, MO). 96-well plates. Tecan Fluorescence was measured using a microplate reader. Excitation was performed at 400 nm with a bandwidth of 30 nm, and emission was detected at 460 nm with a bandwidth of 40 nm. A standard curve was created by plotting the fluorescence intensity versus free amines, and the amount of free amines in the mTet sample was found by solving for free amines using the fluorescence intensity values ​​and the standard curve equation.

[0102] Binding of mTet-labeled proteins to TCO-conjugated beads. TCO agarose beads were purchased from Click Chemistry Tools (Scottsdale, AZ). To test buffer conditions for preventing nonspecific binding, two stock solutions of unlabeled and 6X mTet-CDM-labeled Cy5-labeled carbonic anhydrase were prepared as described above, but with the following changes. After adding mTet-CDM, the samples were incubated at room temperature for 15 min, then 0.83 nmol of Cy5-NHS was added, and then incubated at 4°C for 1 hour. In a separate test tube, 0.15 μg of unlabeled or 6X mTet-labeled carbonic anhydrase (mTet-CA) was added to 100 μL of each of the following buffers: (A) 0.3M NaCl and 100mM HEPES, pH 8.0, (B) 1% SDS and 100mM HEPES, pH 8.0, (C) 0.3M NaCl, 1% SDS and 100mM HEPES, pH 8.0. 50 μL of TCO beads balanced in the same buffer were added to each buffer and incubated for 1 hour with rotation upside down at room temperature. Load controls for each sample were prepared by incubating the corresponding carbonic anhydrase in the corresponding buffer without adding TCO beads. Binding was assessed by running the supernatant and load controls of each sample on a 4-20% SDS-PAGE gel at 130V for 1 hour. Images were acquired using a built-in imager. ImageJ is used to quantify the amount of unbound labeled or unlabeled carbonic anhydrase via the pixel density of the fluorescent image. The loaded sample containing only the labeled or unlabeled carbonic anhydrase of the corresponding amount is used as a baseline. The amount of all other carbonic anhydrases is calculated as the percentage of loading. The amount combined with the TCO beads is calculated by subtracting the amount of free carbonic anhydrase in the solution from 100%.

[0103] In order to test the amount of time required for mTet-tagged proteins to fully bind to TCO beads, a binding time course was performed. TCO beads were balanced in 1% SDS, 0.3M NaCl, 100mM HEPES pH 8.0, and 30 μL of balanced beads were incubated with 25 μg 6XmTet-CA, with a final total volume of 80 μL. The reaction solution was incubated at 4 ° C with 1400rpm shaking for 0min, 5min, 15min, 30min or 60min. After the specified incubation time, the beads were centrifuged into a precipitate, and the supernatant was removed. In the absence of TCO beads, a loading control was prepared by suspending the same amount of 6X mTet-CA in the same amount of buffer. Binding was assessed by running the supernatant and loading control of each sample on a 4-20% SDS-PAGE gel for 1 hour at 130V. Images were acquired using a built-in imager. The amount of mTet-CA carbonic anhydrase that remained unbound was quantified using ImageJ via the pixel density of the fluorescent image. The loaded samples containing only the corresponding amount of mTet-CA were used as baseline. All other mTet-CA amounts were calculated as percentages of loading. The amount bound to the TCO beads was calculated by subtracting the amount of mTet-CA remaining free in solution from 100%.

[0104] The mTet-labeled proteins were eluted from the TCO beads. The mTet-labeled proteins were eluted from the TCO beads using the elution buffer (20mM citrate pH 3.0, 7M urea, 2M thiourea, 10mM DTT, 4% CHAPS) for two-dimensional difference gel electrophoresis (2D-DIGE). As previously described, mTet-CA was bound to the beads and 100 μL of binding buffer containing 10mM HEPES pH 8.0 was used instead of 100mM HEPES pH 8.0 for washing 3 times. Reducing the concentration of the HEPES buffer during washing facilitates the elution step. Proteins were eluted from the beads by incubation at room temperature with 50 μL of elution buffer at 1400 rpm. The supernatant was transferred to a new test tube, and the elution step was repeated three more times. The elution incubation time was 15 min, 30 min, 60 min and 120 min. The yield of each incubation time from the beads was evaluated, and the protein elution of each individual wash was also analyzed. A loading control for each sample was prepared by incubating a starting amount of mTet-CA in elution buffer. The percentage of CA released was assessed using SDS-PAGE and image analysis as described previously. All other amounts of carbonic anhydrase were calculated as a percentage of the load to obtain the yield percentage. For each individual wash, the pixel counts were summed to obtain the total pixels of carbonic anhydrase throughout the elution process. Each lane pixel density was then calculated as a percentage of the total concentration to determine how much protein was eluted in each wash step.

[0105] The entire yeast proteome was captured, washed and released using 1D and 2D gel electrophoresis. Yeast lysates were prepared using strain JWY6147 leu2 his3 trp1 ura3 provided by John Woolford's lab at Carnegie Mellon University. The clumped yeast cells were resuspended in lysis buffer, subjected to 2D-DIGE, and glass beads were added, equal to half of the total volume. The cells were vortexed and lysed six times with glass beads at 30 second intervals, each time on ice for 30 seconds. After lysis, cell debris was precipitated by centrifugation at 7,000 rpm for 10 min, and the supernatant was transferred to a new test tube. Protein concentration was obtained using standard Bradford analysis.

[0106] After quantification, the proteome was 6X labeled with mTet-CDM and labeled with Cy5 as described above. In a LoBind Eppendorf tube, 200 μL TCO beads were washed 3 times with 200 μL lysis buffer. Next, 100 μg of 6X mTet-labeled proteome was added to the beads and incubated at room temperature for 1 hour with shaking at 1400 rpm. The supernatant was removed and discarded, and the beads were washed 3 times with 200 μL lysis buffer. After washing, proteins were eluted from the beads by adding 50 μL 2D-DIGE elution buffer and mixing and incubating at 1400 rpm for 30 min. After elution, the supernatant was removed and transferred to a new tube. This operation was repeated twice. After all the eluates were combined together, the yield was first tested by running the sample on a 4-20% SDS-PAGE gel at 130 V for 1 hour. The eluate was run with a loading sample containing the starting amount of proteome used in this experiment. Yields were quantified as described previously.

[0107] In order to carry out 2D-DIGE analysis to the eluted proteome, as previously described, 20 μg of unlabeled proteome was labeled with Cy3 in 2D-DIGE lysis buffer for use as a control. After labeling, 57 μL control samples were merged with 57 μL eluted samples. 2.4 μL 3-10NL IPG buffer (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania) was added to the sample, and the sample was run using 2D-DIGE. IPG test paper (18 cm, pH 3-10NL) was purchased from GE Healthcare Bio-Sciences (Pittsburgh, Pennsylvania), and rehydrated for 16 hours with a rehydration buffer containing 0.2% 3-10NL IPG buffer. Isoelectric focusing (IEF) was performed using the Protean IEF instrument purchased from Bio-Rad (Hercules, CA). Balance was performed using the previously described in-gel method. Two-dimensional SDS-PAGE was performed on a Protein II xi electrophoresis instrument purchased from Bio-Rad (Hercules, CA) using 12% separating gel. Gel images were acquired using the built-in imager. Comparative stacks were created using ImageJ. First, the brightness and contrast were set to comparative levels using the guide stars on the gel. Then, the color of each gel was changed using the lookup table function. The Cy3 of the control was set to green, while the Cy5 of the eluate was set to red. Each image type was then changed to RGB color and the paste control was set to add. From there, all control images were selected and added to the eluate image.

[0108] Labeling of individual proteins with mTet-CDM. After purifying mTet-CDM, we next wanted to know how much mTet-CDM was needed to efficiently label primary amines on proteins. We labeled the protein carbonic anhydrase with mTet-CDM in the presence of 10 mM HEPES pH 8.0 or 100 mM HEPES pH 8.0 and analyzed the extent of labeling using a fluorescamine assay. The labeling efficiency was much higher in 100 mM HEPES pH 8.0 than in 10 mM HEPES pH 8.0 ( Figure 8). While labeling in 10 mM HEPES pH 8.0 equilibrated at about 30%, labeling in 100 mM HEPES equilibrated at about 80%. The higher HEPES concentration likely helps stabilize the slightly alkaline pH, thereby ensuring the proper protonation state of the required primary amines on the protein and opening of the maleic anhydride ring. The need for higher HEPES concentrations has also been demonstrated by previous students working with biotin-CDM. Based on this, all labeling in this study was performed in 100 mM HEPES pH 8.0 using 6X labeling.

[0109] Optimizing buffer conditions and incubation times for binding of mTet-tagged proteins to TCO beads. To optimize conditions for binding of mTet-tagged proteins to TCO beads, we first established appropriate buffer conditions to prevent nonspecific binding. We knew from previous experience with avidin beads that proteins tend to adhere to the beads nonspecifically, resulting in lower protein yields. We also knew that high SDS and high salt present in the buffer could help prevent nonspecific binding. To find buffer conditions that would prevent nonspecific binding to TCO beads, we incubated mTet-tagged and unlabeled carbonic anhydrase with TCO beads in the presence of 0.3 M NaCl, 1% SDS, or both. We found that 0.3 M NaCl alone did not prevent nonspecific binding of unlabeled protein to the beads, with an 83% loss of unlabeled protein on the beads ( Fig. 9 ). Surprisingly, while 1% SDS alone did prevent nonspecific binding, it also greatly reduced the binding of the mTet-tagged protein to the TCO beads, to only 52% binding. The buffer containing both 0.3M NaCl and 1% SDS showed the best results, with no detectable nonspecific binding and up to 88% binding of the tagged protein to the TCO beads. This is consistent with previous work using biotin-CDM and avidin beads, where a buffer containing 0.3M NaCl + 1% SDS also gave the best results. For all future assays in this chapter, use the buffer used for binding to the TCO beads, which contains 0.3M NaCl, 1% SDS, and 100mM HEPES pH 8.0.

[0110] To determine the best buffer conditions to prevent non-specific binding to TCO beads, mTet-labeled or unlabeled carbonic anhydrase was incubated with TCO beads in different binding buffers. Proteins without mTet tags represent any binding via non-specific interactions, while mTet-labeled samples represent binding via specific interactions. Samples lacking TCO beads are the load used to calculate the % binding to TCO beads. Buffers containing 0.3M NaCl and 1% SDS performed best in preventing non-specific binding of unlabeled carbonic anhydrase to TCO beads, while still allowing mTet-labeled carbonic anhydrase to bind to TCO beads.

[0111] We next tested the incubation time required for complete binding of mTet-tagged proteins to TCO beads. A time course was performed using a range of mTet-CA and TCO beads and binding was measured over the course of 1 h. After only 30 min, 94% of mTet-CA was bound to the TCO beads ( Fig.10 ). After this time point, binding was almost unchanged, increasing by 3% after 1 hour to reach 97% binding. Based on this time course, all future binding of mTet-tagged proteins was performed using an incubation time of 30 min.

[0112] Optimize elution incubation time and number of washes. To optimize the elution of proteins from TCO beads, two factors need to be considered: the incubation time in elution buffer and the number of washes in elution buffer required for complete removal from the beads. Because there is a certain amount of dead volume in agarose beads, proteins cannot be removed in just one elution step by removing the supernatant after incubation in elution buffer. Instead, the beads must be incubated in elution buffer and then the proteins collected in multiple washes of the beads to ensure that no protein is lost to the dead volume of the beads. However, each wash in elution buffer dilutes the final protein concentration, and excessive dilution is undesirable for proteomics techniques such as 2D-DIGE and MS, especially when low-abundance proteins are required. Finding the right balance between incubation time and number of washes is important to ensure that proteins are eluted quickly and in a concentrated manner from the TCO beads.

[0113] mTet-CA was allowed to bind to the TCO beads and then eluted from the beads using four washes and increasing incubation times. The yield for each incubation time was calculated and compared. All elution times showed a yield greater than 80%, with the highest yield (94%) at 30 min ( Fig.11(A)). This is consistent with previous work showing that dimethylmaleic anhydride can be recycled in as little as five minutes. Protein elution from each wash was analyzed to determine the point at which all protein had eluted from the beads. In the first two washes, over 85% of the eluted protein came off the beads, while three washes resulted in 99% of all eluted protein ( Fig.11 (B)). Based on these results, elutions for the remainder of this study used three washes with incubations of 30 min each.

[0114] Capture, wash, and release of the entire yeast proteome using mTet-CDM and TCO beads.

[0115] With capture and release of individual proteins producing yields exceeding 90%, we next sought to apply this technology to capture, wash, and release of the entire proteome. Yeast cells were lysed and cell debris removed from solution. The proteome was 6X labeled with mTet-CDM and captured, washed, and released using previously established parameters. The eluate was then run on an SDS-PAGE gel for quantification. After the entire sample preparation workflow using mTet-CDM and TCO beads, more than 80% of the proteome was recovered ( Fig.12 ). This yield is much higher than sample preparation methods currently used in the art, suggesting that this technique has promise for proteome sample preparation. However, this yield is lower than that observed for individual proteins and may be due to the presence of interfering molecules in the lysate. More work is needed to optimize the workflow for whole proteome purification.

[0116] The eluted yeast proteome was also analyzed using 2D-DIGE. While 2D-DIGE cannot be used for quantification, it can show specific protein losses and make any systematic biases in our sample preparation methods apparent. The eluted proteins were labeled with Cy5 dye and the loading control was labeled with Cy3 dye. These samples were combined and then separated by their isoelectric point and molecular weight. The gel was then imaged using Cy3 and Cy5 filters. The two images were overlaid to show any protein loss between the two samples. If there was a protein loss bias in the workflow, there would be specific spots that appeared only in the Cy3 loaded sample and not in the eluted Cy5 sample. Upon comparing the loaded and eluted proteomes, it was observed that most proteins overlapped well with each other ( Fig.13). It should be noted that the eluted proteome was first labeled with mTet-CDM, which absorbs the same primary amine used to attach the Cy dye. This means that labeling with the Cy5 dye is much darker than the loading control used for comparison, which may make it appear as if spots are present in the load that are not present in the elution, when the spots are simply not visible. Although these results are encouraging, dye labeling with thiol groups rather than primary amines would prevent differences in spot brightness between loading and elution, allowing quantification. Future experiments should explore alternatives to dye labeling or comparison of separate gels using silver staining as well as the use of reciprocal labeling if dye labeling is still used to ensure that dye-dependent changes are not observed.

[0117] Example 4

[0118] Burkholderia cenocepacia lysate was cleaned up using spin columns.

[0119] The bacterial culture pellet of Burkholderia cepacia was dissolved by boiling in a solution of 100mM HEPES pH 8.0, 2% SDS for 5 minutes. The viscous solution was briefly sonicated for about 15 seconds at 30% power with a probe sonicator. The concentration of the resulting bacterial cell lysate was 120mg / ml. The reaction was carried out for 1 hour at room temperature (RT) with a mixture of 153.3μl H2O, 20μl 1M HEPES pH 8, 10μl 10mg / ml Burkholderia cepacia lysate and 16.7μl 30mg / ml mTet-CDM. 150μl TCO magnetic beads (purchased from Click Chemistry Tools (Scottsdale, AZ)) were washed 3 times in 500μl 100mM HEPES pH 8+10% ACN.

[0120] Process samples as follows:

[0121] 1. Remove 20 μl of the reaction for loading control;

[0122] 2. Add 180 μl of reaction solution to the beads in the cold room - mix by inversion for 1 hour (Note: the solution turns from pink to colorless in about 15 minutes)

[0123] 3. Wash beads 3x 400 μl WB [7M urea, 2M thiourea, 4% CHAPS, 10 mM DTT] + 1 mM HEPES pH 8

[0124] 4. Elution 2x 50μl WB + 5mM formic acid

[0125] 5. Run 10% of each step on SDS-PAGE. Prepare mock eluate with lysate in WB + 5mM formic acid

[0126] 6. Combine the eluates and run, make alkaline with 10 mM TEAB, label with Cy3-NHS, and run on 2DE gel and 2D electrophoresis gel ( Fig.14 and 15 ).

[0127] Example 5

[0128] The following evaluates how much labeling is required to achieve near 100% recovery of a peptide mixture containing alcohol dehydrogenase, carbonic anhydrase lysozyme, and bovine serum albumin. Table A provides the reaction mixtures (μl) tested.

[0129] Table A

[0130]

[0131] The samples were processed as follows:

[0132] Set up labeling reaction as above and incubate for 1 hour at room temperature.

[0133] During the incubation, 225 μl of TCO beads were washed 3X per reaction with 400 μl of 100 mM HEPES pH 8.0, 0.2% SDS.

[0134] Wash the beads in the column frit.

[0135] The beads were dried completely before adding the diluted labeling reaction.

[0136] The labeling reaction volume was adjusted to 500 μl using 100 mM HEPES pH 8.0, 0.2% SDS.

[0137] Add the diluted labeling reaction to the washed TCO beads in the open-end column frit.

[0138] The columns were taped into Eppendorf tubes and used as adapters for the rotisserie.

[0139] Incubate in a cold room oven for 1 hour.

[0140] The supernatant was recovered.

[0141] The beads were washed twice with 10 mM HEPES pH 8.0, 25% ACN.

[0142] The beads were washed 2 times with water.

[0143] Add 120 μl 0.1% formic acid and incubate with stirring (1500 RPM) for 1.5 hours at 8°C (build block set to 4°C, only reaches 7°C).

[0144] The eluate was recovered.

[0145] Add 120 μl of 0.1% formic acid 50% ACN and incubate at 7°C with agitation (1500 RPM) for 1.5 hours.

[0146] The eluate was recovered.

[0147] Add 120 μl of 0.1% formic acid 50% ACN and incubate at 7°C with agitation (1500 RPM) for 1 hour.

[0148] The eluate was recovered.

[0149] Four standard curves were set up to match the various buffers in the samples.

[0150] Input standard curve: 100 mM HEPES pH 8.0, 30% ACN.

[0151] Supernatant standard curve: 100 mM HEPES pH 8.0, 1.35% SDS

[0152] Elution 1 Standard Curve: 0.1% FA

[0153] Elution 2,3 Standard curve: 0.1% FA, 50% ACN

[0154] Sample preparation for the fluorescamine assay is as follows:

[0155] Input: 17.5 μl of 70 μl labeling reaction + 182.5 μl 100 mM HEPES pH 8.0.

[0156] Supernatant: 125 μl of 500 μl supernatant + 75 μl of 100 mM HEPES pH 8.0.

[0157] Elution: 30 μl of 120 μl Elution Buffer + 170 μl of 100 mM HEPES pH 8.0.

[0158] To each sample, 50 μl of 3 mg / ml fluorescamine in acetone was added, mixed thoroughly, and then 200 μl was aliquoted into 96-well plates.

[0159] Results (Table B, Fig.16 )

[0160] Table B

[0161] enter Total elution %enter 0X 26.52 2.60 9.81 21X 13.81 52.06 45X 16.03 60.47 75X 24.79 93.48

[0162] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be appreciated by those skilled in the art that various substitutions, modifications or combinations may be made to any of the exemplary embodiments without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed. Sequence Listing <110> Carnegie Mellon University A. Lucas J. Minden <120> Protein and Peptide Purification Methods <130> 6526-1902651 <150> US 62 / 763,298 <151> 2018-06-08 <150> US 62 / 919,469 <151> 2019-03-14 <160> 1 <170> PatentIn 3.5 version <210> 1 <211> 231 <212> PRT <213> Pig (Sus scrofa) <400> 1 Phe Pro Thr Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Thr Cys Ala 1 5 10 15 Ala Asn Ser Ile Pro Tyr Gln Val Ser Leu Asn Ser Gly Ser His Phe 20 25 30 Cys Gly Gly Ser Leu Ile Asn Ser Gln Trp Val Val Ser Ala Ala His 35 40 45 Cys Tyr Lys Ser Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Asp 50 55 60 Val Leu Glu Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys Ile Ile Thr 65 70 75 80 His Pro Asn Phe Asn Gly Asn Thr Leu Asp Asn Asp Ile Met Leu Ile 85 90 95 Lys Leu Ser Ser Pro Ala Thr Leu Asn Ser Arg Val Ala Thr Val Ser 100 105 110 Leu Pro Arg Ser Cys Ala Ala Ala Gly Thr Glu Cys Leu Ile Ser Gly 115 120 125 Trp Gly Asn Thr Lys Ser Ser Gly Ser Ser Tyr Pro Ser Leu Leu Gln 130 135 140 Cys Leu Lys Ala Pro Val Leu Ser Asp Ser Ser Cys Lys Ser Ser Tyr 145 150 155 160 Pro Gly Gln Ile Thr Gly Asn Met Ile Cys Val Gly Phe Leu Glu Gly 165 170 175 Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn 180 185 190 Gly Gln Leu Gln Gly Ile Val Ser Trp Gly Tyr Gly Cys Ala Gln Lys 195 200 205 Asn Lys Pro Gly Val Tyr Thr Lys Val Cys Asn Tyr Val Asn Trp Ile 210 215 220 <h2 style=";text-align:left;direction:ltr">Gln Gln Thr Ile Ala Ala Asn<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 225 230

Claims

1. A coupling compound having the following structure:

2. A method for purifying a polypeptide, comprising: Mixing a sample containing a polypeptide or a polypeptide digested by a protease at an alkaline pH with a certain amount of the coupling compound of claim 1; coupling the polypeptide in the sample to a second member of a bioorthogonal coupling pair attached to a matrix; optionally washing the polypeptide bound to the matrix to remove any unbound material from the matrix-bound polypeptide; and The polypeptide is eluted from the matrix in an elution solution at an acidic pH.

3. The method of claim 2, wherein the substrate is a bead or a solid surface. The method according to claim 3 , wherein the substrate is magnetic beads.

5. The method of claim 3, wherein the matrix is ​​beads contained in a chromatography column or a spin column.

6. The method according to any one of claims 2 to 5, wherein the substrate is a porous substrate.

7. The method of any one of claims 2-5, wherein the first member of the bioorthogonal coupling pair is a tetrazine group and the second member of the bioorthogonal coupling pair is a strained cyclic olefin.

8. The method of claim 7, wherein the strained cyclic olefin is trans-cyclooctene.

9. The method of claim 2, wherein the polypeptides in the sample are digested with a modified trypsin polypeptide, the modified trypsin polypeptide comprising: Trypsin peptides; a biotin moiety attached to the trypsin polypeptide; and a group comprising a member of a bioorthogonal coupling pair attached to the trypsin polypeptide, The ratio of the group comprising a member of a bioorthogonal coupling pair on the trypsin polypeptide to the biotin moiety is 1:4 to 1:

6.

10. A protein isolation kit comprising the compound of claim 1 in a container.

11. The protein isolation kit according to claim 10, further comprising in the package a matrix comprising side groups, the side groups forming bioorthogonal anti-electron demand Diels-Alder conjugation pairs with the tetrazine groups of the compound.

12. The protein isolation kit according to claim 11, wherein the side group that forms a bioorthogonal anti-electron demand Diels-Alder conjugation pair with the tetrazine group of the compound is a strained cyclic olefin moiety.

13. The protein separation kit according to claim 12, wherein the strained cyclic olefin moiety is trans-cyclooctene.

14. The protein separation kit according to any one of claims 11 to 13, wherein the matrix is ​​a bead, a solid surface or a porous matrix.

15. The protein separation kit according to any one of claims 11 to 13, wherein the matrix is ​​a bead or a porous matrix contained in a chromatography column or a spin column.

16. The protein separation kit according to claim 14, wherein the matrix is ​​a bead or a porous matrix contained in a chromatography column or a spin column.

17. The protein separation kit according to any one of claims 11 to 13, wherein the matrix is ​​magnetic beads.

18. The protein separation kit according to any one of claims 10 to 13, wherein the container is a separate compartment in a cassette for an automated protein separation system.

19. The protein separation kit according to claim 14, wherein the container is a separate compartment in a cassette for an automated protein separation system.

20. The protein separation kit according to claim 15, wherein the container is a separate compartment in a cassette for an automated protein separation system.

21. The protein separation kit of claim 16, wherein the container is a separate compartment in a cassette for an automated protein separation system.

22. The protein separation kit of claim 17, wherein the container is a separate compartment in a cassette for an automated protein separation system.

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