Antibody-independent ubiquitination enrichment and detection methods

Abstract

The present invention discloses a non-antibody-dependent protein ubiquitination enrichment method, including whole protein extraction, complete blocking of free amino groups at the protein level, hydrolysis of ubiquitin chains on substrate proteins, labeling of free amino groups at ubiquitination sites with enrichable label reagents, protein enzymatic cleavage, and enrichment of labeled ubiquitinated peptides. The present invention also relates to a detection method, i.e., liquid phase-mass spectrometry detection of the enriched ubiquitinated peptides. The present invention does not require the use of antibodies to recognize the ubiquitin residual motifs produced after enzymatic cleavage, and can achieve indiscriminate enrichment and identification of protein ubiquitination.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an antibody-independent ubiquitination modification enrichment method and a detection method. Background Art

[0002] Protein post-translational modification plays an important role in biological responses to various stimuli and in regulating biological processes quickly and accurately. Ubiquitination is a widely existing reversible protein post-translational modification that can regulate a variety of basic characteristics of substrate proteins, including stability, activity or localization. Ubiquitin is a highly conserved small protein containing 76 amino acids in eukaryotes. The C-terminal glycine of ubiquitin is covalently linked to the lysine residues on the substrate protein through the E1-E2-E3 cascade reaction, and the process can be reversed by deubiquitinase. A single ubiquitin molecule modifies a single lysine residue on the substrate protein to form a monoubiquitination modification. Multiple ubiquitin molecules modify multiple lysine residues on the substrate protein to form a polyubiquitination modification. Since the ubiquitin molecule itself contains 7 lysine residues (K6, K11, K27, K29, K33, K48 and K63), the 7 lysine residues carried by the ubiquitin molecule that has been covalently modified on the substrate protein can continue to be covalently modified by other ubiquitin molecules to form a ubiquitin chain. In addition, the methionine at the N-terminus of the ubiquitin molecule protein also has a free amino group (M1), which can also be covalently modified by other ubiquitin molecules to form a linear ubiquitin chain. Therefore, monoubiquitination, polyubiquitination, and 8 different forms of homogeneous ubiquitin chain modifications can exist in cells. If a ubiquitin chain binds to different lysine residues of ubiquitin itself to form a mixed or branched ubiquitin chain, the complexity of the ubiquitin chain connection type will be further increased. Ubiquitin chains of different connection types are recognized by effector proteins with different ubiquitin binding domains, mediating different signaling pathways. In addition, the dynamic changes of ubiquitination modification control many important biological processes, such as cell division, fate regulation, and migration. The abnormal function or regulation of ubiquitinases leads to disordered protein ubiquitination modification levels, which are closely related to a variety of diseases, such as cancer and neurodegeneration. The identification of ubiquitinated proteins and their modification sites is a prerequisite for studying what biological functions ubiquitination modification plays. Therefore, deep coverage of protein ubiquitination modification analysis is of great significance for studying the molecular mechanism of its biological function. Currently, mass spectrometry-based proteomics is a very effective method for studying protein post-translational modifications. However, protein ubiquitination substrates are difficult to analyze directly due to their low content. In order to improve the identification sensitivity and coverage of protein ubiquitination substrates, researchers have developed a series of enrichment methods to enrich ubiquitinated proteins or peptides, such as using ubiquitin binding domains (UBDs), ectopic ubiquitin expression (Ectopic Expression) and stable tagged ubiquitin exchange (StableTagged Ubiquitin Exchange, StUbEx) to enrich ubiquitinated proteins or peptides. However, these enrichment strategies are difficult to provide accurate information on ubiquitination sites or are limited in the types of samples they are applicable to.Since the ubiquitination modification sites will retain double glycine-modified lysine (K-ε-GG) after trypsin hydrolysis of ubiquitinated proteins, accurate identification of ubiquitination modification sites can be achieved through anti-K-ε-GG pan-antibody enrichment and mass spectrometry analysis. This experimental strategy has been widely used in the study of ubiquitination modification (however, the trypsin hydrolyzed peptides of the modified substrates of ubiquitin-like proteins NEDD8 and ISG15 also contain K-ε-GG structures, so it is difficult for anti-K-ε-GG pan-antibodies to distinguish ubiquitinated modified peptides from modified peptides of ubiquitin-like proteins. After Vyacheslav et al. used the intracellular protease Lys-C to enzymatically digest the protein, thirteen amino acids of lysine (K-ε-ESTLHLVLRLRGG) remained at the ubiquitination modification site, avoiding the interference of the modified substrates of ubiquitin-like proteins NEDD8 and ISG15. However, the thirteen amino acids remaining at the ubiquitination site are relatively long, which will affect the effective identification of protein ubiquitination sites by mass spectrometry. In addition, antibodies have a preference for substrate sequence recognition, different batches of antibodies have different specificities, and the cost of antibody enrichment methods is also high. Therefore, it is of great significance to establish an enrichment method that is not antibody-dependent. Summary of the invention

[0003] One of the purposes of the present invention is to provide an antibody-independent protein ubiquitination modification enrichment method.

[0004] The technical solutions for achieving the above-mentioned purpose include the following.

[0005] A method for enriching protein ubiquitination modification, comprising the following steps:

[0006] S1. extracting and obtaining a whole protein sample from the biological sample to be tested;

[0007] S2, using a chemical modification method to block the free amino groups in the released whole protein sample to obtain substrate protein;

[0008] S3, hydrolyzing the ubiquitin chain on the blocked substrate protein: adding a deubiquitinating enzyme to the substrate protein for enzymatic cleavage to obtain an S3 sample in which the ε amino group of the lysine at the ubiquitination site modification is exposed;

[0009] S4, labeling the ε-amino site of ubiquitinated lysine with an enrichable label reagent to obtain an S4 sample;

[0010] S5, digesting the S4 sample with a proteolytic enzyme to obtain an S5 sample;

[0011] S6. Corresponding to the enrichable tags in S4, the ubiquitinated peptides labeled in the S5 sample are enriched.

[0012] Another object of the present invention is to provide a method for detecting protein ubiquitination.

[0013] A method for detecting protein ubiquitination comprises obtaining enriched ubiquitinated modified peptides according to the above method, and detecting the enriched ubiquitinated modified peptides using liquid chromatography-mass spectrometry technology.

[0014] In some of the embodiments, the dosage of the USP family enzyme is 1 / 50-1 / 500 (mass ratio) of the substrate protein, and the enzyme digestion is carried out at 25° C.-45° C. for 1-12 hours.

[0015] In some embodiments, the deubiquitinating enzyme family includes USP family enzyme, CYLD deubiquitinating enzyme, or OTUD3, or vOTU.

[0016] Furthermore, the USP family enzyme includes one or more of USP1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP15, USP16, USP20, USP21, USP25, USP27x, and USP28.

[0017] In some embodiments, the whole protein sample extraction includes: resuspending the sample to be tested using 7-9M urea or 3-5% SDC (sodium deoxycholate) or 5-7M GuaHCl (guanidine hydrochloride) lysis solution, and then further lysing and releasing the whole protein in the cells or tissues by ultrasound, and taking the supernatant after centrifugation.

[0018] In some of the embodiments, in step S2: adding an amino-active reagent at a concentration of 10-200 mM to the whole protein sample, adding sodium cyanoborohydride at a concentration of 5-100 mM, blocking at 16° C.-80° C. for 0.25-12 h, to obtain a substrate protein.

[0019] In some embodiments, in step S4: adding a reagent containing an enrichment tag at a concentration of 5 mM-50 mM to the S3 sample, labeling for 0.25-12 h at 16° C.-80° C., and removing the residual reaction reagent in the labeled sample using an ultrafiltration tube or molecular exclusion chromatography to obtain a sample D;

[0020] In some of the embodiments, in step S5: a protease is added to the S4 sample, the enzyme dosage is 1:20-1:200 of the protein, and the enzymatic digestion is carried out at 16°C-45°C for 2-24 hours to obtain the S5 sample; the protease is preferably one or more of trypsin, clostripain Arg-C, endoproteinase Glu-C, metalloproteinase Lys-N, chymotrypsin, papain, elastase, and pepsin.

[0021] Reagents that can enrich tags include NHS-SS-Biotin, NHS-Biotin, and NHS-PEG4-Biotin.

[0022] In some of the embodiments, in step S6: add 10 μL-100 μL of a material that can enrich the enrichment tag (for example, a streptavidin matrix material, such as streptavidin gel balls, streptavidin magnetic beads) to the S5 sample, incubate for 0.5-12 hours, obtain a material bound to the modified peptide segment, and use an eluent to elute the enriched peptide segment from the material.

[0023] In some embodiments, the reagent used to block the free amino groups in step S2 is one or more of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acetic anhydride, propionic anhydride, butyric anhydride, acetic acid-N-succinimide, propionic acid-N-succinimide, butyric acid-N-succinimide, 1H-pyrazole-1-carboxamidine hydrochloride, preferably formaldehyde.

[0024] In some of the embodiments, the reagent capable of enriching the label in step S4 is an amino-active reagent with a biotin label (such as NHS-SS-Biotin, NHS-Biotin, NHS-PEG4-Biotin), and the material for enriching the labeled peptides in step S6 is a streptavidin matrix material.

[0025] For biotin-amino active reagents containing disulfide bonds (such as NHS-SS-Biotin), the eluent is 20mM DTT, the reaction is carried out at 16℃-50℃ for 0.5-2h, and finally 50mM IAA is added for alkylation under dark conditions;

[0026] For biotin-amino-active reagents without disulfide bonds (such as NHS-Biotin, NHS-PEG4-Biotin), the elution solution is 10 mM EDTA (pH 8.2) and 95% formamide, and the reaction is carried out at 90°C for 10 minutes.

[0027] In some of the embodiments, the enriched peptides are subjected to mass spectrometry analysis, and the detector is Orbitrap, iontrap, tof, or quadrupole.

[0028] In some embodiments, the biological sample to be detected is one or more of fungi, cells, tissues, blood and body fluids.

[0029] The present invention utilizes the deubiquitinating enzyme family to construct a set of simple, practical, easy-to-operate non-antibody-dependent protein ubiquitination modification enrichment methods and detection methods. The method can be used to study specific types of ubiquitination modification patterns, supplementing the shortcomings of the current antibody recognition ubiquitination modification enrichment method that cannot study specific ubiquitin chain modified substrates and has sequence recognition preferences.

[0030] 1. The present invention relates to an antibody-independent ubiquitination enrichment method, which can enrich ubiquitinated modified peptides without the need for specific antibodies;

[0031] 2. The present invention can identify specific types of ubiquitination substrates and their modification sites for different types of deubiquitinating enzymes. For example, the deubiquitinating enzymes of the USP family (USP1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP15, USP16, USP20, USP21, USP25, USP27x, USP28) have low specificity and can basically hydrolyze all types (M1, K6, K11, The CYLD deubiquitinase can specifically hydrolyze K63 and M1-linked ubiquitin chains, the OUTD3 deubiquitinase can specifically hydrolyze K6 and K11-linked ubiquitin chains, the OTUB1 deubiquitinase can highly specifically hydrolyze K48-linked ubiquitin chains, OTULIN can highly specifically hydrolyze MI-linked ubiquitin chains, and AMSH and BRCC3 deubiquitinases can highly specifically hydrolyze K63-linked ubiquitin chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 , detection of the ubiquitin chain removal effect of deubiquitinating enzyme USP2 in different reaction systems.

[0033] Figure 2 , flow chart of the antibody-independent ubiquitination modification enrichment method described in Example 2 of the present invention.

[0034] Figure 3 , protein ubiquitination modification detection results in HeLa cells.

[0035] Figure 4 , flow chart of the antibody-independent ubiquitination modification enrichment method described in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0039] In some embodiments of the present invention, a non-antibody-dependent protein ubiquitination modification enrichment method is involved, and the entire ubiquitinated peptide enrichment process includes the following parts: (S1) first lyse cells or tissues to extract proteins; (S2) then use an amino-active reagent to block the free amino groups of the protein; (S3) use a deubiquitinating enzyme that can remove ubiquitin chains to hydrolyze the ubiquitin chains on the substrate protein, and new free amino groups will be generated at the protein ubiquitination modification site; (S4) use an amino-active reagent NHS-SS-Biotin containing an enrichment tag to label the newly generated lysine side chain amino group; (S5) use a proteolytic enzyme to enzymatically treat the protein; (S6) use streptavidin balls to enrich the labeled peptides to obtain ubiquitinated modified peptides.

[0040] Afterwards, the enriched peptides can be detected and analyzed using liquid chromatography-mass spectrometry to obtain information on protein ubiquitination modification sites.

[0041] In some embodiments, the antibody-independent protein ubiquitination modification enrichment method and detection method include the following steps:

[0042] 1. Whole protein extraction

[0043] Prepare 8 M urea, 0.1 M HEPES, pH 8.0 lysis buffer, add 10 times the volume of lysis buffer to the cell or tissue sample, and further fully lyse by ultrasound or homogenizer, centrifuge at 20000g, 4°C, and collect the supernatant to obtain sample A.

[0044] 2. Blocking of free amino groups at the protein level

[0045] The free amino groups of the total protein sample are blocked. An amino-active reagent with a concentration of 10-200 mM is added to sample A, and sodium cyanoborohydride with a concentration of 5-100 mM is added. The sample is blocked at 16°C-80°C for 0.25-12 hours, and the residual reaction reagent in the blocked sample is removed by ultrafiltration tube or molecular exclusion chromatography to obtain sample B.

[0046] The amino-active reagent for fully blocking the free amino groups of the protein is one or more of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acetic anhydride, propionic anhydride, butyric anhydride, acetic acid-N-succinimide, propionic acid-N-succinimide, butyric acid-N-succinimide, and 1H-pyrazole-1-carboxamidine hydrochloride.

[0047] 3. Ubiquitin chain removal

[0048] 0.1M HEPES, pH 8.0, was added to sample B to dilute the urea concentration to below 2M, and an enzyme for removing ubiquitinated modified chains was added at an enzyme dosage of 1 / 50-1 / 200 of the protein. Under conditions of 25°C-45°C, the enzyme was digested for 1-12 hours to obtain sample C.

[0049] The enzymes used to remove ubiquitin modification belong to the deubiquitinase family of proteins.

[0050] 4. NHS-SS-Biotin reagent containing enrichable groups labels the free amino groups at the ubiquitination modification sites of proteins

[0051] Add NHS-SS-Biotin reagent with a concentration of 5mM-50mM to sample C, label for 0.5-2h at 16°C-50°C, and use ultrafiltration tube or molecular exclusion chromatography to remove residual reaction reagents in the sample to obtain sample D.

[0052] 5. Protein digestion

[0053] Add 0.1M HEPES, pH 8.0, to sample D to dilute the urea concentration in the solution to below 2M, add protease according to the mass of the protein, the amount of protease is 1 / 50-1 / 200 of the protein, and digest at 16°C-45°C for 2-24h to obtain sample E.

[0054] The enzyme used for protein enzymatic cleavage is one of trypsin, clostripain Arg-C, endoproteinase Glu-C, metalloproteinase Lys-N, chymotrypsin, papain, elastase, pepsin, or a combination of more than one of them.

[0055] 6. Biotin-pullown enrichment of biotin-labeled ubiquitinated peptides

[0056] a. Add 20mM Tris-HCl, pH 7.6, 1mM EDTA, and 0.4% Triton X-100 to the enzyme-cleaved peptide solution;

[0057] b. Take 40 μL of streptavidin matrix beads, centrifuge at 5,000 g for 3 min, remove the supernatant, and wash three times with PBS;

[0058] c. Add the enzyme-cleaved peptide solution to the streptavidin matrix ball and incubate at room temperature for 2 hours;

[0059] d. Centrifuge at 5,000 g for 3 min, remove the supernatant, and use washing buffer (20 mM Tris-HCl, pH

[0060] 7.6, 1 mM EDTA, 0.4% Triton X-100, 600 mM NaCl) to wash the streptavidin matrix beads bound to the biotin-labeled ubiquitinated peptides 4 times, and then wash them 3 times with PBS;

[0061] e. Add 20 mM dithiothreitol to resuspend the streptavidin matrix beads bound to the biotin-labeled ubiquitinated peptides and incubate at 50°C for 30 min;

[0062] f. Add iodoacetamide at a final concentration of 50 mM and incubate at room temperature in the dark for 45 min;

[0063] Centrifuge at 5,000 g for 5 min and take the supernatant to obtain the enriched ubiquitinated peptide sample F;

[0064] 7. Liquid chromatography-mass spectrometry analysis

[0065] Sample F was desalted using a C18 StageTip and then subjected to liquid chromatography-mass spectrometry analysis. The generated mass spectrometry data was analyzed by software to obtain protein ubiquitination modification site information.

[0066] The detectors used for mass spectrometry analysis of the enriched peptides are one or more of Orbitrap, ion trap, tof, quadrupole.

[0067] In order to facilitate the understanding of the present invention, the present invention will be described in more detail below. The following examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and basic ideas of the present invention should be regarded as equivalent replacement methods and should be included in the protection scope of the present invention.

[0068] Example 1

[0069] In order to verify the feasibility of the method described in the present invention, we first purified the low-specificity chain-selective deubiquitinating enzymes USP2 and USP21, and evaluated the ubiquitin chain removal efficiency in different reaction systems.

[0070] The deubiquitinating enzyme USP2 or USP21 was incubated with HeLa lysate in different reaction systems, and then the changes in the ubiquitin chain modification levels in HeLa protein were detected by western blot.

[0071] The experiment includes the following steps:

[0072] S1: Using cervical cancer HeLa cells as samples, the sample was divided into 3 parts, and ①8M urea, 100mM HEPES, pH8.0; ②4% sodium deoxycholate, 100mM HEPES, pH 8.0; ③6M guanidine hydrochloride, 100mM HEPES, pH 8.0 were used as lysis buffer to lyse HeLa cells and extract proteins, and the BCA kit was further used to quantify the protein in the HeLa lysate.

[0073] S2: 100 μg of each cell lysate was taken and diluted 1-fold, 2-fold, 4-fold and 8-fold with 100 mM HEPES, pH 8.0 buffer.

[0074] S3: Add dithiothreitol at a final concentration of 5 mM, add deubiquitinating enzyme USP2 or USP21 at a ratio of 1:200 (mass ratio) of enzyme to substrate, and incubate at 37°C for 2 h.

[0075] S4: Further western blotting experiments were performed using P4D1 antibody to detect the signal intensity of ubiquitinated modified proteins in different reaction systems.

[0076] like Figure 1The results showed that after USP2 was incubated with sodium deoxycholate cell lysate or guanidine hydrochloride cell lysate diluted at different times, the ubiquitination signal intensity was very high, and there was no significant change compared with the samples incubated without USP2, indicating that the USP2 enzyme activity was very low in the sodium deoxycholate and guanidine hydrochloride reaction system and could not hydrolyze the ubiquitin chain on the ubiquitinated protein; after USP2 was incubated in urea cell lysate diluted 1 times (8M) and 2 times (4M), the ubiquitination signal intensity was also very high, and there was no significant change compared with the samples incubated without USP2. When the urea concentration in the cell lysate was diluted 4 times (2M) and 8 times (1M) and then incubated with USP2, the ubiquitination signal intensity was very weak, and there was no significant change compared with the samples incubated without USP2. The activity of USP2 in the samples incubated with 1% urea was significantly reduced, indicating that the enzyme activity of USP2 was very high in urea concentration of 2M and below (the experimental results showed that if the reaction system contained sodium deoxycholate or guanidine hydrochloride, even if it was diluted to a relatively low concentration, the deubiquitinating enzymes USP2 and USP21 would be inactivated, while if the reaction system contained urea, the deubiquitinating enzymes USP2 and USP21 could maintain high activity when the urea concentration was diluted to 2M and below.), which could significantly remove the ubiquitin chain modification on HeLa protein. The experimental results of USP21 were completely consistent with those of USP1, indicating that the method of the present invention could successfully expose the lysine ε amino group at the ubiquitin chain modification site of the protein through the deubiquitinating enzyme, and provide the possibility for subsequent further labeling with enrichable tags.

[0077] Embodiment 2:

[0078] The antibody-independent protein ubiquitination modification enrichment method of this embodiment comprises the following steps:

[0079] S1: Using cervical cancer Hela cells as samples, Figure 2 Technical route. 8M urea, 100mM HEPES, pH8.0 was used as lysis solution to lyse Hela cells and extract proteins. The BCA kit was further used to quantify the protein in the HeLa lysate. According to the BCA quantitative results, the protein concentration of the HeLa lysate was diluted to 1mg / mL.

[0080] S1: Take 1 mg of HeLa protein solution, add sodium cyanoborohydride with a final concentration of 20 mM and formaldehyde with a final concentration of 40 mM, incubate at 37°C for 2 h, then add Tris-HCl with a final concentration of 100 mM, pH 8.0 to terminate the reaction;

[0081] S2: The dimethylated HeLa protein lysate was transferred to a 10 kDa ultrafiltration tube, centrifuged at 14,000 g for 30 min, and the flow-through was discarded; then, the tube was washed three times with an 8 M urea aqueous solution, and then 3 volumes of 100 mM HEPES, pH 8.0, were added to dilute the urea concentration in the solution to 2 M;

[0082] S3: Add dithiothreitol at a final concentration of 5 mM, and hydrolyze the ubiquitin chain on the substrate protein according to the mass ratio of deubiquitinase USP2 to protein of 1:200, digest at 37°C for 2 h; centrifuge at 14,000 g for 30 min, and discard the flow-through;

[0083] S4: Then use 8M urea aqueous solution to wash three times. Add NHS-SS-Biotin reagent with a final concentration of 10mM, react at 30℃ for 2h, then add Tris-HCl with a final concentration of 100mM, pH 8.0 to terminate the reaction. Centrifuge at 14,000g for 30min, discard the flow-through; then use 8M urea aqueous solution to wash three times, and then use 100mM HEPES, pH 8.0 to wash three times;

[0084] S5: Digest the protein at a trypsin to protein mass ratio of 1:50 at 37°C for 12 h. Centrifuge at 14,000 g for 30 min and collect the flow-through, i.e., the peptide solution after digestion.

[0085] S6: Use streptavidin beads to enrich the peptides containing biotin labeling, and incubate at room temperature for 2 hours. Finally, add dithiothreitol at a final concentration of 20mM for elution, react at 50℃ for 30 minutes to open the disulfide bond between the biotin labeling reagent and the peptide, and further add 50mM iodoacetamide, incubate at room temperature in the dark for 30 minutes, and desalt and dry the eluted peptides.

[0086] S7: The dried peptides were subjected to LC-MS / MS analysis and MaxQuant search of the original files to identify the ubiquitinated peptides.

[0087] like Figure 3 As shown, using MaxQuant software retrieval, three technical repeat experiments identified 1167, 1115, and 1138 ubiquitination modification sites, respectively, corresponding to 896, 847, and 897 ubiquitinated peptides and 666, 649, and 672 ubiquitinated proteins, respectively, indicating that the technical route of the present invention can effectively identify protein ubiquitination modification. At the same time, the numbers of ubiquitination modification sites, peptides, and proteins identified in the three technical repeat experiments are comparable, indicating that the method of the present invention has high experimental stability and can be well applied to the research of life science issues.

[0088] Embodiment 3:

[0089] The non-antibody-dependent method for enriching protein linear M1-linked ubiquitination modification in this embodiment comprises the following steps:

[0090] S1: Using cervical cancer Hela cells as samples, Figure 2Technical route. Use 8M urea, 100mM HEPES, pH8.0 as lysis solution to lyse Hela cells and extract protein. Further use BCA kit to quantify the protein in HeLa lysate. According to the BCA quantification results, the protein concentration of HeLa lysate was diluted to 1mg / mL;

[0091] Take 1 mg of HeLa protein solution, add sodium cyanoborohydride with a final concentration of 20 mM and 40 mM acetaldehyde, incubate at 37 °C for 2 h, then add Tris-HCl with a final concentration of 100 mM, pH 8.0 to terminate the reaction;

[0092] S2: The dimethylated HeLa protein lysate was transferred to a 10 kDa ultrafiltration tube, centrifuged at 14,000 g for 30 min, and the flow-through was discarded; then, the tube was washed three times with an 8 M urea aqueous solution, and then 3 volumes of 100 mM HEPES, pH 8.0, were added to dilute the urea concentration in the solution to 2 M;

[0093] S3: Add dithiothreitol at a final concentration of 5 mM, and hydrolyze the ubiquitin chain on the substrate protein according to the mass ratio of deubiquitinating enzyme OTULIN to protein of 1:200, digest at 37°C for 2 h; centrifuge at 14,000 g for 30 min, and discard the flow-through;

[0094] S4: Then use 8M urea aqueous solution to wash three times. Add NHS-SS-Biotin reagent with a final concentration of 10mM, react at 30℃ for 2h, then add Tris-HCl with a final concentration of 100mM, pH 8.0 to terminate the reaction. Centrifuge at 14,000g for 30min, discard the flow-through; then use 8M urea aqueous solution to wash three times, and then use 100mM HEPES, pH 8.0 to wash three times;

[0095] S5: Digest the protein at a trypsin to protein mass ratio of 1:50 at 37°C for 12 h. Centrifuge at 14,000 g for 30 min and collect the flow-through, i.e. the peptide solution after digestion;

[0096] S6: Use streptavidin beads to enrich the peptides containing biotin labeling, and incubate at room temperature for 2 hours. Finally, add dithiothreitol at a final concentration of 20mM for elution, react at 50℃ for 30 minutes to open the disulfide bond between the biotin labeling reagent and the peptide, and further add 50mM iodoacetamide, incubate at room temperature in the dark for 30 minutes, and desalt and dry the eluted peptides;

[0097] S7: The dried peptides were subjected to LC-MS / MS analysis and MaxQuant search of the original files to identify the ubiquitinated peptides.

[0098] Embodiment 4:

[0099] The antibody-independent protein ubiquitination modification enrichment method of this embodiment comprises the following steps:

[0100] S1: Human plasma was used as the sample. Figure 2 Technical route. 8M urea, 100mM HEPES, pH 8.0 lysis buffer was added to dilute the plasma and denature the plasma protein. The plasma sample was further quantified using a BCA kit, and the plasma protein concentration was further diluted to 1mg / mL based on the BCA quantitative results;

[0101] Take 1 mg of plasma protein solution, add sodium cyanoborohydride with a final concentration of 20 mM and formaldehyde with 40 mM, incubate at 37 °C for 2 h, then add Tris-HCl with a final concentration of 100 mM, pH 8.0 to terminate the reaction;

[0102] S2: The diethyl labeled plasma protein lysate was transferred to a 10 kDa ultrafiltration tube, centrifuged at 14,000 g for 30 min, and the flow-through was discarded; then, 8 M urea aqueous solution was used for washing three times, and then 3 volumes of 100 mM HEPES, pH 8.0, were added to dilute the urea concentration in the solution to 2 M;

[0103] S3: Add dithiothreitol at a final concentration of 5 mM, and hydrolyze the ubiquitin chain on the substrate protein according to the mass ratio of deubiquitinase USP2 to protein of 1:200, digest at 37°C for 2 h; centrifuge at 14,000 g for 30 min, and discard the flow-through;

[0104] S4: Then use 8M urea aqueous solution to wash three times. Add NHS-SS-Biotin reagent with a final concentration of 10mM, react at 30℃ for 2h, then add Tris-HCl with a final concentration of 100mM, pH 8.0 to terminate the reaction. Centrifuge at 14,000g for 30min, discard the flow-through; then use 8M urea aqueous solution to wash three times, and then use 100mM HEPES, pH 8.0 to wash three times;

[0105] S5: Digest the protein at a trypsin to protein mass ratio of 1:50 at 37°C for 12 h. Centrifuge at 14,000 g for 30 min and collect the flow-through, i.e. the peptide solution after digestion;

[0106] S6: Use streptavidin beads to enrich the peptides containing biotin labeling, and incubate at room temperature for 2 hours. Finally, add dithiothreitol at a final concentration of 20mM for elution, react at 50℃ for 30 minutes to open the disulfide bond between the biotin labeling reagent and the peptide, and further add 50mM iodoacetamide, incubate at room temperature in the dark for 30 minutes, and desalt and dry the eluted peptides;

[0107] S7: The dried peptides were subjected to LC-MS / MS analysis and MaxQuant search of the original files to identify the ubiquitinated peptides.

[0108] Embodiment 5:

[0109] The antibody-independent protein ubiquitination modification enrichment method of this embodiment comprises the following steps:

[0110] S1: Human colorectal cancer tissue was used as sample. Figure 4 The tissue was minced, 8M urea, 100mM HEPES, pH 8.0 were added, and the tissue was lysed using a homogenizer. The plasma sample was further quantified using a BCA kit, and the protein concentration of the human colorectal cancer tissue protein extract was diluted to 1mg / mL based on the BCA quantitative results.

[0111] Take 1 mg of tissue protein solution, add sodium cyanoborohydride with a final concentration of 20 mM and formaldehyde with 40 mM, incubate at 37 °C for 2 h, then add Tris-HCl with a final concentration of 100 mM, pH 8.0 to terminate the reaction;

[0112] S2: The dimethylated labeled tissue protein lysate was transferred to a 10 kDa ultrafiltration tube, centrifuged at 14,000 g for 30 min, and the flow-through was discarded; then, 8 M urea aqueous solution was used for washing three times, and then 3 volumes of 100 mM HEPES, pH 8.0, were added to dilute the urea concentration in the solution to 2 M;

[0113] S3: Add dithiothreitol at a final concentration of 5 mM, and hydrolyze the ubiquitin chain on the substrate protein according to the mass ratio of deubiquitinase USP21 to protein of 1:200, digest at 37°C for 2 h; centrifuge at 14,000 g for 30 min, and discard the flow-through;

[0114] S4: Then use 8M urea aqueous solution to wash three times. Add NHS-Biotin reagent with a final concentration of 10mM, react at 30℃ for 2h, then add Tris-HCl with a final concentration of 100mM, pH 8.0 to terminate the reaction. Centrifuge at 14,000g for 30min, discard the flow-through; then use 8M urea aqueous solution to wash three times, and then use 100mM HEPES, pH 8.0 to wash three times;

[0115] S5: Digest the protein at a trypsin to protein mass ratio of 1:50 at 37°C for 12 h. Centrifuge at 14,000 g for 30 min and collect the flow-through, i.e. the peptide solution after digestion;

[0116] S6: Use streptavidin beads to enrich the biotin-labeled peptides and incubate at room temperature for 2 hours. Finally, add 10mM EDTA (pH 8.2) and 95% formamide for elution at 90°C for 10 minutes, and desalt and dry the eluted peptides.

[0117] S7: The dried peptides were subjected to LC-MS / MS analysis and MaxQuant search of the original files to identify the ubiquitinated peptides.

[0118] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for enriching protein ubiquitination modification, characterized in that: The following steps are involved: S1. Extracting a whole protein sample from the biological sample to be tested: resuspending the sample to be tested with 7-9M urea, further lysing and releasing the whole protein in the cells or tissues by ultrasound, and taking the supernatant after centrifugation to obtain the whole protein sample; S2. Add 10-200 mM amino-active reagent to the whole protein sample, add 5-100 mM sodium cyanoborohydride, and block for 0.25-12 h at 16°C-80°C to obtain substrate protein; dilute the urea concentration in the solution to 2 M or less; S3, hydrolyzing the ubiquitin chain of the blocked substrate protein: adding a deubiquitinating family enzyme to the substrate protein, performing enzymatic cleavage, and obtaining an S3 sample in which the epsilon amino group of lysine at the ubiquitination site modification is exposed; the deubiquitinating family enzyme includes a USP family enzyme; the USP family enzyme includes one or more of USP1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP15, USP16, USP20, USP21, USP25, USP27x, and USP28; S4, labeling the ε amino site of ubiquitinated lysine with an enrichable label reagent to obtain an S4 sample; S5, digesting the S4 sample with a proteolytic enzyme to obtain an S5 sample; S6, enriching the ubiquitinated peptides labeled in the S5 sample in a manner corresponding to the enrichable tags in S4; Wherein, the reagent capable of enriching the label in step S4 is an amino-active reagent with a biotin label, and the material for enriching the labeled peptide segment in step S6 is a streptavidin matrix material.

2. The protein ubiquitination enrichment method according to claim 1, characterized in that: The mass ratio of the USP family enzyme to the substrate protein is 1:50-500, and the enzyme cleavage is performed for 1-12 hours at 25°C-45°C.

3. The protein ubiquitination enrichment method according to claim 1, characterized in that: The concentration of urea in S1 is 8M.

4. The protein ubiquitination enrichment method according to claim 1, characterized in that: In step S2: add 40 mM amino-active reagent to the whole protein sample, add 20 mM sodium cyanoborohydride, and block for 0.25-12 h at 37° C. to obtain substrate protein.

5. The protein ubiquitination enrichment method according to claim 1, characterized in that: In step S4: add a reagent containing an enrichment tag at a concentration of 5mM-50mM to the S3 sample, label it at 16°C-80°C for 0.25-12h, and use an ultrafiltration tube or molecular exclusion chromatography to remove the residual reaction reagent in the labeled sample to obtain sample D.

6. The protein ubiquitination enrichment method according to claim 1, characterized in that: In step S5: add proteolytic enzyme to the S4 sample, the mass ratio of enzyme to protein is 1:20-1:200, and perform enzymatic digestion for 2-24 hours at 16°C-45°C to obtain the S5 sample.

7. The protein ubiquitination enrichment method according to claim 6, characterized in that: The protein hydrolase is one or more of trypsin, clostripain Arg-C, endoproteinase Glu-C, metalloproteinase Lys-N, chymotrypsin, papain, elastase, and pepsin.

8. The protein ubiquitination enrichment method according to claim 1, characterized in that: In step S6: add 10 μL-100 μL of a reagent that can enrich the enrichment tag to the S5 sample, incubate for 0.5-12 hours, obtain a material bound to the modified peptide segment, and use an eluent to elute the enriched peptide segment from the material.

9. The protein ubiquitination modification enrichment method according to any one of claims 1 to 8, characterized in that: In step S2, the reagent for blocking the free amino group is one or more of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, acetic anhydride, propionic anhydride, butyric anhydride, acetic acid-N-succinimide, propionic acid-N-succinimide, butyric acid-N-succinimide, and 1H-pyrazole-1-carboxamidine hydrochloride.

10. The protein ubiquitination enrichment method according to any one of claims 1 to 8, characterized in that: The biological sample to be detected is one or more of fungi, cells, tissues, blood and body fluids.

11. A method for detecting protein ubiquitination, characterized in that: The method comprises obtaining enriched ubiquitinated modified peptides according to the method according to any one of claims 1 to 10, and then detecting the enriched ubiquitinated modified peptides by liquid chromatography-mass spectrometry.

Images