Method for identifying modified enzyme substrate based on photoreaction activity probe

Abstract

The invention discloses a method for identifying a modified enzyme substrate based on a photoreaction activity probe, and particularly relates to a method established by utilizing different substrate recognition capabilities of a wild-type modified enzyme and a mutant-type modified enzyme. The probes are used for respectively reacting with wild type and mutant type modified enzymes to obtain the wild type and mutant type modified enzyme probes with photoreaction activity. The method comprises the following steps: respectively incubating a wild type modified enzyme probe and a mutant modified enzyme probe with a biological sample, then carrying out photo-crosslinking, respectively enriching cross-linked substrates, and quantitatively comparing the substrates enriched by the wild type modified enzyme probe and the mutant modified enzyme probe to specifically identify the modified substrates.

Description

Technical Field

[0001] The invention belongs to the research field of substrate identification in the research direction of chemical biology, and specifically relates to a method for specifically identifying a modified substrate by comparing the difference between the enriched substrates of a wild-type and a mutant-type modified enzyme probe. Background Art

[0002] Modification of substrates mediated by modifying enzymes plays an important regulatory role in a variety of biological processes, so effectively identifying the substrates of modifying enzymes is of great significance for revealing the regulatory mechanism of modifying enzymes. Analogs of donors can label substrates of modifying enzymes (Reference 1. Wang, R.; Zheng, WH; Yu, HQ; Deng, HT; Luo, MK, Labeling substrates of protein arginine methyltransferase with engineered enzymes and matched S-adenosyl-L-methionine analogues. Journal of the American Chemical Society, 2011, 133: 7648-7651.), but most modifying enzymes cannot be effectively modified using analogs, which limits their use. Affinity purification coupled with mass spectrometry (AP-MS) is an effective method for identifying interactions, and is also used to identify substrates of modifying enzymes (Reference 2. Dansu, DK; Liang, JL; Selcen, I.; Zheng, HY; Moore, DF; Casaccia, P., PRMT5 interacting partners and substrates in oligodendrocyte lineage cells. Frontiers in Cellular Neuroscience, 2022, 16: 820226.), however, strong interactions will inhibit transient and weak interactions, and cannot effectively identify weak substrates.The proximity labeling technology Biotin identification (BioID) is also used to identify substrates (Reference 3. Wei, HH; Fan, XJ; Hu, Y.; Tian, ​​XX; Guo, M.; Mao, MW; Fang, ZY; Wu, P.; Gao, SX; Peng, C.; Yang, Y.; Wang, ZF, A systematic survey of PRMTinteractomes reveals the key roles of arginine methylation in the globalcontrol of RNA splicing and translation. Science Bulletin, 2021, 66: 1342-1357.), however, this strategy cannot distinguish between substrates and non-substrates. Current methods are inefficient in identifying substrates of modifying enzymes, so effective methods are urgently needed to identify modified substrates.

[0003] In order to effectively distinguish substrates from non-substrates, the present invention specifically identifies modified substrates by comparing substrates enriched by wild-type and mutant modified enzyme photoreactive activity probes. Summary of the invention

[0004] The object of the present invention is to provide a method for specifically identifying a modified enzyme substrate which is simple, low-cost and has good repeatability.

[0005] The method provided by the present invention is a method established by utilizing the difference in substrate recognition ability between the wild-type modification enzyme probe and the mutant modification enzyme probe. The abundance of the substrate enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe is monitored, and the difference in the abundance of the substrate enriched by the two probes is compared to specifically identify the modification substrate.

[0006] The specific steps of the method for identifying modified enzyme substrates based on photoreactive probes are as follows:

[0007] (1) reacting a wild-type modification enzyme and a mutant modification enzyme with a probe to obtain a wild-type modification enzyme probe and a mutant modification enzyme probe with photoreactivity, respectively;

[0008] (2) incubating the wild-type modification enzyme probe and the mutant modification enzyme probe with the biological sample, photocrosslinking, and enriching the crosslinked substrate;

[0009] (3) detecting the abundance of the enriched substrate in the wild-type modification enzyme probe and the mutant modification enzyme probe respectively;

[0010] (4) The ratio of the abundance of the substrate enriched by the wild-type modification enzyme probe / the mutant modification enzyme probe is greater than 1 (preferably greater than 1.2, most preferably greater than 1.5), which means that it is the substrate of the wild-type modification enzyme.

[0011] The modification enzyme can be one or more of a protein modification enzyme, a DNA modification enzyme, an RNA modification enzyme, etc., but is not limited to these modification enzymes; 1-3 amino acid residues on the active center where the wild-type modification enzyme binds to the substrate (the site in the wild-type modification enzyme that can directly bind to the substrate and catalyze the chemical reaction of the substrate; the active center is divided into a binding site, a part that binds to the substrate, and a catalytic site, a site that catalyzes the chemical reaction of the substrate; the amino acid residues in the binding site are mutated, and the wild-type modification enzyme loses the ability to bind to the substrate) are mutated (1-3 amino acids in the binding site of the wild-type modification enzyme are changed into other amino acids different from it), and mutations that do not destroy the spatial conformation of the modification enzyme are preferably selected.

[0012] The probe comprises a reactive group, a linker, a photoreactive group and an enrichment group; the reactive group, the photoreactive group and the enrichment group are connected by a linker; the linker is one or more of polyethylene glycol (degree of polymerization 200-8000), alkane (chain length 3-20 carbon atoms), etc., but not limited to these groups; the reactive group can react with one or more of the amino group, carboxyl group, etc. on the modified enzyme, which can be one or more of N-hydroxysuccinimide, hydroxyl group, guanidine group, etc., but not limited to these groups; the photocrosslinking group is one or more of benzophenone, azidophenyl, diaziridine, etc., but not limited to these groups; the enrichment group is one or more of alkynyl, azide, biotin, etc., but not limited to these groups.

[0013] The wild-type modification enzyme and the mutant modification enzyme are expressed and purified respectively, and reacted with the probe in a buffer respectively to obtain the wild-type modification enzyme probe and the mutant modification enzyme probe; the buffer includes one or more of PBS, hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, etc., but is not limited to these buffers; the molar ratio of the modification enzyme to the probe is 1:1-1:100 (preferably 1:10), and the preferred molar ratio is to maintain the spatial conformation of the modification enzyme; the reaction time of the probe and the modification enzyme is 1-120s (preferably 60s), and the preferred reaction time is that one modification enzyme protein molecule reacts with only one probe.

[0014] The biological sample includes one or more proteins, DNA, RNA, etc., one or more mixtures thereof; one or more mixtures thereof include one or more tissue and / or cell extracts; the tissues or cells are one or more tissues or cells derived from bacteria, animals, plants or humans; the tissue and / or cell extracts are obtained by lysing the tissues and / or cells using a buffer, and the natural spatial conformation of the proteins, DNA or RNA, etc. in the biological sample should be maintained at this time; the preferred buffer contains 100-500mM sodium chloride and a 5-100mM tris(hydroxymethyl)aminomethane aqueous solution with a volume concentration of 0.5-2% ethylphenyl polyethylene glycol, and the tissues and / or cells are ultrasonically crushed and lysed in the buffer.

[0015] Incubation process: the wild-type modification enzyme probe and the mutant modification enzyme probe are incubated with the biological sample maintaining the natural conformation, the optimal time for the maximum binding amount of the modification enzyme and the substrate is 1-4 hours, and a mixture of the modification enzyme and the substrate is obtained; the photocrosslinking process: the natural conformation of the modification enzyme under ultraviolet irradiation is maintained, and the mixture of the modification enzyme and the substrate is ultraviolet irradiated for 10-60 minutes to obtain a mixture of the modification enzyme and the substrate after ultraviolet crosslinking; the enrichment process: the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe are pulled down by using the enrichment material, and the enrichment material is incubated with the mixture of the modification enzyme and the substrate after ultraviolet crosslinking for 1-5 hours (preferably 2 hours) to obtain the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe and the non-specific adsorption of the enrichment material; the enrichment material includes one or more of hydrazide resin, Avidin agarose beads, etc., but is not limited to these enrichment materials; the non-specific adsorption of the enrichment material is washed according to The concentration of the washing buffer is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and volume concentration of 0.1-10% sodium dodecyl sulfate; the washing buffer II is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and 0.1-10 M urea; the washing buffer III is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and 0.1-10 M sodium chloride; after washing the non-specific adsorption of the enriched material, the substrate is retained on the enriched material to obtain a substrate enriched with the wild-type modification enzyme probe and the mutant modification enzyme probe; the substrates enriched with the wild-type modification enzyme probe and the mutant modification enzyme probe are respectively enzymolyzed with a protease to obtain an enzymolysis solution, and the impurities in the enzymolysis solution are respectively removed with a C18 solid phase extraction column to facilitate subsequent mass spectrometry analysis to obtain the abundance of the wild-type modification enzyme probe and the mutant modification enzyme probe enriched substrate, but the method is not limited to the above method.

[0016] The detection of substrate abundance includes substrates enriched by wild-type modification enzyme probes and substrates enriched by mutant modification enzyme probes; the abundance (Intensity) of the substrate includes one or more of the relative content of the molecule, the peak area of ​​gas chromatography, the peak area of ​​liquid chromatography, etc., but is not limited to these; the method for detecting the abundance of the modification enzyme probe enriched substrate includes one or more of the techniques of Southern blot, Northern blot, Western blot, quantitative proteomics, etc., but is not limited to the above methods; the quantification method of proteins or peptides in quantitative proteomics includes one or two of label-free quantification or labeled quantification; wherein the labeled quantification method includes one or more of isotope labeling methods such as SILAC, dimethyl labeling, ITRAQ or TMT.

[0017] The screening criteria for modification enzyme substrates are that the abundance ratio of the wild-type modification enzyme probe / mutant modification enzyme probe enriched substrate is greater than 1 (preferably greater than 1.2, most preferably greater than 1.5), that is, the screened wild-type modification enzyme substrate; the threshold value can be appropriately adjusted according to different methods for detecting substrate abundance (the optimal threshold value is determined by the maximum sensitivity and specificity values).

[0018] Advantages of the present invention:

[0019] The present invention is based on the strategy of photoreactive probes to specifically identify the transient substrate of the modification enzyme, so that the substrate and the modification enzyme are covalently linked, which is conducive to the removal of non-specific adsorption; by comparing the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe, the modification substrate can be specifically identified; the present invention has a wide range of applications, and the method can be extended to various types of modification enzymes. By simply changing the probe skeleton, the interaction between other enzymes and substrates can be effectively identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The flowchart of the method for identifying the substrate of the modified enzyme is shown in FIG. 1. (A) The chemical structure of the photoreactive modified enzyme probe. (B) The wild-type modified enzyme probe and the mutant modified enzyme probe are incubated with the biological sample respectively, and then UV cross-linked, and the cross-linked substrates are enriched respectively, and the substrates enriched by the wild-type and mutant modified enzyme probes are quantitatively compared.

[0021] Figure 2 Flow chart for identifying substrate proteins of the PRMT1 methyltransferase. (A) Chemical structure of the photoreactive PRMT1 probe. (B) The wild-type PRMT1 methyltransferase probe and the mutant PRMT1 methyltransferase probe were incubated with low-methylation-modified cell lysate, followed by UV cross-linking, enrichment of cross-linked proteins, LC-MS / MS analysis of enzymatic peptides, and quantitative comparison of substrate proteins enriched by the wild-type and mutant PRMT1 methyltransferase probes.

[0022] Figure 3 The modified enzyme substrate identification method is applied to the identification of PRMT1 methyltransferase substrate proteins. (A) Differentially enriched proteins of wild-type and mutant PRMT1 methyltransferase probes. (B) Identified substrate proteins of PRMT1 methyltransferase.

[0023] Figure 4 Verification of PRMT1 methyltransferase substrate protein. (A) Western blotting analysis of methyltransferase probe pull-down substrate protein. (B) In vitro methylation experiment of substrate protein.

[0024] Figure 5 LC-MS / MS analysis of substrate protein methylation sites for in vitro methylation experiments. DETAILED DESCRIPTION

[0025] This example takes the identification of PRMT1 methyltransferase substrate protein as an example. By comparing the proteins enriched by the wild-type PRMT1 methyltransferase probe and the mutant PRMT1 methyltransferase probe, the substrate protein can be specifically identified. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0026] Example

[0027] The specific steps of the method for identifying the substrate protein of PRMT1 methyltransferase are as follows:

[0028] (1) 1×10 cells cultured in 10 mL of RPMI-1640 medium 7 Adenosine dialdehyde (Sigma, A7154) was added to each HeLa cell at a final concentration of 20 μM, cultured at 37°C for 48 h, washed three times with 1 mL of 10 mM PBS buffer (pH 7.4), and then 1 mL of a buffer solution containing 50 mM tris(hydroxymethyl)aminomethane, pH 7.4, and 150 mM sodium chloride was added, and a final concentration of 1% ethylphenyl polyethylene glycol and a final concentration of 1% protease inhibitor cocktail (Sigma, p8340) were added;

[0029] (2) ultrasonication was performed for 3 cycles at 150 W, each cycle for 6 s, and centrifuged at 15,000 g for 10 min to obtain a low-methylation modified cell lysate;

[0030] (3) The two acidic amino acids in the substrate-binding active center of the mouse wild-type PRMT1 methyltransferase (PRMT1 structural information PDBID: 1OR8) (Reference 4. Zhang, X.; Cheng, XD, Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides. Structure, 2003, 11: 509-520.) were mutated into basic amino acids (aspartic acid at position 46 was mutated into lysine, and glutamic acid at position 50 was mutated into arginine) (Reference 5. Lee, DY; Ianculescu, I.; Purcell, D.; Zhang, X.; Cheng, XD; Stallcup, MR, Surface-scanning mutational analysis of protein arginine methyltransferase 1: Roles of specific aminoacids in methyltransferase substrate specificity, oligomerization, and coactivator function. Molecular Endocrinology,2007,21:1381-1393.), and the mutant PRMT1 methyltransferase was obtained; the sequences of wild-type and mutant PRMT1 methyltransferase were fully synthesized by Nanjing Baode Biotechnology Co., Ltd., and 5 μl of wild-type (unmutated mouse PRMT1 methyltransferase, including the mouse PRMT1 methyltransferase from the 11th to the 353rd amino acid, uniprot: Q63009) and 5 μl of mutant PRMT1 methyltransferase sequence (sequence length is consistent with the aforementioned wild-type PRMT1 methyltransferase (including the mouse PRMT1 methyltransferase from the 11th to the 353rd amino acid,), only 2 amino acid residues were mutated) and 10 μl pET28a vector (Biovector) were used to incubate the cells with 1 μl EcoRⅠ (15 U / μl) and 1 μl BamHI (15U / μl), (Takara, 1040S and 1010S) were used for double digestion at 30℃ for 12h, and the digestion products were subjected to nucleic acid electrophoresis to recover the sequences of wild-type and mutant PRMT1 and pET28a vector digestion. 5μl of wild-type and 5μl of mutant PRMT1 methyltransferase sequences obtained by digestion were connected with 5μl pET28a vector (Biovector) using 1μl DNA ligase (Takara, 2011A) and reacted at 37℃ for 12h; 10μl of the connected plasmids were mixed with 100μl BL21 (DE3) Escherichia coli competent cells, incubated at 4℃ for 30min, 42℃ for 90s, and 4℃ for 2min, and 1mL of fresh LB medium was added to the Escherichia coli competent cells containing wild-type and mutant PRMT1 plasmids, respectively, and incubated at 37℃ for 2h. The cells were centrifuged at 4000 g for 5 min, and 200 μl of the bacterial solution was spread on a plate containing LB medium containing 50 μg / mL kanamycin (Sangon Biotech (Shanghai) Co., Ltd., A506636-0025) and incubated at 37°C overnight. The next day, single clones were picked and cultured in 1 mL of LB medium containing 50 μg / mL kanamycin (Sangon Biotech (Shanghai) Co., Ltd., A506636-0025). When the OD600 value of the bacterial solution reached 0.5, 1 mL of the expression strain containing the wild-type and mutant PRMT1 plasmids was added to 200 mL of Continue to culture in LB medium. When the OD600 value of the bacterial solution reaches 0.8, add isopropyl-β-D-thiogalactoside (Takara, 9030) with a final concentration of 0.4 mM to 200 mL LB medium containing the expression strain of wild-type and mutant PRMT1 plasmids, induce expression at 16°C for 16 h, and centrifuge at 15000g for 30 min to obtain 200 mL bacterial solution expressing wild-type and mutant PRMT1 proteins.

[0031] (4) 10 mL of lysis buffer solution (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, final volume concentration of 0.5% polyethylene glycol octylphenyl ether, final mass concentration of 1 mg / L lysozyme, 10 mM imidazole, final volume concentration of 1% protease inhibitor cocktail, in water) was added to the bacterial solution obtained in step (3), and the mixture was ultrasonicated at a power of 150 W for 30 cycles, each cycle for 6 s, and centrifuged at 15000 g for 30 min to obtain lysates expressing wild-type and mutant PRMT1 proteins, respectively;

[0032] (5) The lysates expressing the wild-type and mutant PRMT1 proteins obtained in step (4) were mixed with 1 mL of nickel agarose beads (GE HealthCare, 17-5318-01), respectively, and added to SPE columns (Merck, specification: 500 mg / 6 ml), respectively, and washed three times with 1 mL of washing buffer solution (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, containing 30 mM imidazole, in water), and then eluted with 1 mL of elution buffer solution (10 mM PBS buffer, pH 7.4, containing 500 mM imidazole) to obtain purified wild-type and mutant PRMT1 proteins, respectively;

[0033] (6) 50 μg of purified wild-type and mutant PRMT1 proteins were reacted with 0.5 μl (10 mg / mL, dissolved in DMSO) of sulfo-N-hydroxysuccinimide-2-(6-[biotinylamino]-2-(p-azidobenzoylamino)-hexanoylamino)ethyl-1,3'-dithiopropionate (Thermo, 33033) with azide photocrosslinking group and biotin enrichment group in 50 μl 10 mM PBS buffer (pH 7.4) for 60 s. The molar ratio of protein to probe was 1:10. The unlabeled probe was removed by ultrafiltration at a centrifugal force of 14,000 g using an ultrafiltration tube with a molecular weight cutoff of 10K (Sartorius, VN01H02). The filtrate was then centrifuged using 1 mL 10 mM PBS buffer (pH 7.4). 7.4) Wash three times, and the proteins retained by the filter membrane are the photoreactive wild-type and mutant PRMT1 probes;

[0034] (7) 50 μg of the photoreactive wild-type and mutant PRMT1 probes were incubated with 300 μl of the low-methylated HeLa cell lysate obtained in step (2) at 4°C for 2 h, followed by UV cross-linking at a wavelength of 365 nm for 30 min. 50 μl of Avidin agarose beads (Thermo, 29201) were mixed with the cross-linked proteins and incubated at 4°C for 2 h. The cells were washed three times with 1 mL of washing buffer solution I (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, final volume concentration of 2% sodium dodecyl sulfate in water), and then washed three times with 1 mL of washing buffer solution II (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, 8 M uric acid, respectively). The beads were washed 3 times with 1 mL of washing buffer solution III (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, 2.5 M sodium chloride in water), and 200 μl of a buffer solution containing 6 M urea and 50 mM tris(hydroxymethyl)aminomethane was added to Avidin agarose beads (Thermo, 29201). A final concentration of 20 mM dithiothreitol was added, and the beads were incubated in a 37°C water bath for 2 h. Then, a final concentration of 40 mM iodoacetamide was added, and the beads were reacted at 25°C in the dark for 40 min. The beads were washed 3 times with 1 mL of washing buffer solution II (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, 8 M urea in water), and 1 mL of 50 The pellet was washed three times with 50 mM tris(hydroxymethyl)aminomethane and a buffer solution with a pH value of 8, and 200 μl of 50 mM tris(hydroxymethyl)aminomethane and a buffer solution with a pH value of 8 was added to the Avidin agarose beads (Thermo, 29201). Then, trypsin (Sigma, T1426) was added at a ratio of 1 / 50 to the protein mass, and enzymolysis was performed in a water bath at 37°C for 16 h to obtain a protein enzymolysis solution.

[0035] (8) Add trifluoroacetic acid to the obtained protein hydrolysate to adjust the pH to 2-3, activate a C18 solid phase extraction column (Waters, 10 mg, 186000383) with 600 μl of methanol, wash away the methanol with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, add 200 μl of the protein hydrolysate to a C18 solid phase extraction column (Waters, 10 mg, 186000383), wash with 600 μl of a final volume concentration of 0.1% trifluoroacetic acid aqueous solution, and then elute with 600 μl of a final volume concentration of 80% acetonitrile / 0.1% trifluoroacetic acid aqueous solution, and freeze-dry the obtained peptide eluate;

[0036] (9) The obtained peptide fragments were respectively redissolved in 20 μl of a final volume concentration of 0.1% acetic acid for RP LC-MS / MS analysis;

[0037] (10) The raw files of mass spectrometry were searched using the Maxquant search engine (Version 1.5.3.30), and quantitative analysis was performed using the standard-free quantitative method. The quantitative results were processed using the Perseus software (Version 1.6.1.1) to quantitatively compare the abundance of proteins enriched by the wild-type PRMT1 probe and the mutant PRMT1 probe (peak area of ​​liquid chromatography). A total of 23 proteins with different abundances were identified, of which 18 had an abundance ratio greater than 1.5 times ( Figure 3 A); Among the 18 proteins with abundance ratios greater than 1.5 times, 15 are known substrate proteins ( Figure 3B) (The BioGRID database collects substrate proteins of PRMT1 (Reference 6. Oughtred, R.; Rust, J.; Chang, C.; Breitkreutz, BJ; Stark, C.; Willems, A.; Boucher, L.; Leung, G.; Kolas, N.; Zhang, F., The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions. Protein Science, 2021, 30: 187-200.); Knockdown of PRMT1 down-regulated the expression of substrate proteins (Reference 7. Hartel, NG; Chew, B.; Qin, J.; Xu, J.; Graham, NA, Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets. Molecular & Cellular Proteomics, 2019, 18: 2149-2164.); the literature has reported that it is a substrate protein of PRMT1 (Reference 8. Akter, KA; Mansour, MA; Hyodo, T.; Ito, S.; Hamaguchi, M.; Senga, T., FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation. Tumor Biology, 2016, 37: 4531-4539.), 3 proteins are unknown substrate proteins including HNRNPA0 (uniprot: Q13151), ILF2 (uniprot: Q12905) and SBSN (uniprot: K7ESC4), the abundance ratio of HNRNPA0 is 6.0, the abundance ratio of ILF2 is 3.7, and the abundance ratio of SBSN is 4.4.

[0038] (11) Nanjing Biode Biotechnology Co., Ltd. produced the following proteins: HNRNPA0 (uniprot: Q13151) and ILF2 (uniprot: Q12905), which have never been reported as substrates of PRMT1, and HNRNPA1 (reference 9. Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, A. P.; Kim, H. J.; Mittag, T.; Taylor, J. P., Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell, 2015, 163: 123-133.) and HNRNPA2B1.

[0039] (Reference 10. Wang, L.; Wen, MY; Cao, XT, Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science, 2019, 365: eaav0758.) sequence was fully synthesized; 5 μl substrate protein sequence and 20 μl pGEX-6P-1

[0040] The (Biovector) vector was double-digested with 1 μl EcoRⅠ (15 U / μl) and 1 μl BamHI (15 U / μl), (Takara, 1040S and 1010S), respectively, at 30°C for 12 h, and the digestion products were subjected to nucleic acid electrophoresis to recover the digestion substrate protein and pGEX-6P-1 vector sequences. 5 μl of the substrate protein sequence obtained by digestion was ligated with 5 μl of pGEX-6P-1 (Biovector) vector using 1 μl DNA ligase (Takara, 2011A), and reacted at 37°C for 12 h; 10 μl of the ligated plasmid was mixed with 100 μl of BL21 (DE3) competent Escherichia coli cells, and incubated at 4°C for 30 min, 42°C for 90 s, and 4°C for 2 min, respectively. 1 mL of fresh LB was added to the competent E. coli cells containing the substrate protein plasmid, incubated at 37°C for 2 h, centrifuged at 4000 g for 5 min, 200 μl of the bacterial solution was spread on the plate containing 100 mg / mL ampicillin (Sangon Biotech (Shanghai) Co., Ltd., A100339-0005) of LB medium, incubated at 37°C overnight, and the next day, single clones were picked and cultured in 1 mL of LB medium containing 100 mg / mL ampicillin (Sangon Biotech (Shanghai) Co., Ltd., A100339-0005) respectively. When the OD600 value of the bacterial solution was 0.5, 1 mL of the expression strain containing the substrate protein plasmid was placed in 200 mL Continue to culture in LB medium. When the OD600 value of the bacterial solution reaches 0.6, add isopropyl-β-D-thiogalactoside (Takara, 9030) with a final concentration of 0.4 mM to 200 mL LB medium of the expression strain containing the substrate protein plasmid, induce expression at 25°C for 5 h, and centrifuge at 15000 g for 30 min to obtain 200 mL bacterial solution expressing the substrate protein.

[0041] (12) adding 10 mL of lysis buffer solution (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, final volume concentration of 0.5% polyethylene glycol octylphenyl ether, final mass concentration of 1 mg / L lysozyme, final volume concentration of 1% protease inhibitor cocktail, in water) to the bacterial solution obtained in step (11), ultrasonically disrupting the solution at a power of 150 W for 30 cycles, each cycle for 6 s, and centrifuging at 15000 g for 30 min to obtain a lysate expressing the substrate protein;

[0042] (13) The lysate of the expressed substrate protein obtained in step (12) was incubated with 1 mL of glutathione S-transferase agarose beads (Thermo, 16100) at 4°C for 2 h, added to an SPE column (Merck, specification: 500 mg / 6 ml), washed three times with 1 mL of a washing buffer solution (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, final volume concentration 0.5% polyethylene glycol octylphenyl ether, in water), and then eluted with 1 mL of an elution buffer solution (50 mM tris(hydroxymethyl)aminomethane, pH 7.4, 20 mM reduced glutathione, in water) to obtain a purified substrate protein;

[0043] (14) 10 μg of purified substrate proteins (HNRNPA0, HNRNPA1, HNRNPA2B1, and ILF2) were reacted with 5 μg of PRMT1 methyltransferase and 1 mM non-radioactive methyl donor SAM (Sigma, A4377) at 37°C for 1 h, and the reaction was terminated at 100°C for 10 min;

[0044] (15) Western blotting analysis of the substrate protein of the PRMT1 photoreactive probe pull-down. The results are as follows Figure 4 As shown in A, only the wild-type PRMT1 photoreactive probe can pull-down the substrate protein; Western blotting also analyzed the reaction products of the in vitro methylation experiment of the substrate protein. The results are shown in Figure 4 As shown in B, PRMT1 can monomethylate and dimethylate these four substrate proteins; in addition, LC-MS / MS analyzed the methylation sites of the substrate protein in vitro methylation reactions, such as Figure 5 As shown, a total of 12 methylation sites were identified.

[0045] Figure 2 Schematic diagram of the photoreactive probe reaction for identifying PRMT1 substrate protein. The N-hydroxysuccinimide of the probe reacts with the primary amine group of the PRMT1 protein to form a photoreactive probe ( Figure 2 A). The azide of the photoreactive probe will cross-link to the surrounding proteins under ultraviolet irradiation. Avidin agarose beads are used to enrich the biotin-tagged proteins, and the cross-linked proteins are enzymatically digested. The peptides produced by enzymatic digestion are analyzed by LC-MS / MS. The substrate proteins enriched by the wild-type methyltransferase probe and the mutant methyltransferase probe are quantitatively analyzed without labeling ( Figure 2 B).

[0046] Figure 3 The results of the identification of PRMT1 methyltransferase substrate proteins are shown in Figure 2. By comparing the abundance of wild-type and mutant PRMT1 probe-enriched proteins, a total of 23 proteins with different abundances were identified, and 18 proteins had an abundance ratio greater than 1.5 times ( Figure 3 A), 15 of which are known substrate proteins ( Figure 3 B), 3 proteins are unknown substrate proteins including HNRNPA0 (uniprot: Q13151), ILF2 (uniprot: Q12905) and SBSN (uniprot: K7ESC4), the abundance ratio of HNRNPA0 is 6.0, the abundance ratio of ILF2 is 3.7, and the abundance ratio of SBSN is 4.4. It shows that the strategy based on photoreactive probe can specifically identify the substrate protein of PRMT1 methyltransferase. WT represents the wild-type PRMT1 probe, and Mutant represents the mutant PRMT1 probe.

[0047] Figure 4 To verify the in vitro methylation of substrate proteins, only the wild-type PRMT1 probe can pull-down the substrate proteins ( Figure 4 A), indicating that only wild-type PRMT1, but not mutant PRMT1, binds to substrate proteins; and PRMT1 can monomethylate and dimethylate these four substrate proteins ( Figure 4 B), indicating that the abundance ratio of the wild-type PRMT1 probe / mutant PRMT1 probe enriched substrate proteins greater than 1.5 times are all PRMT1 substrate proteins; WT represents the wild-type PRMT1 probe, and Mutant represents the mutant PRMT1 probe.

[0048] Figure 5 For LC-MS / MS analysis of the methylation sites of substrate proteins in the in vitro methylation experiment, PRMT1 can methylate arginine 189 of HNRNPA0, arginine 140, 194, 206, 218, 336 and 371 of HNRNPA1, arginine 153, 325, 350 and 352 of HNRNPA2B1, and arginine 24 of ILF2, indicating that the strategy based on photoreactive probes has a high degree of credibility in identifying PRMT1 methyltransferase substrate proteins.

[0049] In summary, the present invention is based on the strategy of photoreactive probes to specifically identify the transient substrates of PRMT1 methyltransferase. A total of 18 proteins with different abundances were identified, 15 of which were substrate proteins, and 4 substrate proteins were successfully verified using pull-down and in vitro methylation experiments, indicating that the strategy based on photoreactive probes can effectively distinguish substrate proteins from non-substrate proteins, and the interaction between other enzymes and substrates can be effectively identified by simply changing the probe backbone.

Claims

1. A method for identifying a modified enzyme substrate based on a photoreactive probe, characterized in that: The method is established by utilizing the difference in substrate recognition ability between the wild-type and mutant modification enzymes; the wild-type and mutant modification enzymes (the binding site of the active center where the wild-type modification enzyme binds to the substrate is mutated) are expressed and purified, and reacted with probes with a reaction group, a photoreaction group and an enrichment group to obtain photoreaction probes of the wild-type and mutant modification enzymes respectively; the wild-type and mutant modification enzyme probes are incubated with biological samples respectively, and then photocrosslinked to enrich the crosslinked substrates respectively; the abundance of the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe is quantitatively compared (relative content of molecules, peak area of ​​gas chromatography, peak area of ​​liquid chromatography, etc.), and the ratio of the abundance of the substrates enriched by the wild-type modification enzyme probe / the abundance of the substrates enriched by the mutant modification enzyme probe is greater than 1 (preferably greater than 1.2, most preferably greater than 1.5), and the corresponding substrate is the screened substrate of the wild-type modification enzyme.

2. The method according to claim 1, characterized in that The steps of this method are: (1) reacting a wild-type modification enzyme and a mutant modification enzyme with a probe to obtain a wild-type modification enzyme probe and a mutant modification enzyme probe with photoreactivity, respectively; (2) incubating the wild-type modification enzyme probe and the mutant modification enzyme probe with the biological sample, photocrosslinking, and enriching the crosslinked substrate; (3) detecting the abundance of the enriched substrate in the wild-type modification enzyme probe and the mutant modification enzyme probe respectively; (4) The ratio of the abundance of the substrate enriched by the wild-type modification enzyme probe / the mutant modification enzyme probe is greater than 1 (preferably greater than 1.2, most preferably greater than 1.5), which means that it is the substrate of the wild-type modification enzyme.

3. The method according to claim 1 or 2, characterized in that: The modifying enzyme may be one or more of protein modifying enzymes, DNA modifying enzymes, RNA modifying enzymes, etc., but is not limited to these modifying enzymes.

4. The method according to claim 1 or 2, characterized in that: 1-3 amino acid residues at the active center where the wild-type modification enzyme binds to the substrate (the site in the wild-type modification enzyme that can directly bind to the substrate and catalyze the chemical reaction of the substrate; the active center is divided into a binding site, a part that binds to the substrate, and a catalytic site, a site that catalyzes the chemical reaction of the substrate; by mutating the amino acid residues at the binding site, the wild-type modification enzyme loses the ability to bind to the substrate) are mutated (1-3 amino acids at the binding site of the wild-type modification enzyme are changed into other amino acids that are different from it), and mutations that do not destroy the spatial conformation of the modification enzyme are preferred.

5. The method according to claim 1 or 2, characterized in that: The probe comprises a reactive group, a linker, a photoreactive group and an enrichment group; the reactive group, the photoreactive group and the enrichment group are connected by the linker; The linker is one or more of polyethylene glycol (degree of polymerization 200-8000), alkane (chain length 3-20 carbon atoms), etc., but is not limited to these groups; The reactive group can react with one or more groups such as amino group and carboxyl group on the modified enzyme, and can be one or more groups such as N-hydroxysuccinimide, hydroxyl group, guanidine group, etc., but is not limited to these groups; The photo-crosslinking group is one or more of benzophenone, phenylazide, diaziridine, etc., but is not limited to these groups; The enrichment group is one or more of alkynyl, azide, biotin, etc., but is not limited to these groups.

6. The method according to claim 1 or 2, characterized in that: The wild-type modification enzyme and the mutant modification enzyme are expressed and purified respectively, and reacted with the probe in a buffer respectively to obtain the wild-type modification enzyme probe and the mutant modification enzyme probe; The buffer solution includes one or more of PBS, hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, etc., but is not limited to these buffer solutions; The molar ratio of the modification enzyme to the probe is 1:1-1:100 (preferably 1:10), and the preferred molar ratio is to maintain the spatial conformation of the modification enzyme; The reaction time between the probe and the modification enzyme is 1-120 s (preferably 60 s), and the preferred reaction time is that one modification enzyme protein molecule reacts with only one probe.

7. The method according to claim 1 or 2, characterized in that: The biological sample includes one or more proteins, DNA, RNA, etc., or a mixture of two or more; A mixture of one or more of protein, DNA, RNA, etc., including one or more of tissue and / or cell extracts; The tissue or cell is derived from one or more of bacteria, animals, plants or humans; The tissue and / or cell extract is obtained by lysing the tissue and / or cells using a buffer, and the natural spatial conformation of proteins, DNA or RNA in the biological sample should be maintained; A preferred buffer solution contains 100-500 mM sodium chloride and a 5-100 mM tris(hydroxymethyl)aminomethane aqueous solution with a volume concentration of 0.5-2% ethylphenyl polyethylene glycol. The tissues and / or cells are ultrasonically disrupted and lysed in the buffer solution.

8. The method according to claim 2, characterized in that: Incubation process: The wild-type modification enzyme probe and the mutant modification enzyme probe are incubated with the biological sample maintaining the natural conformation respectively. The optimal time for the maximum binding amount of the modification enzyme and the substrate is 1-4 hours to obtain a mixture of the modification enzyme and the substrate. Photo-crosslinking process: maintaining the natural conformation of the modified enzyme under ultraviolet irradiation, and irradiating the mixture of the modified enzyme and the substrate with ultraviolet light for 10-60 minutes to obtain a mixture of the modified enzyme and the substrate after ultraviolet crosslinking; Enrichment process: using the enrichment material to pull down the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe, respectively, the enrichment material is incubated with the mixture of the modification enzyme and the substrate after UV cross-linking for 1-5 hours (preferably 2 hours) to obtain the substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe and the non-specific adsorption of the enrichment material; The enrichment materials include one or more of hydrazide resin, Avidin agarose beads, etc., but are not limited to these enrichment materials; The nonspecific adsorption of the washed enriched material is appropriately adjusted according to the strength of the binding force; Preferably, the washing buffer I is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and volume concentration of 0.1-10% sodium dodecyl sulfate; the washing buffer II is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and 0.1-10 M urea; the washing buffer III is 5-100 mM tris(hydroxymethyl)aminomethane, pH 7-10, and 0.1-10 M sodium chloride; After washing the non-specific adsorption of the enrichment material, the substrate is retained on the enrichment material to obtain the substrate enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe; The substrates enriched by the wild-type modification enzyme probe and the mutant modification enzyme probe are respectively enzymatically hydrolyzed by protease to obtain enzymatic hydrolysates, and impurities in the enzymatic hydrolysates are respectively removed by C18 solid phase extraction columns to facilitate subsequent mass spectrometry analysis to obtain the abundance of the enriched substrates of the wild-type modification enzyme probe and the mutant modification enzyme probe, but the method is not limited to the above method.

9. The method according to claim 1 or 2, characterized in that: The detection of substrate abundance includes substrates enriched by wild-type modifying enzyme probes and substrates enriched by mutant modifying enzyme probes; The abundance of the substrate includes one or more of the relative content of the molecule, the peak area of ​​the gas chromatography, the peak area of ​​the liquid chromatography, etc., but is not limited to these; Methods for detecting the abundance of the enriched substrate of the modification enzyme probe include one or more of Southern blot, Northern blot, Western blot, quantitative proteomics and the like, but are not limited to the above methods; The quantitative methods of proteins or peptides in quantitative proteomics include one or both of label-free quantification and label-based quantification; The labeling quantification method includes one or more isotope labeling methods such as SILAC, dimethyl labeling, ITRAQ or TMT.

10. The method according to claim 1 or 2, characterized in that: The screening criteria for modification enzyme substrates are that the abundance ratio of the wild-type modification enzyme probe / mutant modification enzyme probe enriched substrate is greater than 1 (preferably greater than 1.2, most preferably greater than 1.5), that is, the screened wild-type modification enzyme substrate; the threshold value can be appropriately adjusted according to different methods for detecting substrate abundance (the optimal threshold value is determined by the maximum sensitivity and specificity values).

Images