A phorbol acylated detection probe, a preparation method and application thereof

Abstract

The application discloses a crotonylation detection probe and a preparation method and application thereof, and the preparation method comprises the following steps: taking an alkyne alcohol (compound II) as raw material, reacting with methoxycarbonylmethylidene triphenyl positive phosphorus under the action of an oxidant to obtain a methyl ester compound (compound III), hydrolyzing the methyl ester compound (compound III) under the action of alkali to obtain an acid (compound IV), and finally reacting the acid (compound IV) with alkali, filtering and freeze-drying to obtain the probe molecule (compound I). The chemical probe (compound I) with an alkyne group is applied in cells, the alkyne group is selectively loaded on target proteins at specific modification sites through metabolic labeling, and through a bio-orthogonal chemical reaction, a fluorescent dye can be orthogonally connected to the modification site, so that the probe is used for fluorescent imaging of crotonylation proteins in cells and identification of crotonylation modification substrates and sites, and by using the probe, crotonylation sites regulated by histone deacetylases (HDACs) can be determined through quantitative chemical proteomics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical synthesis, and particularly relates to a tigloylation detection probe, a preparation method and application thereof. BACKGROUND

[0002] Lysine acylation influences chromatin structure, gene expression, enzyme activity, and protein-protein interactions. More than ten types of lysine acylation have been discovered, including lactylation, tigloylation, malonylation, and succinylation. Tigloylation was discovered in 2011 and is a covalent modification formed by adding a four-carbon group containing a double bond to the lysine side chain amino group. Tigloylation has been shown to be associated with a variety of physiological and pathological processes, including cell differentiation, cancer development, kidney disease, neurological disease, and metabolic disorders.

[0003]

[0004] There are two ideas for detecting tigloylation at present. The first idea is based on the enrichment technology of pan-antibody. This technology requires specific antibodies, and thus has the disadvantages of high cost, sequence preference, and difficulty in dynamically monitoring the turnover of tigloylation (Cell, 2011, 146, 1015-1027). The second detection idea is to use the Michael addition reaction of tris(2-carboxyethyl)phosphonium hydrochloride with the α, β unsaturated acid of tigloylation (J. Am. Chem. Soc., 2018, 140, 4757-4760.) for the detection of tigloylated proteins. However, this method is only applied to the single protein level, but due to the low detection throughput, it is difficult to realize the identification of high-throughput substrate proteins and modification sites. Due to the above-mentioned defects in the detection of tigloylated protein substrates and modification sites at present, it is urgent to develop an efficient, economical and comprehensive method for studying tigloylated protein substrates and their functions in mammalian systems. In recent years, chemical proteomics strategies have become a powerful alternative method for studying protein post-translational modifications (PTMs).

[0005] Due to the above-mentioned defects in the detection of tigloylated protein substrates and modification sites at present, it is urgent to develop an efficient, economical and comprehensive method for studying tigloylated protein substrates and their functions in mammalian systems. In recent years, chemical proteomics strategies have become a powerful alternative method for studying protein post-translational modifications (PTMs). Incubating / administering chemical probes with biological ortho-functional groups (such as alkyne and azido) in cells or animals can selectively load these functional groups onto target proteins at specific modification sites through metabolic labeling. Through biological ortho-chemical reactions (such as copper(I)-catalyzed alkyne-azido cycloaddition reaction, CuAAC), fluorescent dyes and / or biotin can be orthogonally linked to the modification sites. This method realizes the selective visualization, enrichment and identification of metabolically labeled proteins. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art, and provide a crotonylation detection probe, a preparation method and application thereof.

[0007] To achieve the above object, the technical scheme provided by the present application is as follows:

[0008] A crotonylation detection probe has a structure shown in formula I:

[0009]

[0010] Wherein, n is 1 or 2.

[0011] A preparation method of a crotonylation detection probe is used to prepare the crotonylation detection probe, and comprises the following steps:

[0012]

[0013] Wherein: n is 1 or 2, in the process of preparing compound III from compound II, the oxidizing agent is DMP or IBX, and the molar ratio of compound II to methoxycarbonylmethylene triphenyl phosphonium and the oxidizing agent ranges from 1:1 to 2:2-3; in the process of preparing compound IV from compound III, the base is sodium hydroxide or potassium hydroxide, and the molar ratio of compound III to the base ranges from 1:5 to 6; in the process of preparing compound I from compound IV, the base is sodium bicarbonate, sodium carbonate or sodium hydroxide, and the molar ratio of compound IV to the base ranges from 1:1 to 1.1.

[0014] The application of the crotonylation detection probe, the probe can be used for fluorescence detection of crotonylation protein.

[0015] The probe can be used for pulling down crotonylation protein from cells and tissues.

[0016] The probe can be used for identifying crotonylation protein.

[0017] The crotonylation protein is divided into histone or non-histone protein, wherein the non-histone protein is NCL (nucleolar protein), HMGB1 (high mobility group protein B1), HMGN2 (high mobility group nucleosome binding domain 2), DDX21 (dead-box rna helicase), HMGB3 (high mobility group protein B3), PARP1 (poly ADP ribose polymerase 1), TMPO (thymopoietin), P53 (p53 tumor protein), SETLP (SET-like protein), WIZ (Wingsless-related zinc finger protein), PI2R (prostaglandin I2 receptor), UBE2T (ubiquitin conjugating enzyme ube2t) or ZN735 (zinc finger protein 735).

[0018] The probe can be used for identifying the modification site of phorbol acylation.

[0019] The method for identifying the modification site of phorbol acylation is as follows: the probe is incubated with cells for protein labeling, then a whole lysate of the cells is prepared, the probe-labeled protein is modified by azide-biotin, and finally the probe-labeled protein is separated from the proteome by using streptavidin-coupled magnetic beads, and the probe-labeled protein is detected and analyzed by mass spectrometry, wherein the azide-biotin is azide-biotin or azide-DADPS-biotin.

[0020] The probe can be used for identifying the phorbol acylation site regulated by phorbol acylation enzyme through quantitative chemical proteomics, and the phorbol acylation enzyme is deacetylase.

[0021] A kit containing a phorbol acylation detection probe.

[0022] Beneficial effects: the application discloses a phorbol acylation detection probe, a preparation method and application thereof, the preparation method comprises the following steps: taking acetylene alcohol (compound II) as raw material, reacting with methoxycarbonylmethyltriphenylphosphonium under the action of an oxidizing agent to obtain a methyl ester compound (compound III), hydrolyzing to obtain an acid (compound IV) under the action of alkali, and finally reacting with alkali, filtering and freeze-drying to obtain the probe molecule (compound I). The chemical probe (compound I) with an acetylene group is applied in cells, the acetylene group is selectively loaded on target proteins at specific modification sites through metabolic labeling, and through a bio-orthogonal chemical reaction, a fluorescent dye can be orthogonally connected to the modification site, so that the probe is used for fluorescent imaging of phorbol acylated proteins in cells and identification of phorbol acylated modification substrates and sites. Not only new phorbol acylation sites are identified, but also new phorbol acylation modification substrates are identified. The probe can be used for determining the phorbol acylation sites regulated by deacetylase (HDACs) through quantitative chemical proteomics.

[0023] The abbreviations of reaction reagents involved in the specification are as follows:

[0024] DMP: des-martin periodinane;

[0025] IBX: 2-iodoxybenzoic acid

[0026] DCM: dichloromethane;

[0027] NaHCO3: sodium bicarbonate;

[0028] NaOH: sodium hydroxide;

[0029] H2O: water;

[0030] HCl: hydrochloric acid;

[0031] EP tube: centrifuge tube. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1A Principle diagram of detecting probe labeled protein for crotonylation;

[0033] Figure 1B Fluorescence situation of gel map of detecting probe labeled protein for crotonylation;

[0034] Figure 1C Fluorescence analysis data of gel map of detecting probe labeled protein for crotonylation;

[0035] Figure 2A Labeling effect of detecting probe for crotonylation with concentration change;

[0036] Figure 2B Labeling effect of detecting probe for crotonylation with time change;

[0037] Figure 2C Labeling effect of detecting probe for crotonylation with cell line type change;

[0038] Figure 3 Fluorescence situation of gel map of competition between crotonic acid and detecting probe for crotonylation;

[0039] Figure 4A Western blot map of detecting probe for crotonylation labeling histone;

[0040] Figure 4B Western blot map of detecting probe for crotonylation labeling non-histone protein HMGB1;

[0041] Figure 4C Western blot map of detecting probe for crotonylation labeling non-histone protein YWHAE;

[0042] Figure 5 Fluorescence imaging map of crotonylated protein;

[0043] Figure 6 Chemical proteomics test flow of detecting probe for crotonylation;

[0044] Figure 7A Statistical diagram of three repeated data of sample labeled by detecting probe for crotonylation;

[0045] Figure 7B Statistical diagram of identifying crotonylation site on histone by detecting probe for crotonylation;

[0046] Figure 7C Identifying crotonylation site on histone by detecting probe for crotonylation;

[0047] Figure 8A Analysis of non-histone modification sites identified by crotonylation detection probe;

[0048] Figure 8B Identification of non-histone modification sites by crotonylation detection probe and its number statistics;

[0049] Figure 8C Identification of TMPO protein by crotonylation detection probe;

[0050] Figure 9A Cellular component analysis of proteins modified by crotonylation detection probe;

[0051] Figure 9B GO analysis of biological processes of proteins modified by crotonylation detection probe;

[0052] Figure 9C Molecular function analysis of proteins modified by crotonylation detection probe;

[0053] Figure 9D PTM enrichment analysis of proteins modified by crotonylation detection probe;

[0054] Figure 9E pLogo analysis of amino acid sequences around crotonylation sites;

[0055] Figure 9F Secondary propensity analysis of modified lysine residues;

[0056] Figure 10 Flow of quantitative chemical proteomics method;

[0057] Figure 11A Heatmap of values quantified by quantitative chemical proteomics in HDAC1 overexpression, normal cells, knockdown cells

[0058] Figure 11B Heatmap of values quantified by quantitative chemical proteomics in HDAC3 overexpression, normal cells, knockdown cells

[0059] Figure 11C Volcano plot of histone-derived crotonylation sites in HDAC1 group;

[0060] Figure 11D Volcano plot of histone-derived crotonylation sites in HDAC1 group;

[0061] Figure 11E Volcano plot of histone-derived crotonylation sites in HDAC3 group;

[0062] Figure 11FVolcano plot of histone-derived crotonylation sites for HDAC3 group. DETAILED DESCRIPTION

[0063] The application is further illustrated by the following specific examples, which are implemented on the premise of the technical scheme of the application. It should be understood that the examples are only used to illustrate the application and not used to limit the scope of the application.

[0064] Synthesis of compound I-1 (Cr-alkyne)

[0065] Synthesis of compound III-1:

[0066]

[0067] Compound II-1 pentynol (2 g, 24 mmol) was dissolved in DCM (2 mL), DMP (20 g, 48 mmol) was added at room temperature and stirred for 30 min, then methoxycarbonylmethyltriphenylphosphonium (16.05 g, 48 mmol) was added. The reaction was carried out for 2 h. Ethyl acetate was extracted 3 times, the organic phase was combined, washed with saturated NaHCO3 (15 mL) and saturated brine (20 mL x 2). Dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (CH2Cl2: EtOAc = 10: 1) to obtain the product compound III-1, colorless oil (2.64 g, yield 80.3%).

[0068] 1 H NMR (400 MHz, Chloroform-d) δ 6.98 (dt, J = 15.7, 6.6 Hz, 1H), 5.96-5.84 (m, 1H), 2.48-2.40 (m, 2H), 2.38-2.33 (m, 2H), 2.03 (t, J = 2.6 Hz, 1H).

[0069] 13 C NMR (101 MHz, CDCl3) δ 166.61, 146.56, 122.04, 82.55, 69.47, 51.40, 30.93, 17.32.

[0070] Synthesis of compound IV-1:

[0071]

[0072] Compound III-1 (1.38 g, 10 mmol) was dissolved in methanol (2 mL), NaOH / H2O (1 g, 50 mmol) was added at room temperature, and the reaction was allowed to proceed overnight. HCl was added to adjust the pH to acidic, and the mixture was extracted with ethyl acetate three times. The organic phase was combined, washed with saturated NaHC03(15 mL) and saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product compound IV-1 was purified by column chromatography (CH2Cl2:EtOAc = 3:1) to obtain a white solid (1.01 g, yield 81%).

[0073] 1 H NMR (400 MHz, Chloroform-d) δ 12.19 (s, 1H), 7.11 (dt, J = 15.7, 6.7 Hz, 1H), 5.91 (d, J = 15.7 Hz, 1H), 2.47 (qd, J = 6.7, 3.2 Hz, 2H), 2.41-2.35 (m, 2H), 2.02 (t, J = 2.6 Hz, 1H).

[0074] 13 C NMR (101 MHz, CDC13) δ 171.95, 149.34, 121.91, 82.45, 69.62, 31.06, 17.28.

[0075] Synthesis of compound I-1 (Cr-alkyne):

[0076]

[0077] Compound IV-1 was transferred to an EP tube, and aqueous NaHC03(equivalent amount) was added dropwise. The mixture was filtered through a 0.45 pm membrane, and the white powder obtained after lyophilization was compound I-1 (Cr-alkyne).

[0078] Synthesis of compound I-2 (Cr-alkyne-2)

[0079] Synthesis of compound III-2:

[0080]

[0081] Compound II-2 hexynol (2 g, 20 mmol) was dissolved in DCM (2 mL), DMP (18 g, 40 mmol) was added at room temperature and stirred for 30 min, then methoxycarbonylmethyltriphenylphosphonium (16.05 g, 48 mmol) was added. The reaction was carried out for 2 h. Ethyl acetate was added for extraction 3 times, the organic phase was combined, washed with saturated NaHC03(15 mL) and saturated brine (20 mL x 2). Dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (CH2Cl2: EtOAc = 10: 1) to obtain the product III-2 as a colorless oil (2.6 g, yield 84%).

[0082] 1 H NMR (400 MHz, Chloroform-d) δ 6.95 (dt, J = 15.6, 7.0 Hz, 1H), 5.87 (dt, J = 15.6, 1.6 Hz, 1H), 2.34 (qd, J = 7.2, 1.5 Hz, 2H), 2.23 (td, J = 7.0, 2.7 Hz, 2H), 1.99 (t, J = 2.6 Hz, 1H), 1.70 (p, J = 7.2 Hz, 2H).

[0083] 13 C NMR (101 MHz, CDC13) δ 166.90, 148.14, 121.64, 83.43, 69.04, 51.41, 30.91, 26.67, 17.81.

[0084] Synthesis of compound IV-2:

[0085]

[0086] Compound III-2 (1.52 g, 10 mmol) was dissolved in methanol (2 mL), NaOH / H20 (1 g, 50 mmol) was added at room temperature and the reaction was carried out overnight. HCl was added to adjust the pH to acidic, and ethyl acetate was added for extraction 3 times, the organic phase was combined, washed with saturated NaHC03(15 mL) and saturated brine (20 mL x 2). Dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (CH2Cl2: EtOAc = 3: 1) to obtain the product compound IV-2 as a colorless solid (1.2 g, yield 87%).

[0087] 1H NMR (400 MHz, Chloroform-d) δ 11.87 (s, 1H), 7.08 (dt, J = 15.6, 7.0 Hz, 1H), 5.87 (dt, J = 15.6, 1.6 Hz, 1H), 2.38 (qd, J = 7.1, 1.5 Hz, 2H), 2.24 (td, J = 7.0, 2.7 Hz, 2H), 1.99 (t, J = 2.7 Hz, 1H), 1.71 (p, J = 7.2 Hz, 2H).

[0088] 13 C NMR (101 MHz, CDC13) δ 172.09, 150.97, 121.42, 83.37, 69.16, 31.04, 26.54, 17.88.

[0089] Synthesis of compound I-2 (Cr-alkyne-2):

[0090]

[0091] Compound IV-2 was transferred to an EP tube and aqueous NaHC03solution (equivalent amount) was added dropwise. The mixture was filtered through a 0.45 pm membrane and lyophilized to obtain compound I-2 (Cr-alkyne-2) as a white powder.

[0092] Example 3 Comparison of labeling efficiency of crotonylated probes Cr-alkyne and Cr-alkyne-2

[0093] After obtaining Cr-alkyne and Cr-alkyne-2, the present application first tested whether these two probes could be modified to proteins by metabolic means in living mammalian cells and evaluated their labeling efficiency. To this end, HEK293T cells were incubated with Cr-alkyne and Cr-alkyne-2, and further lysed with a highly denaturing lysis buffer to maximize protein extraction. Then, the resulting cell lysate was subjected to click chemistry reaction of CuAAC with azidorhodamine to covalently bind the probe-labeled proteins with the rhodamine dye for in-gel fluorescence analysis. The labeling principle is shown in Figure 1A .

[0094] The results are shown in Figure 1B -C shows that at a concentration of 0.5 mM or 5 mM, Cr-alkyne containing a shorter chain labels proteins in living cells more efficiently than Cr-alkyne-2. The reason for this result is that we consider that Cr-alkyne has one less carbon than Cr-alkyne-2, has stronger hydrophilicity, and thus has stronger binding ability in the hydrophilic pocket of the protein. Therefore, the present application selected Cr-alkyne for subsequent research.

[0095] Example 4 Evaluation of Cr-alkyne labeling

[0096] The labeling efficiency of Cr-alkyne was evaluated with different concentrations, time and cell lines. The results of in-gel fluorescence analysis are shown in Figure 2A Figure 4A: The labeling efficiency of Cr-alkyne was evaluated with different concentrations. It was found that the labeling intensity of Cr-alkyne increased with the increase of concentration. Figure 2A Figure 4B: The labeling efficiency of Cr-alkyne was evaluated with different labeling time. It was found that the labeling intensity of Cr-alkyne changed little from 2h to 8h. Figure 2B Figure 4C: The labeling efficiency of Cr-alkyne was evaluated with different cell lines. It was found that Cr-alkyne had good labeling efficiency in different cell lines, and the labeling pattern was different. Figure 2C Therefore, in the following experiments, Hek293T cells were treated with 5mM Cr-alkyne for 8h.

[0097] Example 4 Test of competition of Cr-alkyne with phorbol acid

[0098] Different concentrations (0-10 times the concentration of Cr-alkyne) of phorbol acid were used to compete with 5mM Cr-alkyne, followed by click reaction with azido rhodamine and in-gel fluorescence analysis. The fluorescence results of the gel map are shown in Figure 3 Figure 5: The labeling intensity of Cr-alkyne decreased with the increase of phorbol acid concentration. This indicates that Cr-alkyne is likely to be metabolized into phorbol acylated proteins, and this metabolism can be competed by phorbol acid.

[0099] Example 5 Test of Cr-alkyne labeling on known phorbol acylated substrate proteins (histones)

[0100] Subsequently, the present application tests whether Cr-alkyne can metabolically label known phorbol acylated proteins. It has been confirmed that histones are highly phorbol acylated modified proteins. Therefore, we extracted core histones from Cr-alkyne labeled HEK293T cells, followed by click reaction with azido rhodamine and in-gel fluorescence analysis. From Figure 4A It can be seen that the histones with characteristic low molecular weight are clearly labeled by Cr-alkyne.

[0101] In addition, HMGB1 and YWHAE were reported as two known non-histone crotonylation substrate proteins. To further confirm the effectiveness of the probe, HEK293T cells expressing FLAG-tagged target proteins were metabolically labeled with Cr-alkyne, and then the extracted proteins were subjected to click chemistry reaction with azide-biotin and enriched by streptavidin beads. As shown in FIG. 6A, Western blot analysis of the immunoprecipitated target proteins showed that Cr-alkyne could be metabolically labeled into HMGB1 and YWHAE. Figure 4B and Figure 4C As shown in FIG. 6B, Western blot analysis of the immunoprecipitated target proteins showed that Cr-alkyne could be metabolically labeled into HMGB1 and YWHAE.

[0102] Example 6 Co-localization test of Cr-alkyne with nucleus

[0103] Further fluorescence visualization of crotonylated proteins was performed in HEK293T cells. PBS-treated or Cr-alkyne-treated HEK293T cells were washed, then subjected to fixation, permeabilization and click chemistry linking with azide-rhodamine. As shown in FIG. 7A, compared with the disordered signal in PBS-treated cells, a stronger rhodamine signal was observed in Cr-alkyne-treated cells, especially in the nucleus. Therefore, the method developed by the present application first realized the fluorescence imaging of crotonylated proteins in cells by Cr-alkyne. These results collectively indicated that Cr-alkyne could be metabolically assembled into crotonylated proteins in mammalian cells and realized a powerful fluorescence detection of protein crotonylation. Figure 5 Example 7 Modification site analysis of Cr-alkyne on histones

[0104] After the fluorescence labeling detection of Cr-alkyne, we wanted to know the exact modification site of Cr-alkyne at the proteome level. Therefore, the whole lysate of Cr-alkyne-labeled HEK293T cells was prepared, combined with the acid-cleavable azide-DADPS-biotin tag by click chemistry, and subjected to affinity purification with streptavidin beads. After further enzymatic cleavage on the beads and acid-mediated cleavage, the Cr-alkyne-modified peptides were enriched and eluted for mass spectrometry analysis. The collected MS / MS spectra were analyzed using the peptide identification tool (MSFragger), setting the Cr-alkyne-labeled lysine residue (K+249.1477 Da) as the variable modification. The chemical proteomics test flow of Cr-alkyne is shown in FIG. 8.

[0105] Figure 6 To enhance the reliability and coverage of site identification, as shown in FIG. 9A, the Cr-alkyne-labeled lysine residues (K+249.1477 Da) were set as the variable modification, and the MS / MS spectra were analyzed using the peptide identification tool (MSFragger). As shown in FIG. 9B, the Cr-alkyne-labeled lysine residues (K+249.1477 Da) were set as the variable modification, and the MS / MS spectra were analyzed using the peptide identification tool (MSFragger).

[0106] To enhance the reliability and coverage of site identification, as shown in FIG. 9A, the Cr-alkyne-labeled lysine residues (K+249.1477 Da) were set as the variable modification, and the MS / MS spectra were analyzed using the peptide identification tool (MSFragger). As shown in FIG. 9B, the Cr-alkyne-labeled lysine residues (K+249.1477 Da) were set as the variable modification, and the MS / MS spectra were analyzed using the peptide identification tool (MSFragger). Figure 7A ​As shown, we performed triplicates for Cr-alkyne labeled samples, and only sites identified in at least two of the three replicates were considered for further analysis. MS / MS analysis indicated that Cr-alkyne was indeed metabolized to the known crotonylation sites in histones. For example, KAVTKCr-alkyneAQKcontaining Cr-alkyne modification at H2B-K20 was identified by MS / MS analysis. Notably, we also identified two new histone crotonylation sites, including H1-K177 and H2B-K24. In addition to the typical histone crotonylation marks (e.g. Figure 7B As shown, we identified 28 crotonylation sites in typical histones in human cells, 18 of which have been reported in the work by Tan et al. (Cell, 2011, 146, 1015-1027.). For example, KAVTKCr-alkyneAQKcontaining Cr-alkyne modification at H2B-K20 was identified by MS / MS analysis. Notably, we also identified two new histone crotonylation sites, including H1-K177 and H2B-K24. In addition to the typical histone crotonylation marks (e.g. Figure 7C As shown) we also identified five new atypical histone crotonylation modifications, including H2AX-K5, H2AX-K9, H2AZ1-K7, H2AZ1-K115 and H2AZ1-K120, as shown in Table 1. Collectively, these data support that Cr-alkyne probes can directly target lysine residues on histones and identify new histone Kcrsites across the proteome.

[0107] Table 1. New modification sites identified using crotonylation probes

[0108]

[0109]

[0110] Example 8. Analysis of Cr-alkyne modification sites on non-histone proteins

[0111] We explored the analysis of Cr-alkyne on modification sites in non-histone proteins. As shown, the number of modification sites in non-histone derived peptides was higher than that in histones in triplicates. Some representative crotonylated substrate proteins, such as NCL, HMGB1, HMGN2, DDX21, HMGB3 and PARP1, were identified by MS / MS analysis, as shown. Figure 8A Figure 8B TMPO protein, which was reported as a crotonylated protein in previous proteomic data, was identified by MS / MS analysis, as shown. Figure 8C ​As shown, some known crotonylated proteins were also identified, and the modification sites were confirmed by pull-down experiments and WB analysis. It is worth noting that there are also some newly identified modification sites for known crotonylated proteins, such as EEFlAl-K457 and NUCKSl-K188. More importantly, several new crotonylated substrate proteins were identified, such as P53, SETLP, WIZ, PI2R, UBE2T and ZN735 (see Table 1).

[0112] Example 9 Bioinformatics analysis of all Cr-alkyne modified proteins

[0113] To better understand the identified crotonylated substrate proteins, we performed a series of bioinformatics analysis at the protein level. Cell component analysis using the Gene Ontology (GO) database found that, as shown in Figure 9A , crotonylated proteins were more highly represented in the nucleus, nucleoplasm and nucleosome. In addition, as shown in Figure 9B , GO analysis of biological processes showed that crotonylated proteins were significantly enriched in processes such as nucleosome assembly, positive regulation of RNA polymerase II promoter and chromatin organization. Molecular function analysis showed that, as shown in Figure 9C , many crotonylated proteins had RNA, DNA and chromatin binding activity. PTM enrichment analysis showed that, as shown in Figure 9D , most crotonylated proteins contained other types of PTMs, with higher enrichment in acetylation, isopeptide bond and methylation.

[0114] Overall, these results suggest that crotonylation mainly occurs on proteins associated with nuclear biological processes, such as chromatin organization, RNA processing / metabolism and DNA repair. To explore the sequence distribution characteristics of crotonylation, we used pLogo to analyze the amino acid sequences around the identified crotonylation sites. The results showed that, as shown in Figure 9E , at many positions, positively charged lysine residues were preferentially selected (e.g., -7, -5, -4, +4, +6 and +7), while negatively charged glutamic acid and aspartic acid residues were generally lacking in the sequence. In addition, alanine and proline were highly enriched at positions -6 / -1 / +1 / +2 / +3 and -1 / +5. We also analyzed the secondary propensity of these modified lysine residues. From the histogram in Figure 9F , we found that crotonylation sites had a preference for loop structures, while H and E were generally lacking. These data suggest that crotonylation has a unique intrinsic reactivity for lysine.

[0115] Example 10: Quantitative chemical proteomics reveals crotonylation sites regulated by histone deacetylases (HDACs).

[0116] This probe can be used to discover crotonylation sites regulated by histone deacetylases (HDACs) through quantitative chemical proteomics. Therefore, we performed tandem orthogonal proteolytic activity-based proteomic ... Figure 10 .

[0117] Therefore, for each lysine identified from the analytical experiment, such as Figure 11A and 11B As shown, we calculated two ratios. The heavy / light ("untreated / knockout") ratio reflects the degree of enhancement of Cr-alkyne markers after HDAC knockout, while the heavy / medium ("untreated / overexpressed") ratio indicates the degree of loss of Cr-alkyne markers after HDAC overexpression. We analyzed the statistical differences for each lysine using t-tests (p-values) and plotted a volcano plot based on p-values ​​and ratios. The results show that many crotonylation sites are regulated by HDACs. Figure 11C-Figure 11F As shown, in the HDAC1 group, histone-derived crotonylation sites, such as H3-K23, H4-K8, H2AZ1-K8, and H2B-K20, exhibit R... H / L The sites are negatively regulated, and the R of these sites H / M The sites are positively regulated. In the HDAC3 regulatory group, the R... H / L The sites are negatively regulated, and the R of these sites H / M The sites are positively regulated. Among these crotonylation sites, H3-K23 has been reported as a site regulated by HDAC1, while H4-K8 is considered to be a site regulated by both HDAC1 and HDAC3, which is consistent with our data and confirms the reliability of using Cr-alkyne for quantitative proteomics.

[0118] The above detailed description is merely descriptive of the application and specific embodiments thereof. It is not intended as a limitation on the scope of the application. Changes, equivalent substitutions, improvements, combinations, and the like, which are apparent to a skilled artisan, are covered by the following claims.

Claims

1. A method for preparing a crotonylation detection probe, characterized in that, Includes the following steps: ， Wherein: n is 1 or 2; in the process of preparing compound III from compound II, the oxidant is DMP or IBX, and the molar ratio of compound II to methoxycarbonylmethylenetriphenylphosphine and the oxidant is in the range of 1:1~2:2~3; in the process of preparing compound IV from compound III, the base is sodium hydroxide or potassium hydroxide, and the molar ratio of compound III to the base is in the range of 1:5~6; in the process of preparing compound I from compound IV, the base is sodium bicarbonate, sodium carbonate or sodium hydroxide, and the molar ratio of compound IV to the base is in the range of 1:1~1.

1.

2. The application of a crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1, characterized in that, The probe can be used for the fluorescence detection of crotonylated protein, and the application does not involve the diagnosis or treatment of disease.

3. The application of the crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1, characterized in that, The probe can be used to pull down crotonylated proteins from cells and tissues, and the application does not involve the diagnosis or treatment of diseases.

4. The application of a crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1, characterized in that, The probe can be used to identify crotonylated proteins, and the application does not involve the diagnosis or treatment of diseases.

5. The application according to claim 4, characterized in that, The crotonylated protein is divided into histones or non-histone proteins, wherein the non-histone proteins are NCL, HMGB1, HMGN2, DDX21, HMGB3, PARP1, TMPO, P53, SETLP, WIZ, PI2R, UBE2T or ZN735.

6. The application of a crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1, characterized in that, The probe can be used to identify crotonylation modification sites, and the application does not involve the diagnosis and treatment of diseases.

7. The application according to claim 6, characterized in that, The method for identifying crotonylation modification sites is as follows: the probe is co-incubated with cells to label the protein, then a complete cell lysis buffer is prepared, the probe-labeled protein is biotinylated with azide biotin, and finally the probe-labeled protein is separated from the proteome using streptavidin-coupled magnetic beads, and the probe-labeled protein is analyzed by mass spectrometry.

8. The application of a crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1, characterized in that, The probe can identify crotonylation sites regulated by crotonylase through quantitative chemical proteomics; wherein, the crotonylase is a deacetylase; the application does not involve the diagnosis and treatment of diseases.

9. A kit comprising a crotonylation detection probe obtained by the preparation method of the crotonylation detection probe according to claim 1.

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