Croton acylation detection probe as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: by taking alkynol (compound II) as a raw material, reacting with methoxycarbonyl methylene triphenyl phosphorus under the action of an oxidizing agent to obtain a methyl ester compound (compound III), hydrolyzing under the action of alkali to obtain acid (compound IV), finally reacting with alkali, and filtering to obtain the crotonyl acylation detection probe. And freeze-drying to obtain the probe molecule (compound I). According to the present invention, a chemical probe (compound I) having an alkynyl group is applied in a cell, the alkynyl group is selectively loaded on a target protein at a specific modification site through a metabolic marker, and a fluorescent dye can be orthogonally linked to the modification site through a bio-orthogonal chemical reaction, therefore, the probe is used for performing fluorescence imaging on crotonyl protein in cells and identifying crotonyl modified substrates and sites, and the crotonyl sites regulated and controlled by histone deacetylase (HDACs) can be determined by utilizing the probe through quantitative chemical proteomics.

Description

Technical Field

[0001] The invention belongs to the field of chemical synthesis, and specifically relates to a crotonylation detection probe and a preparation method and application thereof. Background Art

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

[0003]

[0004] There are currently two approaches to detecting crotonylation. The first approach is based on pan-antibody enrichment technology. This technology requires specific antibodies, so there are disadvantages such as high cost during detection, sequence preference, and difficulty in dynamically monitoring the turnover of crotonylation (Cell, 2011, 146, 1015-1027). The second detection approach is to use the Michael addition reaction of tri(2-carbonylethyl)phosphine hydrochloride with crotonyl α, β-unsaturated acid (J.Am.Chem.Soc., 2018, 140, 4757-4760.) for the detection of crotonyl proteins. However, this method is only applied to the single protein level, but due to the low detection flux, it is difficult to achieve high-throughput identification of substrate proteins and modification sites. Due to the above-mentioned defects in the current detection of crotonyl protein substrates and modification sites, it is urgent to develop efficient, economical and comprehensive methods for studying crotonyl substrate proteins and their functions in mammalian systems. In recent years, chemical proteomics strategies have become a powerful alternative to study post-translational modifications (PTMs) of proteins.

[0005] Due to the above-mentioned deficiencies in the current detection of crotonylated protein substrates and modification sites, there is an urgent need to develop efficient, economical and comprehensive methods for studying crotonylated substrate proteins and their functions in mammalian systems. In recent years, chemical proteomics strategies have become a powerful alternative for studying protein post-translational modifications (PTMs). Chemical probes with bioorthogonal functional groups (such as alkynyl and azido groups) are incubated / administered in cells or animals, and these functional groups can be selectively loaded onto target proteins at specific modification sites through metabolic labeling. Fluorescent dyes and / or biotin can be orthogonally attached to modification sites through bioorthogonal chemical reactions (such as copper (I)-catalyzed alkynyl-azido cycloaddition reaction, CuAAC). This method enables selective visualization, enrichment and identification of metabolically labeled proteins. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a crotonylation detection probe and a preparation method and application thereof.

[0007] To achieve the above purpose, the technical solution provided by the present invention 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 method for preparing a crotonylation detection probe, for preparing the crotonylation detection probe, comprises the following steps:

[0012]

[0013] 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 to 2:2 to 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 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 is in the range of 1:1 to 1.1.

[0014] An application of the crotonylation detection probe, wherein the probe can be used for fluorescence detection of crotonylation proteins.

[0015] The probe can be used to pull down crotonylated proteins from cells and tissues.

[0016] The probe can be used to identify crotonylated proteins.

[0017] The crotonylated proteins are divided into histones or non-histones, wherein the non-histones are 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 associated zinc finger protein), PI2R (prostaglandin I2 receptor), UBE2T (ubiquitin conjugating enzyme ube2t) or ZN735 (zinc finger protein 735).

[0018] The probe can be used to identify the modification site of crotonylation.

[0019] The method for identifying the crotonylation modification site is as follows: the probe is co-incubated with cells for protein labeling, then a whole cell lysate is prepared, and then the probe-labeled protein is biotin-modified using azide-biotin, and finally the probe-labeled protein is separated from the proteome using streptavidin-coupled magnetic beads, and the probe-labeled protein is subjected to mass spectrometry detection and analysis, wherein the azide-biotin is azide-biotin or azide-DADPS-biotin.

[0020] The probe can identify crotonylation sites regulated by crotonylase by quantitative chemical proteomics; wherein the crotonylase is a deacetylase.

[0021] A kit comprising a crotonylation detection probe.

[0022] Beneficial effects: The present invention discloses a crotonylation detection probe and its preparation method and application. The preparation method comprises: using alkynol (Compound II) as a raw material, reacting with methoxycarbonyl methylene triphenylphosphine under the action of an oxidant to obtain a methyl ester compound (Compound III), hydrolyzing under the action of a base to obtain an acid (Compound IV), and finally reacting with a base, filtering, and freeze-drying to obtain a probe molecule (Compound I). A chemical probe (Compound I) having an alkynyl group is applied in cells, and the alkynyl group is selectively loaded onto a target protein at a specific modification site through metabolic labeling. Through a bioorthogonal chemical reaction, a fluorescent dye can be orthogonally connected to the modification site, so that the probe is used in cells for fluorescent imaging of crotonyl proteins and identification of crotonylation modification substrates and sites, not only identifying new crotonylation sites, but also identifying new crotonylation modification substrates. The probe can be used to determine the crotonylation sites regulated by deacetylases (HDACs) through quantitative chemical proteomics.

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

[0024] DMP: Dess-Martin periodinane;

[0025] IBX: 2-Iodoacylbenzoic 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 THE DRAWINGS

[0032] Figure 1A Schematic diagram of protein labeling with crotonylation detection probe;

[0033] Figure 1B The fluorescence of the gel image of the protein labeled with the crotonylation detection probe;

[0034] Figure 1C Fluorescence analysis data of gel images of proteins labeled with crotonylation detection probes;

[0035] Figure 2A To detect the labeling effect of the crotonylation probe as the concentration changes;

[0036] Figure 2B To detect the labeling effect of the crotonylation probe over time;

[0037] Figure 2C The labeling effect of the crotonylation detection probe varies with the cell line type;

[0038] Figure 3 The fluorescence of the gel image is the competition between crotonic acid and crotonylation detection probe;

[0039] Figure 4A To test the labeling of histones by crotonylation detection probe;

[0040] Figure 4B Western blot image of non-histone protein HMGB1 labeled with crotonylation detection probe;

[0041] Figure 4C Western blot image of non-histone protein YWHAE labeled with crotonylation detection probe;

[0042] Figure 5 This is the fluorescence imaging of crotonylated protein;

[0043] Figure 6 Chemical proteomics experimental process for crotonylation detection probe;

[0044] Fig. 7A The statistical graph is the data of three replicates of samples labeled with crotonylation detection probe;

[0045] Figure 7B Statistical diagram of the data for identifying crotonylation sites on histones for crotonylation detection probes;

[0046] Figure 7C Identify the crotonylation sites on histones for crotonylation detection probes;

[0047] Fig. 8A Assays to identify modification sites in non-histone proteins for crotonylation detection probes;

[0048] Figure 8B Identification and quantitative statistics of modification sites in non-histone proteins by crotonylation detection probes;

[0049] Figure 8C For identification of TMPO protein using crotonylation detection probe;

[0050] Fig. 9A Analysis of cellular components of proteins modified with crotonylation detection probes;

[0051] Fig. 9B GO analysis of biological processes of proteins modified by crotonylation detection probes;

[0052] Fig. 9C Molecular functional analysis of proteins modified with crotonylation detection probes;

[0053] Fig.9D PTM enrichment analysis of proteins modified with crotonylation detection probes;

[0054] Fig.9E The amino acid sequence surrounding the crotonylation site was analyzed for pLogo;

[0055] Fig.9F Secondary propensity analysis for modified lysine residues;

[0056] Fig.10 The process for quantitative chemical proteomics methods;

[0057] Fig.11A Heat map of the values ​​quantified by quantitative chemical proteomics in HDAC1 overexpression, normal cells, and knockdown cells

[0058] Fig. 11B Heat map of the values ​​quantified by quantitative chemical proteomics in HDAC3 overexpression, normal cells, and knockdown cells

[0059] Fig. 11C The volcano plot is of histone-derived crotonylation sites in the HDAC1 group;

[0060] Fig.11D The volcano plot is of histone-derived crotonylation sites in the HDAC1 group;

[0061] Fig.11E The volcano plot is of histone-derived crotonylation sites in the HDAC3 group;

[0062] Fig.11FVolcano plot of histone-derived crotonylation sites in the HDAC3 group. DETAILED DESCRIPTION

[0063] The present invention is further illustrated below in conjunction with specific examples. The present examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

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

[0065] Synthesis of compound III-1:

[0066]

[0067] Compound II-1 pentynol (2g, 24mmol) was dissolved in DCM (2mL), and DMP (20g, 48mmol) was added at room temperature and stirred for 30min, followed by addition of methoxycarbonylmethylenetriphenylphosphine (16.05g, 48mmol). The mixture was reacted for 2h. Ethyl acetate was added for extraction 3 times, and the organic phases were combined and washed with saturated NaHCO3 (15mL) and saturated brine (20mL×2). The mixture was 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, a colorless oil (2.64g, yield 80.3%).

[0068] 1 H NMR (400MHz, Chloroform-d) δ6.98 (dt, J=15.7, 6.6Hz, 1H), 5.96-5.84 (m, 1H), 2.48-2.40 (m, 2H), 2.38-2.33 (m, 2H), 2.03 (t, J=2.6Hz, 1H).

[0069] 13 C NMR (101MHz, CDCl3) δ166.61, 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), and NaOH / H2O (1 g, 50 mmol) was added at room temperature and reacted overnight. HCl was added to adjust the pH to acidic, and ethyl acetate was extracted 3 times. The organic phases were combined, and washed with saturated NaHCO3 (15 mL) and saturated brine (20 mL×2). Drying over anhydrous sodium sulfate, filtering, evaporation, and column chromatography purification (CH2C12: EtOAc = 3: 1) gave the product compound IV-1 as a white solid (1.01 g, yield 81%).

[0073] 1 H NMR (400MHz, Chloroform-d) δ12.19 (s, 1H), 7.11 (dt, J=15.7, 6.7Hz, 1H), 5.91 (d, J =15.7Hz, 1H), 2.47 (qd, J=6.7, 3.2Hz, 2H), 2.41-2.35 (m, 2H), 2.02 (t, J=2.6Hz, 1H).

[0074] 13 C NMR (101MHz, CDCl3) δ171.95, 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 an aqueous NaHCO3 solution (equivalent amount) was added dropwise. The mixture was filtered through a 0.45 μm membrane and freeze-dried to obtain a white powdery compound I-1 (Cr-alkyne).

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

[0079] Synthesis of compound III-2:

[0080]

[0081] Compound II-2 hexynol (2g, 20mmol) was dissolved in DCM (2mL), and DMP (18g, 40mmol) was added at room temperature and stirred for 30min, followed by addition of methoxycarbonylmethylenetriphenylphosphine (16.05g, 48mmol). The reaction was continued for 2h. Ethyl acetate was added for extraction 3 times, and the organic phases were combined and washed with saturated NaHCO3 (15mL) and saturated brine (20mL×2). The mixture was dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and purified by column chromatography (CH2Cl2: EtOAc = 10: 1) to obtain product III-2 as a colorless oil (2.6g, yield 84%).

[0082] 1 H NMR (400MHz, Chloroform-d) δ6.95 (dt, J=15.6, 7.0Hz, 1H), 5.87 (dt, J=15.6, 1.6Hz, 1H), 2.34 ( qd, J=7.2, 1.5Hz, 2H), 2.23 (td, J=7.0, 2.7Hz, 2H), 1.99 (t, J=2.6Hz, 1H), 1.70 (p, J=7.2Hz, 2H).

[0083] 13 C NMR (101MHz, CDCl3) δ166.90, 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), and 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 ethyl acetate was added to extract 3 times, and the organic phases were combined, and washed with saturated NaHCO3 (15 mL) and saturated brine (20 mL×2). Drying over anhydrous sodium sulfate, filtering, evaporation, and column chromatography purification (CH2Cl2: EtOAc = 3: 1) gave the product compound IV-2 as a colorless solid (1.2 g, yield 87%).

[0087] 1H NMR (400MHz, Chloroform-d) δ11.87 (s, 1H), 7.08 (dt, J=15.6, 7.0Hz, 1H), 5.87 (dt, J=15.6, 1.6Hz, 1H) , 2.38 (qd, J=7.1, 1.5Hz, 2H), 2.24 (td, J=7.0, 2.7Hz, 2H), 1.99 (t, J=2.7Hz, 1H), 1.71 (p, J=7.2Hz, 2H).

[0088] 13 C NMR (101MHz, CDCl3) δ172.09, 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 an aqueous NaHCO3 solution (equivalent amount) was added dropwise. The mixture was filtered through a 0.45 μm membrane and freeze-dried to obtain a white powdery compound I-2 (Cr-alkyne-2).

[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 invention first tested whether these two probes can be modified to proteins through metabolism 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 a CuAAC click chemistry reaction with rhodamine azido to covalently bind the probe-labeled protein to the rhodamine dye for in-gel fluorescence analysis. The labeling principle is as follows Figure 1A shown.

[0094] The results are as follows Figure 1B -C showed that at a concentration of 0.5mM or 5mM, Cr-alkyne with a shorter chain was more effective than Cr-alkyne-2 in labeling proteins in living cells. The reason for this result is that we consider that Cr-alkyne has one less carbon than Cr-alkyne-2 and has stronger hydrophilicity, thus having stronger binding ability in the hydrophilic pocket of the protein. Therefore, the present invention selected Cr-alkyne for subsequent research.

[0095] Example 4 Cr-alkyne labeling evaluation

[0096] Evaluation of the labeling effect of Cr-alkyne with changes in concentration, time, and cell line type. The results of in-gel fluorescence analysis are as follows: Figure 2A -C: Using different concentrations of Cr-alkyne for labeling, it was found that the labeling intensity of Cr-alkyne continued to increase with the increase of concentration ( Figure 2A As the labeling time increases, the labeling intensity does not change much from 2h to 8h ( Figure 2B ). Different MCF-7, LX2 and Raw264.7 cell lines were labeled and it was found that Cr-alkyne had good labeling efficiency and the labeled patterns were different ( Figure 2C Therefore, Hek293T cells were subsequently treated with a labeling concentration of 5 mM and a labeling time of 8 h.

[0097] Example 4 Testing of competition of crotonic acid against Cr-alkyne

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

[0099] Example 5 Cr-alkyne labeling test on known crotonylated substrate proteins (histones)

[0100] Subsequently, the present invention examined whether Cr-alkyne can metabolically label known crotonylated proteins. It has been confirmed that histones are highly crotonylated proteins. Therefore, we extracted core histones from Cr-alkyne-labeled HEK293T cells, followed by click reactions with azidrhodamine and in-gel fluorescence analysis. Figure 4A It can be seen that 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 the extracted proteins were then subjected to click chemistry using azide-biotin and enriched by streptavidin beads. Figure 4B and Figure 4C As shown, Western blot analysis of immunoprecipitated target proteins showed that Cr-alkyne could be metabolically labeled to HMGB1 and YWHAE.

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

[0103] We further performed fluorescence visualization of crotonylated proteins in HEK293T cells. PBS-treated or Cr-alkyne-treated HEK293T cells were washed and then fixed, permeabilized, and subjected to click chemistry conjugation of azide-rhodamine. Figure 5 As shown, stronger rhodamine signals were observed in Cr-alkyne-treated cells, especially in the nucleus, compared with the disordered signals in PBS-treated cells. Therefore, the method developed in the present invention has realized the fluorescence imaging of crotonylated proteins in cells by Cr-alkyne for the first time. Taken together, these results indicate that Cr-alkyne can metabolically assemble into crotonylated proteins in mammalian cells and achieve robust fluorescence detection of protein crotonylation.

[0104] Example 7 Analysis of modification sites of Cr-alkyne on histones

[0105] After detecting the fluorescent labeling of Cr-alkyne, we wanted to understand the exact modification sites of Cr-alkyne at the proteomic level. Therefore, whole lysate of Cr-alkyne-labeled HEK293T cells was prepared, coupled to the acid-cleavable azide-DADPS-biotin tag by click chemistry, and affinity purified with streptavidin beads. After further enzymatic cleavage and acid-mediated cleavage on the beads, 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), and the Cr-alkyne-labeled lysine residues (K+249.1477Da) were set as variable modifications. The chemical proteomics experimental process of Cr-alkyne is as follows: Figure 6 shown.

[0106] To enhance the reliability and coverage of site identification, e.g. Fig. 7AAs shown, we performed three replicates of Cr-alkyne labeling samples, and only sites that were identified at least twice in the three replicates were considered for further analysis. MS / MS analysis showed that Cr-alkyne was indeed metabolized to known crotonylation sites in histones. Figure 7B As shown in Figure 2, we identified 28 crotonylation sites in typical histones of human cells, 18 of which have been reported in the work of Tan et al. (Cell, 2011, 146, 1015-1027). For example, KAVTKCr-alkyneAQK containing Cr-alkyne modification at H2B-K20 was identified by MS / / MS analysis. It is worth noting that we also identified two new histone crotonylation sites, including H1-K177 and H2B-K24. In addition to typical histone crotonylation marks (such as Figure 7C In addition to the above, we also identified five new atypical histone crotonylation modifications, as shown in Table 1, including H2AX-K5, H2AX-K9, H2AZ1-K7, H2AZ1-K115 and H2AZ1-K120. These data together support that the Cr-alkyne probe can directly target lysine residues on histones and identify new histone Kcr sites in the whole proteome.

[0107] Table 1 Newly identified modification sites using crotonylation probes

[0108]

[0109]

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

[0111] We explored the analysis of Cr-alkyne modification sites in non-histone proteins. Fig. 8A As shown in Figure 2, in the three replicates, the number of modified sites of non-histone derived modified peptides was higher than that of histones. Some representative crotonylation substrate proteins, such as NCL, HMGB1, HMGN2, DDX21, HMGB3 and PARP1, were identified by MS / MS analysis. Figure 8B TMPO proteins, which were reported as crotonylated proteins in previous human omics data, are shown in Figure 2. Figure 8CAs shown in the figure, it can also be identified this time and confirmed by protein interaction detection (pull-down) experiments and WB analysis. It is worth noting that for known crotonylated proteins, there are also some newly identified modification sites, such as EEF1A1-K457 and NUCKS1-K188. More importantly, several new crotonylated substrate proteins were also 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 crotonylation substrate proteins, we performed a series of bioinformatics analyses at the protein level. Cellular component analysis using the Gene Ontology (GO) database revealed that Fig. 9A As shown in Figure 2, crotonylated proteins are more abundant in the nucleus, nucleoplasm, and nucleosomes. Fig. 9B As shown in Figure 2, GO analysis of biological processes showed that crotonylated proteins were significantly enriched in processes such as nucleosome assembly, positive regulation of RNA polymerase II promoters, and chromatin organization. Molecular function analysis showed that, Fig. 9C As shown in Figure 2, many crotonylated proteins have RNA, DNA, and chromatin binding activities. PTM enrichment analysis showed that, for example, Fig.9D As shown, most crotonylated proteins contained other types of PTMs, with high enrichment in acetylation, isopeptide bonds, and methylation.

[0114] Overall, these results suggest that crotonylation occurs primarily on proteins associated with nuclear biological processes, such as chromosome organization, RNA processing / metabolism, and DNA repair. To explore the sequence distribution characteristics of crotonylation, we used pLogo to analyze the amino acid sequences surrounding the identified crotonylation sites. Fig.9E As shown, positively charged lysine was preferentially selected at many positions (e.g., -7, -5, -4, +4, +6, and +7), while negatively charged glutamate and aspartate 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 preferences of these modified lysine residues. We calculated the different preferences of crotonylation sites on the three major secondary structures: α-helix (H), β-sheet (E), and loop (L). From Fig.9F In the histogram of , it was found that the crotonylation site had a preference for the ring structure, while H and E were generally lacking. These data suggest that crotonylation has a unique intrinsic reactivity towards lysine.

[0115] Example 10 Quantitative chemical proteomics to discover crotonylation sites regulated by histone deacetylases (HDACs)

[0116] This probe can be used to discover crotonylation sites regulated by histone deacetylases (HDACs) by quantitative chemical proteomics. Therefore, we performed a tandem orthogonal proteolytic activity-based protein profiling (rdTOP-ABPP) experiment to quantitatively identify specific lysine modifications perturbed by HDACs. HDAC1 / 3 knockout, overexpression, and untreated cells were treated with the Cr-alkyne probe, and the extracted proteomes were then conjugated to an acid-cleavable azido-biotin label by CuAAC. After enrichment with streptavidin beads and on-bead trypsin digestion, probe-bound peptides from knockout, overexpression, and untreated cells were isotopically labeled using light, medium, and heavy dimethylation reagents using the rdTOP-ABPP procedure. The mixed samples were acid-cleaved, and the released peptides were analyzed by LC-MS / MS and quantified. The workflow of the quantitative chemical proteomics method is detailed in the following. Fig.10 .

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

[0118] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above are only specific implementation methods of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent substitutions, improvements, etc. made on the basis of the technical solutions of the present application should be included in the protection scope of the present application.

Claims

1. A crotonylation detection probe, characterized in that: It has the structure shown in Formula I: Wherein, n is 1 or 2.

2. A method for preparing a crotonylation detection probe, for preparing the crotonylation probe according to claim 1, characterized in that: The steps include: 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 to 2:2 to 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 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 is in the range of 1:1 to 1.

1.

3. An application of the crotonylation detection probe according to claim 1, characterized in that: The probe can be used for fluorescence detection of crotonylated proteins.

4. A use 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.

5. A use of the crotonylation detection probe according to claim 1, characterized in that: The probe can be used to identify crotonylated proteins.

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

7. A use of the crotonylation detection probe according to claim 1, characterized in that: The probe can be used to identify the modification site of crotonylation.

8. The use according to claim 7, characterized in that: The method for identifying the crotonylation modification site is as follows: the probe is co-incubated with cells for protein labeling, then a whole cell lysate is prepared, and then the probe-labeled protein is biotin-modified using azide-biotin, and finally the probe-labeled protein is separated from the proteome using streptavidin-coupled magnetic beads, and the probe-labeled protein is subjected to mass spectrometry detection and analysis.

9. A use of the crotonylation detection probe according to claim 1, characterized in that: The probe can identify crotonylation sites regulated by crotonylase by quantitative chemical proteomics; wherein the crotonylase is a deacetylase. 10 . A kit comprising the crotonylation detection probe according to claim 1 .

Images