Protein post-translational modification detector based on GST fusion histone H3 fragment mixture as well as preparation method and application of protein post-translational modification detector

Abstract

The invention discloses a protein post-translational modification detector based on a GST fusion histone H3 fragment mixture as well as a preparation method and application of the protein post-translational modification detector. The protein post-translational modification detector comprises 9 segmented fragments H3F1-H3F9 of histone H3. The prepared GST is fused with an H3 fragment (H3F1-H3F9) mixed plasmid; the mixed plasmid is transformed into BL21-DE escherichia coli; GST fusion H3 fragment mixture expression is carried out; purification is carried out; after the mixture is combined with GSH resin, incubation with a cell lysis solution is carried out for PTM; the GST label of the mixture after PTM is cut off by using precession TM protease, and the H3F1-H3F9 mixture is released into a solution; if a signal peak of the corresponding molecular weight is detected in the mass spectrum, PTM of the organic acid can be confirmed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a protein post-translational modification detector based on a GST-fused histone H3 fragment mixture, and a preparation method and application thereof. Background Art

[0002] Post-translational modifications (PTMs) of proteins refer to the covalent reactions of specific small proteins or functional groups with protein side chains after mRNA is translated into protein. These modifications include small protein modifications such as ubiquitination, sumoylation, nedidylation, and isoglycation, as well as phosphorylation, glycosylation, nitrosylation, methylation, and various acylation modifications (such as acetylation, malonylation, hydroxyisobutyrylation, crotonylation, benzoylation, lactylation, palmitoylation, and succinylation). PTMs play a key role in regulating protein activity, localization, and interactions with other molecules (such as proteins, nucleic acids, lipids, and cofactors), thereby influencing replication, transcription, translation, and other important intracellular molecular events. Abnormalities in PTMs are closely associated with the occurrence and development of various diseases. For example, there are hundreds of PTM sites on histones, which are referred to as the "histone code." Some of these acylation sites have been shown to have important functions. Histone acylation regulates chromatin accessibility by altering the tightness of histone binding. Furthermore, the functions of non-histone acylation are mainly divided into two categories: enhancing protein activity and stability, or reducing protein activity and stability. These acylation / deacylation processes are regulated by acylases / deacylases, and the activities of these modification enzymes themselves are also regulated by PTMs.

[0003] In PTM research, PTM identification is one of the most important tasks. PTM identification can be divided into two categories: 1) identification of PTM sites in the whole proteome or specific proteins (such as histones) for known types of PTMs (such as crotonylation); 2) discovery of new PTMs. The identification of known PTMs usually uses pan-PTM antibodies or specific protein antibodies to enrich PTM proteins, and then uses proteases (such as trypsin, chymotrypsin, aspartic proteases, glutamic proteases and lysine proteases) to enzymatically cut the proteins into small peptide fragments. Then, high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) is used to identify PTM sites and quantify their abundance. Finally, professional software (such as MaxQuant, PTMap) is used to search the protein database. For the discovery of new PTMs, researchers usually need to have a strong background in mass spectrometry analysis and use customized software. For example, Zhao Yingming's team successfully identified the crotonylation site of histones using mass spectrometry technology (Cell. 2011 Sep 16; 146(6): 1016-28.). However, these methods require researchers to have high mass spectrometry analysis capabilities, which limits their application among a large number of researchers.

[0004] Therefore, developing a simple and efficient method for identifying novel PTMs is of great significance. Considering that many of the novel modifications discovered are acylation modifications, the reaction process generally involves the reaction of an organic acid (such as lactic acid, crotonic acid, and benzoic acid) with coenzyme A to form acyl-CoA, followed by the transfer of the acyl group to the side-chain amino group of a protein lysine residue by an acyltransferase. For short peptides (6-15 amino acids) of a given molecular weight, the molecular weight before and after acylation can be calculated. Based on this, we proposed a method for detecting unknown novel PTMs using short histone H3 fragments. Because short peptides can be directly read by LC-MS, we simply incubate the purified H3 short fragments with cell or tissue lysate containing the predicted PTM acyl donor (organic acid), skip the protease digestion step, and directly analyze the PTM-modified H3 peptides by mass spectrometry. By providing the mass spectrometry company with amino acid composition and molecular weight information, we can target the corresponding signal peaks in the mixed peptide signal peak graph.

[0005] Based on this hypothesis, we fragmented histone H3 into nine fragments (H3F1-H3F9), each approximately 15 amino acids long, and constructed these fragments into the GST-fusion protein expression plasmid pGEX-6P1. Following mixed expression and purification, these fragments were incubated with mouse ovary, testis, and 3T3 cell lysates for PTM modification. The GST tag was then cleaved with Precision protease, releasing the H3F1-H3F9 mixture for mass spectrometry identification. The results showed that all fragments were successfully identified, but with significant variations in abundance (1.13%-23.4%). Compared to the H3 crotonylation site reported by Zhao Yingming's group (87.3% coverage, Cell 2011), our method identified five new crotonylation sites (sites 14, 37, 64, 115, and 122) in mouse ovary lysates, with coverage approaching 100%.

[0006] Furthermore, a mixture of GST-tagged histone H3 fragments can be used to identify unknown novel PTMs. Because this method does not require protease digestion of the H3 fragments, the molecular weight of the PTM-modified H3 fragment can be calculated by adding the molecular weight of the organic acid to the molecular weight of the H3 fragment minus 18 (the molecular weight of H2O). By sending the PTM-modified H3 fragments to a mass spectrometry company and providing molecular weight information before and after the PTM, the spectra can be targeted to identify signal peaks of relevant molecular weights. Summary of the Invention

[0007] Technical Problems Solved: The present invention provides a protein post-translational modification detector based on a mixture of GST-fused histone H3 fragments, and its preparation method and application. The prepared GST-fused H3 fragment (H3F1-H3F9) mixed plasmid is transformed into BL21-DE Escherichia coli → the GST-fused H3 fragment mixture is expressed → purified → the mixture is bound to GSH resin and incubated with cell lysate for PTM → the mixture after PTM is purified by precipitant TM The protease removes the GST tag, releasing the H3F1-H3F9 mixture into solution. The solution is then sent to a mass spectrometry company for targeted searches for H3 fragments with PTMs of corresponding molecular weights. If a signal peak of the corresponding molecular weight is detected in the mass spectrum, it can be confirmed that the organic acid can indeed undergo PTMs.

[0008] Technical solution: A protein post-translational modification detector based on a mixture of GST-fused histone H3 fragments, containing nine segments of histone H3, H3F1-H3F9, whose amino acid sequences are:

[0009] H3F1:MARTKQTARKSTGGKA;

[0010] H3F2:PRKQLATKAARKSAP;

[0011] H3F3:STGGVKKPHRYRPGT;

[0012] H3F4:VALREIRRYQKSTEL;

[0013] H3F5:LIRKLPFQRLVREIA;

[0014] H3F6:QDFKTDLRFQSAAIG;

[0015] H3F7:ALQEASEAYLVGLFE;

[0016] H3F8:DTNLCAIHAKRVTIM;

[0017] H3F9:PKDIQLARRIRGERA.

[0018] Each fragment was expressed through GST tag fusion, and the lysine residue of the potential PTM site in each fragment was located in the middle position of the sequence.

[0019] The amino acid sequence of the above GST tag is:

[0020] MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYI

[0021] DGDVKLTOSMAIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK

[0022] VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPK

[0023] LVCFKKRIEAIPOIDKYLKSSKYIAWPLOGWOATFGGGDHPPKSDLEVLFOGPLGSPFPGRLERPH.

[0024] The preparation method of the protein post-translational modification detector based on the GST-fused histone H3 fragment mixture comprises the following steps: (a) cloning the DNA sequences of H3F1-H3F9 into the pGEX-6P1 plasmid respectively to construct 9 GST fusion expression plasmids; (b) mixing and transforming the 9 plasmids in step (a) into DH5α Escherichia coli at one time, randomly picking colonies with at least 7 times the number of plasmids for amplification, and extracting the mixed plasmid; (c) transforming the mixed plasmid into BL21-DE-plys Escherichia coli, randomly picking colonies with at least 30 times the number of plasmids for induction of expression; (d) purifying the GST fusion protein using glutathione agarose resin, and performing pre-cleavage. TM The GST tag was removed by protease to obtain a mixture of free H3F1-H3F9 fragments.

[0025] The conditions for inducing expression in step (c) are: culturing in LB medium containing 0.1 mmol / L IPTG at 16° C. and 200 RPM shaking for 16 hours.

[0026] The method for identifying protein post-translational modification (PTM) sites using the protein post-translational modification probe based on the GST-fused histone H3 fragment mixture comprises the following steps: (a) binding the purified GST-fused histone H3 fragment mixture to glutathione resin and then incubating with a solution containing a PTM donor to be detected; (b) using PreScission TM The GST tag is removed by protease, releasing the H3F1-H3F9 fragment; (c) The molecular weight of the PTM-modified fragment is detected by liquid chromatography-mass spectrometry (LC-MS / MS), and the signal peak is targeted for analysis to confirm the PTM site.

[0027] The PTM is acylated, and the PTM donor to be tested is at least one selected from lactic acid, crotonic acid, and benzoic acid.

[0028] The above-mentioned protein post-translational modification detector based on the GST-fused histone H3 fragment mixture is used in the screening of novel PTMs. By calculating the molecular weight difference before and after PTM modification, ΔM = organic acid molecular weight - 18Da, the mass spectrometry signal peak is targeted for detection to confirm the presence of the novel PTM.

[0029] The detection of the above novel PTM covers the PTM sites of the entire sequence of histone H3, with a coverage rate of ≥95%.

[0030] The application of the above-mentioned protein post-translational modification probe based on the GST-fused histone H3 fragment mixture in comparing PTM differences in different tissues or disease models includes the following steps: (a) incubating the mixture with cell or tissue lysates from different sources; (b) analyzing the molecular weight differences of each group of PTM modifications by mass spectrometry to screen for specific PTM sites.

[0031] The above-mentioned protein post-translational modification probe based on a mixture of GST-fused histone H3 fragments is used to prepare a DNA damage repair kit. The kit evaluates the role of PTM in DNA damage repair by identifying the effect of the loss of the H3-K115 crotonylation site on rH2AX levels and gene expression profiles.

[0032] Benefits: 1. Significantly simpler than traditional methods for identifying unknown PTMs. Since H3 can undergo a variety of known PTMs, we chose to use H3 fragments as PTM detectors. Since protease digestion of H3 fragments is not required, the molecular weights of each fragment before and after the PTM are completely predictable. This is significantly simpler than inferring PTMs based on mass spectrometry knowledge combined with software.

[0033] 2. Significantly more efficient than traditional methods for identifying unknown PTMs. Traditional PTM identification methods require protease digestion of proteins before LC-MS analysis. Due to the varying fragment sizes, software searches (Mascot, PTMap) to identify signal peaks can also take a long time. However, our PTM-derived fragments eliminate the need for protease digestion and can be directly targeted for signal peaks, resulting in a sample testing time reduction of at least one week. Furthermore, the raw data volume is expected to be several dozen times smaller, resulting in an estimated one-week reduction in data analysis time.

[0034] 3. Significantly improved coverage compared to traditional histone H3 PTM site identification. Histone H3 PTMs, as a key epigenetic modification, play a crucial role in various physiological processes and have been confirmed by numerous high-level studies. Therefore, the H3 mixed fragments described in this application can not only serve as a detector for unknown novel PTMs, but can also be used to fully identify H3 PTM sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 We successfully ligated H3F1-F9 fragments into pGEX6P1 individually but mixed them into DH5α at once and extracted the plasmids: all nine plasmids were detected in 63 colonies randomly picked at 7 times the number of plasmids.

[0036] Figure 2We successfully performed a one-time mixed prokaryotic expression, PTM modification, and full-coverage crotonylation mass spectrometry identification of H3F1-F9: A. H3F1-F9 was transformed into protein expression-specific E. coli at one time, and the presence of individual fragments was checked by PCR. B. Schematic diagram of the mixed expression, purification, and mass spectrometry identification of H3F1-F9 fragments. C. Colonies (265) with 30 times the number of fragments were randomly selected, shaken, expressed, purified, and the GST-H3 mixed fragment was purified and then identified by SDS-PAGE and staining. TM After protease removal of the GST tag, H3F1-F9 is not visible on the gel due to its small molecular weight, but can be detected by mass spectrometry. D. Ratio of each fragment in the mass spectrometry data.

[0037] Figure 3 .Compared with the current highest level of identification, we identified 6 new H3 crotonylation sites: We found that compared with the H3 crotonylation sites reported by Zhao Yingming's team (87.3% coverage, Cell 2011), our method newly identified 6 crotonylation sites in ovarian lysate, namely sites 14, 36, 37, 64, 115 and 122 (indicated by arrows).

[0038] Figure 4 We have preliminarily discovered that the H3-K115 crotonylation site has an important function: A and B. Plasmids containing H3-WT, -K14A, -K64A, and the inactive -K115A mutant were overexpressed in parallel in cells. Green fluorescence and Strep II blotting revealed efficient overexpression of the plasmids. All plasmids were fused with EGFP and Strep II, and the same applies hereafter. B and C. Overexpression of H3-K115A significantly increased rH2AX levels by over 10-fold, suggesting that H3-K115 crotonylation is essential for cellular DNA damage repair.

[0039] Figure 5 We found that both activating and inactivating H3-K115 crotonylation mutants significantly altered gene expression profiles: A to C. We found that overexpression of H3-K115Q (activating mutation) and H3-K115A (inactivating mutation) plasmids in MCA205 cells resulted in significantly different gene expression patterns compared to overexpression of H3-WT plasmids compared to control plasmids. D. These differentially expressed genes participate in multiple important signaling pathways, suggesting that H3-K115 crotonylation must maintain an adaptive level in cells.

[0040] Figure 6.We found that the H3-K115 crotonylation-inactivating mutation significantly altered the spectrum of interacting proteins: A and B. We overexpressed H3-K115A and H3-WT plasmids in cells and performed IP-mass spectrometry identification using Strep II antibody. We also performed IP-mass spectrometry identification using Strep II antibody in untransfected cell lysate as a control (the captured proteins were non-specific proteins). We found that the interacting protein spectrum of H3-K115A was significantly different from that of H3-WT. DETAILED DESCRIPTION

[0041] Example 1

[0042] The DNA sequences corresponding to the 9 H3 fragments H3F1-H3F9 were connected to pGEX-6P1, and the mixture was transformed into DH5alpha Escherichia coli (for plasmid construction) at one time. 7 times the number of plasmids were picked (63 to ensure that all 9 plasmids were present), and the mixed plasmid was extracted at one time. Next, the mixed plasmid was transformed into BL21-DE-Plys Escherichia coli (for prokaryotic protein expression), 30 times the number of plasmids were picked (270 to ensure that all 9 plasmids were present), and added to 100mL LB culture medium to proliferate. The bacterial solution was added to 4L LB culture medium at a volume ratio of 1:50, and the culture was expanded in a shaker at 200RPM and 37°C to OD 600 =0.6, 0.1 mmol / L IPTG was added, and the cells were induced at 16°C, 200 RPM overnight for 16 hours. The cells were collected by centrifugation at 3500 rpm for 10 minutes at 4°C, washed with ice-cold PBS at pH 7.3, and lysed in 25 mL of lysis buffer (PBS, pH 7.3, 1 mM DTT, 1 mM PMSF, 1% Triton X-100, 1:100 InStab TM Protease inhibitor cocktail (Yeasen), 1:100 InStab TM Lyse in a phosphatase inhibitor cocktail (Yeasen). After standing on ice for 15 minutes, the cells were disrupted with a high-pressure crusher and the supernatant (containing the GST fusion protein) was collected by centrifugation at 16000RPM at 4°C. The fusion protein was captured with 1mL glutathione agarose resin at 4°C for 30 minutes. Next, the resin bound to the GST fusion polypeptide was transferred to a 10mL chromatography column and the resin was washed three times with 20mL of washing buffer. Finally, the GST fusion polypeptide was eluted with elution buffer (1×PBS, 1mM MgSO4, 0.5mM DTT, 10mM Glutathione, pH 7.4). To remove the GST tag, the GST fusion polypeptide was transferred to the PreScission column by centrifugation using a size-exclusion spin column. TMProtease digestion buffer (50mM Tris, pH 8.0, 0.5mM EDTA, 1mM DTT) was added and re-bound to the GSH resin. TM Use protease to remove the GST tag (1:25-1:100 U enzyme / μg fusion peptide), releasing the mixed H3 fragments into the solution. Since the molecular weight of the mixed fragments is too small to estimate purity and concentration by SDS-PAGE, a Nanodrop can be used to directly measure protein concentration (using a short peptide of known concentration as a reference).

[0043] Example 2

[0044] Metabolic abnormalities are an important feature of various chronic diseases or sub-health conditions. Many differential metabolites are organic acid molecules that may induce PTMs and thus affect protein stability, gene expression, etc. To determine whether they induce PTMs, the PTM detector in this application can be used to achieve simple, rapid and efficient detection. For example, we conducted a full coverage detection of H3 crotonylation sites. We used a GST-fused H3F1-H3F9 fragment mixed plasmid ( Figure 1 ; Figure 2 :A) One-time transformation into BL21DE-Plys for one-time expression of H3 mixed fragments, purification, binding to GSH resin and incubation with organic acid treated cells or tissues, using PreScission TM The protease cleaves the GST tag and releases the mixed H3 fragments into the solution ( Figure 2 : BD), and finally sent to the company for LCMS detection, targeting the signal peak of the molecular weight to be measured in the mixed signal peak. We used this method to achieve 100% coverage of histone H3 crotonylation sites, screening out 6 more new crotonylation sites than the reported by Zhao Yingming's team (87.3% coverage, Cell, 2011) ( Figure 3 ).

Claims

1. A protein post-translational modification detector based on a mixture of GST-fused histone H3 fragments, characterized in that: Contains 9 segments of histone H3, H3F1-H3F9, whose amino acid sequences are: H3F1:MARTKQTARKSTGGKA; H3F2:PRKQLATKAARKSAP; H3F3:STGGVKKPHRYRPGT; H3F4:VALREIRRYQKSTEL; H3F5:LIRKLPFQRLVREIA; H3F6:QDFKTDLRFQSAAIG; H3F7:ALQEASEAYLVGLFE; H3F8:DTNLCAIHAKRVTIM; H3F9:PKDIQLARRIRGERA; each fragment is expressed through GST tag fusion, and the lysine residue of the potential PTM site in each fragment is located in the middle position of the sequence.

2. The protein post-translational modification detector based on a GST-fused histone H3 fragment mixture according to claim 1, characterized in that: The amino acid sequence of the GST tag is: MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTOSMAIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDF LSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPOIDKYLKSSKYIAWPLOGWOATFGGGDHPPKSDLEVLFOGPLGSPEFPGRLERPH.

3. A method for preparing a protein post-translational modification detector based on a mixture of GST-fused histone H3 fragments according to claim 1, characterized in that: The following steps are involved: (a) The DNA sequences of H3F1-H3F9 were cloned separately into the pGEX-6P1 plasmid to construct nine GST fusion expression plasmids. (b) The nine plasmids from step (a) were mixed and transformed into DH5α Escherichia coli at once. Colonies with at least 7 times the number of plasmids were randomly selected for amplification, and the mixed plasmid was extracted. (c) The mixed plasmid was transformed into BL21-DE-plys Escherichia coli, and colonies with at least 30 times the number of plasmids were randomly selected for induction of expression. (d) The GST fusion protein was purified using glutathione agarose resin, and the GST tag was removed using PreScission™ protease to obtain a mixture of free H3F1-H3F9 fragments.

4. The preparation method according to claim 3, characterized in that The conditions for inducing expression in step (c) are: culturing in LB medium containing 0.1 mmol / L IPTG at 16° C. and 200 RPM shaking for 16 hours.

5. A method for identifying protein post-translational modification (PTM) sites using the protein post-translational modification detector based on the GST-fused histone H3 fragment mixture according to claim 1, characterized in that: The following steps are involved: (a) A mixture of purified GST-fused H3 fragments is bound to glutathione resin and incubated with a solution containing the PTM donor to be tested. (b) The GST tag is removed using PreScission™ protease, releasing the H3F1-H3F9 fragments. (c) The molecular weight of the PTM-modified fragments is determined by liquid chromatography-mass spectrometry (LC-MS / MS), and the signal peaks are targeted for analysis to confirm the PTM sites.

6. The method according to claim 5, characterized in that The PTM is acylated, and the PTM donor to be tested is selected from at least one of lactic acid, crotonic acid, and benzoic acid.

7. A use of the protein post-translational modification detector based on the GST-fused histone H3 fragment mixture according to claim 1 in screening for novel PTMs, characterized in that: By calculating the molecular weight difference before and after PTM modification, ΔM = organic acid molecular weight - 18 Da, targeted detection of mass spectrometry signal peaks was performed to confirm the presence of new PTMs.

8. The use according to claim 7, characterized in that The detection of the novel PTM covers PTM sites in the entire sequence of histone H3, with a coverage rate of ≥95%.

9. A use of the protein post-translational modification probe based on the GST-fused histone H3 fragment mixture according to claim 1 for comparing PTM differences in different tissues or disease models, characterized in that: The following steps are involved: (a) The mixture is incubated with cell or tissue lysates from different sources. (b) The molecular weight differences of each PTM modification group are analyzed by mass spectrometry to screen for specific PTM sites.

10. Use of the protein post-translational modification probe based on the GST-fused histone H3 fragment mixture according to claim 1 in the preparation of a DNA damage repair kit, wherein the kit evaluates the role of PTM in DNA damage repair by identifying the effect of the loss of the H3-K115 crotonylation site on rH2AX levels and gene expression profiles.

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