CN116283677B - Small molecular chemical cross-linking agent and preparation method and application thereof - Google Patents

Small molecular chemical cross-linking agent and preparation method and application thereof Download PDF

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CN116283677B
CN116283677B CN202310163905.XA CN202310163905A CN116283677B CN 116283677 B CN116283677 B CN 116283677B CN 202310163905 A CN202310163905 A CN 202310163905A CN 116283677 B CN116283677 B CN 116283677B
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唐士兵
杨兵
吴婷
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Zhejiang University ZJU
Guangzhou Institute of Biomedicine and Health of CAS
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Abstract

The invention provides a small molecular chemical cross-linking agent, a preparation method and application thereof, wherein the structure of the small molecular chemical cross-linking agent is shown as follows; wherein R is selected from any one of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 amido, and substituted or unsubstituted C6-C12 aryl; the substituted substituent is selected from halogen, hydroxy, cyano, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl. The small molecule chemical crosslinking agent provided by the invention can effectively crosslink the combination of the protein and the substrate thereof, and provides an effective tool for researching the functional property and application of the protein.

Description

Small molecular chemical cross-linking agent and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a small molecular chemical cross-linking agent, and a preparation method and application thereof.
Background
Recent developments in biological profiling technology have made it important to play an important role in protein structure and function research, providing important support for related research. Chemical cross-linking mass spectrometry (Chemical cross-linking of proteins coupled with mass spectrometry, CXMS) is a new technology developed in recent years to study protein structure and interactions. CXMS the chemically active small molecule cross-linking agent is used to link amino acids with close spacing in the protein or protein complex by covalent bonds to form covalent bonds within or between the protein. The cross-linked protein is degraded by pancreatin to obtain cross-linked polypeptide and other polypeptides, and then the sequence and cross-linked site of the cross-linked polypeptide are obtained through mass spectrometry, so that the structural information of the protein is obtained, and the analysis of the folding state of the protein in a three-dimensional space and the approximate region of protein-protein interaction is facilitated. CXMS has low requirements on the quantity and purity of protein samples, can capture the dynamic conformation of the protein in the solution, has no special requirements on the uniformity of the samples and whether the protein can be crystallized or not, and has the advantages of simple and quick operation and the like, so that the method has wide application in recent years, such as helping hand analysis of the fine structure of large-scale protein complex, searching of direct interaction areas between proteins and the like.
Affinity purification mass spectrometry (Affinity Purification-Mass Spectrometry, APMS) is a standard means of studying protein-protein interactions. Protein samples are usually lost in transient, weak-force protein-protein interactions during affinity purification rinsing of the beads, and are therefore difficult to detect by mass spectrometry. On the other hand, in the list of proteins obtained by mass spectrometry after affinity purification, researchers cannot distinguish whether these proteins interact directly or indirectly with the target protein. At this point, if chemical cross-linking is introduced in the APMS experiment, the chemical cross-linking agent converts non-covalent bonds between proteins into covalent bonds, facilitating capture of transient and weak protein-protein interactions. Therefore, when protein-protein interactions are studied, the combination of chemical crosslinking and APMS technology can significantly improve experimental sensitivity, and can identify direct interaction proteins and acquire more abundant information such as protein interaction interfaces. In particular, membrane receptor proteins of paramount importance for drug development often require the use of relatively harsh cell lysis and rinsing conditions during the processing of such proteins using affinity purification techniques, and thus tend to disrupt the interactions between the proteins. If the membrane receptor protein and the interaction protein are immobilized using a cross-linking agent prior to affinity purification, the loss of the interaction protein can be significantly reduced during later sample manipulations, thus significantly improving the efficiency of identifying membrane protein ligands and membrane-bound proteins in mass spectrometry.
In recent years, as researchers have intensively studied in the fields of fine structural analysis of proteins, research on protein-protein interactions, and the like, some chemical cross-linking agents have been developed and widely used in research on protein chemistry and biology. Meanwhile, with the rapid development of the biological mass spectrometry technology, the technology for identifying crosslinked polypeptides and crosslinked sites in protein samples after treatment by chemical crosslinking agents is more and more mature, so that CXMS technology plays an increasingly important role in biological research and even disease pathology research.
Although there are a few chemical cross-linking agents on the market, the existing commercial agents have some disadvantages: a) The existing cross-linking agent is easy to degrade when used in protein cross-linking solution, has poor cell membrane permeability and is rarely applied to living cells; b) The intra-molecular crosslinking ratio of the protein is high, and the crosslinking ratio between the protein and the protein is low; c) Crosslinking agents tend to be single-functional, whereas multifunctional crosslinking agents tend to be difficult to synthesize and not commercialized, or even very expensive. Therefore, aiming at the defects of the commercialized crosslinking agent, the chemical crosslinking agent with strong functions, low cost and easy availability is developed, and the biological application of the chemical crosslinking agent is shown in various biological scenes such as in-vitro proteins, living cells and the like, so that the chemical crosslinking agent has very important significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a small molecular chemical cross-linking agent, and a preparation method and application thereof. The small molecule chemical crosslinking agent provided by the invention can effectively crosslink the combination of the protein and the substrate thereof, and provides an effective tool for researching the functional property and application of the protein.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a small molecule chemical crosslinker having the structure shown below:
wherein R is selected from any one of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 amido, and substituted or unsubstituted C6-C12 aryl.
The substituted substituent is selected from halogen, hydroxy, cyano, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.
The compound with the specific structure can effectively crosslink the combination of the protein and the substrate thereof, and provides an effective tool for researching the property of the protein.
Wherein, C1-C6 respectively refer to a structure comprising one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms or six carbon atoms, and the like, C1-C6 alkyl can be methyl, ethyl, propyl, n-butyl, and the like, C2-C6 alkenyl can be ethenyl, propenyl, and the like, and C2-C6 alkynyl can be ethynyl, propynyl, and the like.
Preferably, R is selected from hydrogen, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 amido.
Preferably, the structure of the small molecule chemical crosslinking agent is as follows:
wherein R has the same defined range as above.
Preferably, the small molecule chemical crosslinking agent is selected from any one of the following structures:
preferably, the small molecule chemical crosslinking agent is selected from any one of the following structures:
in a second aspect, the present invention provides a method for preparing a small molecule chemical crosslinker as described above, comprising the steps of:
And (3) carrying out condensation reaction on the phenylenediamine compound and 2-chloroethane sulfonyl chloride to obtain the micromolecular chemical cross-linking agent.
The reaction route is as follows:
wherein R has the same defined range as above.
Preferably, the reaction is carried out in the presence of a base comprising any one or a combination of at least two of 4-dimethylaminopyridine, pyridine, triethylamine, diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, potassium carbonate or cesium carbonate.
Preferably, the temperature of the reaction is 20-30 ℃.
Preferably, the reaction time is 10-18 hours.
The reaction temperature may be 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃ or the like, and the reaction time may be 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours or the like, but the reaction temperature is not limited to the above-listed values, and other values not listed in the above-listed value ranges are equally applicable.
In a third aspect, the invention provides the use of a small molecule chemical crosslinker as described above for studying enzyme and substrate interactions.
In a fourth aspect, the invention also provides the use of a small molecule chemical cross-linker as described above in protein analysis.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a compound with a specific structure, which can effectively capture interaction proteins through chemical crosslinking and provides an effective tool for researching the functional properties of the proteins.
Drawings
FIG. 1 is a graph of Western blot analysis results of in vitro PDES after chemical cross-linking of Trx1 with PAPR or PAPR C239S mutants;
FIG. 2 is a complex structure of Trx1 and PAPR;
FIG. 3 is a graph showing Western blot analysis results of PDES in E.coli after chemical cross-linking of Trx1 with PAPR;
FIG. 4 is a graph showing Western blot analysis results of PDES in E.coli after chemical cross-linking of Trx1 and PAPR C239S mutants;
FIG. 5 is a graph of Western blot analysis of PDES in 293T cells capturing TXN1 direct interaction protein by chemical cross-linking;
FIG. 6 is a graph of Western blot analysis of the capture of TXN1 direct interaction proteins by chemical cross-linking of ePDES1 in 293T cells;
FIG. 7 is a graph of the results of a mass spectrometry assay for chemical click and biotin enrichment following the capture of TXN1 direct interaction protein by ePDES in 293T cells;
FIG. 8 is a graph of Western blot analysis of the capture of TXN1 direct interaction proteins by chemical cross-linking of ePDES within 293T cells;
FIG. 9 is a graph of the results of a mass spectrometry assay performed for chemical click and biotin enrichment after ePDES.sup.2 capture of TXN1 direct interaction protein in 293T cells.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
Synthesis of PDES compound
M-phenylenediamine (541 mg5.0 mmol 1) was dissolved in 60mL of methylene chloride, and 2-chloroethanesulfonyl chloride (2037.5 mg,12.5 mmol 1) and pyridine (1977.5 mg,25.0 mmol 1) were added at 0℃and then moved to 25℃and stirred for 12 hours. The reaction mixture was diluted with water and extracted with ethyl acetate (150 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. And purified by silica gel chromatography to give 795.6mg PDES (55% yield) as a white solid.
1H NMR(400MHz,DMSO-d6)δ＝10.08(s,2H),7.19(t,J＝8.1,1H),7.05(s,1H),6.81(dd,J＝8.1,1.8,2H),6.74(dd,J＝16.4,9.9,2H),6.13(d,J＝16.4,2H),6.06(d,J＝9.9,2H).13C NMR(126MHz,DMSO)δ＝138.58,135.98,129.73,128.00,114.46,109.95、
HRMS (ESI) M/z: [ m+h ] +C10H16N3O4S2 = 306.0577; it was found that 306.0573,
Example 2
Synthesis of Compound ePDES1
To 5-iodo-1, 3-phenylenediamine (4638 mg,2.0mmol,1.0 eq), diphenylphosphine palladium dichloride (34 mg,0.048mmol,2.4 mol%), cuprous iodide (13 mg,0.068mmo1,3.4 mol%) and trimethylsilylacetylene (1.57 g,16.0mmol,8.0 eq) were added 2.0mL of triethylamine and 8.0mL of tetrahydrofuran, and stirred at 25℃for 12 hours. After completion of the reaction, the supernatant was washed with tetrahydrofuran, and the organic phases were combined and concentrated. The target product compound 1 was obtained by silica gel column chromatography and found 204.82 as a brown solid (370mg,90.69％).1H NMR(500MHz,CDCl3)δ6.24(2H,br d,J＝1.6Hz),5.99(1H,br t,J＝1.6Hz),0.22(9H,s).MS(ESI)C11H17N2Si[M+H]+＝205.12,.
Compound 1 (369 mg,1.81mmol,1.0 eq.) pyridine (760 mg,9.61mmol,5.3 eq.) and a catalytic amount of 4-dimethylaminopyridine (47 mg,0.38mmol,0.2 eq.) are dissolved in dichloromethane and 2-chloroethanesulfonyl chloride (689 mg,4.23mmol,2.3 eq.) is added dropwise to the reaction solution at 0deg.C. The reaction was carried out at 25℃and stirred for 12h. Washing the mixture with 1M hydrochloric acid (2 times) and sodium bicarbonate solution, washing with saturated solution of ketone salt, drying with anhydrous sodium sulfate, spin drying, and separating with silica gel column chromatography to obtain compound 2 as yellow oily substance (360mg,51.72％).1H NMR(500MHz,DMSO-d6)δ10.22(2H,br s),7.10(1H,br t,J＝1.9Hz),6.84(2H,d,J＝1.9Hz),6.78(2H,dd,J＝16.4,9.9Hz),6.14(2H,d,J＝16.4Hz),6.09(2H,d,J＝9.9Hz),0.22(9H,s).
Compound 2 was dissolved in 4.4mL of N, N-dimethylformamide, and 0.88mL of water and potassium fluoride (119 mg,2.05mmol,2.2 equivalents) were added at 25 ℃. The reaction was carried out at 25℃for 4 hours. Saturated ammonium chloride and ethyl acetate were added for extraction, the organic phases were combined, dried, spin-dried, and chromatographed on silica gel to give compound 3 (i.e., ePDES 1) as a yellow oil (360mg,51.72％).1H NMR(500MHz,CDCl3)δ7.06(1H,bt t,J＝1.9Hz),7.00(2H,d,J＝1.9Hz),6.82(2H,br s),6.56(2H,dd,J＝16.5,9.9Hz),6.37(2H,d,J＝16.5Hz),6.05(2H,d,J＝9.9Hz),3.11(1H,s).13C NMR(125MHz,CDCl3)δ137.7,134.7,129.4,124.5,119.4,111.7,81.9,79.0.HRMS(ESI)C12H13N2O4S2[M+H]+＝313.0311, as 313.0293.
Example 3
Synthesis of Compound ePDES2
Methyl 3, 5-diaminobenzoate (1.0g,6.02mm ol,1.0 eq), pyridine (2.38 g,30.10mmol,5.0 eq) and catalytic amount of 4-dimethylaminopyridine (147mg,1.20mm ol,0.2 eq) were dissolved in 30mL of dichloromethane, cooled to 0 ℃ and 2-chloroethanesulfonyl chloride (2.16 g,13.24mmol,2.2 eq) was added dropwise to the reaction solution, which was then moved to 25 ℃ and stirred for 12h. The mixture was washed with 1M hydrochloric acid (2 times) and sodium bicarbonate solution, and finally with saturated brine, dried over anhydrous sodium sulfate, and dried by spin-drying. Separating by silica gel column chromatography to obtain yellow solid 4(1.28g,61.54％).1H NMR(500MHz,DMSO-d6)δ10.34(2H,s),7.43(2H,d,J＝1.9Hz),7.30(1H,t,J＝1.9Hz),6.77(2H,dd,J＝16.4,9.9Hz),6.14(2H,d,J＝16.4Hz),6.08(2H,d,J＝9.9Hz),3.82(3H,s).MS Calcd for C12H15N2O6S2 347.04[M+H]+,found 347.00.
Compound 4 is dissolved in 18.5mL of tetrahydrofuran, cooled to 0deg.C, and then 18.5mL of lithium hydroxide solution (354 mg,14.78mmol,4.0 eq.) is added dropwise. The reaction was carried out at 25℃and stirred for 12h. Dropwise adding 1M hydrochloric acid solution at 0deg.C, adjusting pH to 3-4, distilling under reduced pressure to remove tetrahydrofuran, extracting aqueous solution with 10% methanol/dichloromethane (5 times), mixing organic phases, drying with anhydrous sodium sulfate, concentrating to dryness to obtain desired pale white solid product 5(1.21g,98.37％).1H NMR(500MHz,DMSO-d6)δ13.09(1H,br s),10.28(2H,s),7.41(2H,d,J＝1.9Hz),7.27(1H,t,J＝1.9Hz),6.76(2H,dd,J＝16.4,9.9Hz),6.14(2H,d,J＝16.4Hz),6.08(2H,d,J＝9.9Hz).
Compound 5 (960 mg,2.89mmol,1.0 eq.), HATU (1.65 g,4.34mmol,1.5 eq.), triethylamine (585mg,5.78mm ol,2.0 eq.) and propargylamine (160 mg,2.89mmol,1.0 eq.) are added to 15: 15m L dry N, N-dimethylformamide and stirred for 12h at 25 ℃. The reaction solution was extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine (3 times), dried over anhydrous sodium sulfate, and dried by spin-drying. Silica gel column chromatography gave product 6 as a yellow oil (i.e. ePDES1, 255mg, yield 23.88％).1H NMR(500MHz,DMSO-d6)δ10.24(2H,s),8.90(1H,t,J＝5.4Hz),7.23(2H,br d),7.18(1H,br t),6.77(2H,dd,J＝16.4,9.9Hz),6.14(2H,d,J＝16.4Hz),6.07(2H,d,J＝9.9Hz),3.98(2H,dd,J＝5.4,2.2Hz),3.12(1H,t,J＝2.2Hz).13C NMR(125MHz,DMSO-d6)δ165.8,138.8,136.4,136.0,128.4,114.0,113.1,81.4,73.1,28.8.MS(ESI)C14H16N3O5S2[M+H]+＝370.0526, found 370.0526.
The compounds provided in examples 1-3 were then tested.
Materials and methods
Protein expression ：pET-22b-Trx1-6×His,pET-22b-Tpx-flag-6×His,pET-22b-PAPR-flag-6×His,pRSF-Duet1-Trx1-6×His-tpx-flag,,pRSFDuet-1-Trx1-6×His-cysh-flag was transformed into BL21 (DE 3) chemocompetent cells, respectively. Transformants were inoculated on LB agar plates containing antibiotics. Single colonies were inoculated into 5mL of 2 XYT containing antibiotics and incubated at 37℃for 12h. 1mL of the cell culture broth was diluted into 100mL of LB containing the antibiotic and shake-cultured. At an OD 600 of about 0.6, protein expression was induced by adding 400. Mu. Mol of IPTG (Sangon) and then incubating for 4h (pET-22 b-Trx1-6 XHis, pET-22b-TPx-FLAG-6 XHis and pET-22b-PAPR-FLAG-6 XHis) and 2h (coexpression plasmid), respectively, at 37 ℃. The cells were collected, centrifuged at 4000 Xg for 30min at 4℃and stored at-80℃for purification or for cross-linking of PDES.
In vitro crosslinking reaction: in a 20. Mu.L reaction, 10. Mu.M Trx1 (in Hepes buffer, pH 8.0) and 10. Mu.M TPX or PAPR (in Hepes buffer, pH 8.0) were crosslinked with 0.5mM PDES at 25℃for 4h, corresponding to a protein/crosslinker molar ratio of 1:50. The crosslinking reaction was terminated by adding 100mM DTT (Sangon) at 25℃and incubating for 20 minutes.
Living cell crosslinking reaction: concentration tests were performed with different crosslinker concentrations (0, 0.25mM, 0.5mM, 1.0mM, 2.0 mM) in 30. Mu.L of E.coli or 293T cell resuspension. PDES was also removed by centrifugation and PBS washing. Cell samples from these 30 μl-grade systems were analyzed by western blot.
Purification of His-Tag protein: cells were suspended in 12mL lysis buffer (50 mM Tris-HCl pH8.0,500mM NaCl,20mM imidazole, 1% v/v Tween 20, protease inhibitor cocktail) and the cell lysates were incubated in an ice-water bath with an sonicator (FISHER SCIENCE,30% Output, 10min, 2s off and 1s on) were used for E.coli cell lysis sonication, followed by centrifugation (21000 Xg, 30min,4 ℃). The soluble fractions were collected and incubated with pre-equilibrated Ni-NTA agarose resin (200. Mu.L/100 mL culture, SMART-Lifesciences) for 1h at 4℃under constant mechanical rotation. The slurry was loaded onto a Poly-Prep column (Sangon Biotech), washed 3 times with 5mL of wash buffer 1 (50 mM Tris-HCl, pH 8.0,500mM NaCl and 20mM imidazole) and eluted 5 times with 200. Mu.L of elution buffer (50 mM Tris-HCl, pH 8.0,500mM NaCl and 250mM imidazole). The eluate was concentrated using an Amicon Ultra column (Millipore) and exchanged to protein storage buffer 1 (50mM Hepes,pH 8.0, and 250mM NaCl) and stored at-80 ℃ for future analysis.
First, the BVSB crosslinkability of Trx1 (thioredoxin) with its known protein binding protein, adenosine 5' -phosphosulfate reductase (PAPR) was studied in vitro. PAPR catalyzes the formation of sulfite from adenosine 5' -phosphate sulfate, the mechanism of which involves binding to Trx1 to restore the active enzyme. To covalently capture the PAPR/Trx1 complex, his-tagged Trx1 and PAPR were incubated with PDES for 4h at 25 ℃. As shown in fig. 1, the crosslinked bands corresponding to the covalent complexes of Trx1/PAPR heterodimers were detected on Western blots, with the crosslinked points being cysteine residues corresponding to the enzymatic active centers of Trx1 and PAPR, which are close to each other in crystal structure (fig. 2). When the active site Cys239 was mutated to Ser, no cross-linked band was detected (fig. 1), highlighting the specificity of PDES capture interacting protein active cysteines. Importantly, although Trx1 binds to reduced forms of PAPR with relatively low binding affinity (Kd-110 μm), PDES successfully captured this weak protein-protein interaction.
Next, the crosslinking activity of PDES was further tested in living e.coli cells by coexpression of His-tagged Trx1 and Flag-tagged PAPR (fig. 3). After viable cell cross-linking with different concentrations of PDES, the cross-linked bands of Trx1 and PAPR heterodimers were seen using both anti-His and anti-FLAG antibodies in western blot analysis (fig. 3). However, when Cys239 of PAPR is mutated to Ser, the crosslinked band of Trx1/PARP is substantially disappeared (fig. 4). These results indicate that PEDS is a cell permeable cross-linker that targets Cys residues well at the binding interface of Trx1 and its interacting protein.
In mammalian cells, thioredoxin (TXN) is involved not only in disulfide exchange reactions but also in reversible nitrosylation (SNO) of cysteine residues. Previous chemical proteomics studies found 3632 potential SNO target proteins in cell extracts after treatment with S-nitrosoglutathione (GSNO), indicating the prevalence of SNO modifications. While Cys32 and 35 residues in TXN1 are important for disulfide exchange reactions, cys73 is thought to catalyze the trans-nitration. Thus, we hypothesize that of the 3632 potential SNO target proteins, those that cross-link Cys73 of TXN1 are potential SNO substrates for TXN 1. To verify this hypothesis, we transfected HEK293T cells with a plasmid expressing His-tagged TXN1 and used PDES for living cell cross-linking. Western blot analysis showed a number of cross-linked bands after cross-linking with PDES (fig. 5), indicating successful capture of the TXN1 binding protein.
Human 293T cells were then grown in DMEM supplemented with 10% fbs and antibiotics and cultured in 5.0% co 2 incubator at 37 ℃ for testing ePDES, ePDES.
For transient transfection, 15. Mu.g of TXN1-6 Xits mammalian expression vector and 30. Mu.L of PEI 25K (BIOHIB) were mixed with 400. Mu.L of DMEM, respectively, and allowed to stand for 5 minutes, then PEI solution and plasmid solution were mixed and allowed to stand for 15 minutes, and then the mixture was added to a 10cm dish.
After 48 hours, the transfected 293T cells were scraped off and washed 3 times with PBS. Finally, the cells were resuspended in 800. Mu.L PBS (final volume about 1.2 mL) and 0.5mM ePDES1/2 3h (slow rotation at 4℃for 1 h to allow the cross-linker to enter the cells and resting at 25℃for 2h for cross-linking). The sample was then centrifuged (500 g,4 ℃ C., 1 min) to remove the supernatant, then washed 3 times with PBS to thoroughly remove excess crosslinker, and 50mM NH 4CO3 was added to quench the reaction.
The above reaction conditions were used for mass spectrometry. Before this, concentration tests were carried out with different crosslinker concentrations (0, 0.25mM, 0.5mM, 1.0mM, 2.0 mM) in 30. Mu.L of cell suspension. ePDES1/2 was also removed by centrifugation and PBS washing. Cell samples from these 30 μl systems were analyzed by western blot.
Purification of TXN1 cross-linked complexes from 293T cells: cell samples were lysed with a configuration buffer (50 mM Tris-HCl pH 8.0, 500mM NaCl,1% v/v Tween 20) supplemented with protease inhibitor (Roche). Ultrasonic instrument used in ice-water bath30% Output, 5min, 2 sec off, 1 sec on) cell lysates were sonicated and then centrifuged (21000 Xg, 30min, 4 ℃). Soluble fractions were collected and pre-equilibrated Ni-NTA agarose resin (200. Mu.L/100 mL culture, intelligent life sciences) was added and incubated at 4℃for 1 hour with constant mechanical rotation. The slurry was loaded onto a Poly-Prep chromatographic column (Sangon Biotech), washed 3 times with 5mL of wash buffer 1 (50 mM Tris-HCl pH 8.0, 500mM NaCl and 20mM imidazole), and eluted 5 times with 200. Mu.L of elution buffer (50 mM Tris-HCl, pH 8.0, 500mM NaCl and 250mM imidazole). The eluate was concentrated using an Amicon Ultra column (Millipore) and the buffer was exchanged into protein storage buffer 1 (50mM Hepes,pH 8.0 and 250mM NaCl) and stored at-80 ℃ for future analysis.
Click chemistry and enrichment: 200 μg of the sample was diluted to 0.5 μg/μl in buffer (50mM Hepes,pH 7.5 and 150mM NaCl), followed by the addition of 1mM biotin-PEG 3-azide, 10mM sodium ascorbate, 5mM TBTA and 10mM CuSO 4 for the CuAAC reaction. The samples were allowed to react at 25 ℃ for 2 hours, rotation and photoprotection. Samples were loaded into 10K filters and buffer replaced 5 times with 8M urea buffer after click chemistry. After 20min reduction with 5mM TCEP (Sigma) and 15 min alkylation with 10mM iodoacetamide (Sigma) in the dark, the buffer was replaced with 50mM NH 4CO3 and digested with trypsin (from Promega, protein: enzyme ratio 50:1) at 37℃for 16 hours, then enriched with streptavidin beads at 20℃for 2 hours in PBS buffer. Streptavidin beads were washed 1 time with 1M KCl, 1 time with PBS, 3 times with 10% acetonitrile, then eluted 3 times with 200. Mu.L buffer (50% acetonitrile, 5% FA), dried and desalted, and analyzed by mass spectrometry.
Mass spectrometry method: the digested peptides were loaded onto an analytical column (75X 15cm, 1.9. Mu. m C18,1 μm tip) of Easy-nLC 1200 system. Samples were analyzed at a flow rate of 300nL min-1 with a 60 minute gradient as follows: 2-8% B for 2 min, 8-27% B for 43 min, 27-35% B for 8 min, 35-100% B for 3 min, 100% B for 4 min. Q Exactive HF-X mass spectrometer was run in data dependent mode, one complete MS scan was performed at r=60 000 (m/z 200), then 20 HCD MS/MS scans were performed, r=15 000, nce=27, isolation width 1.6m/z. AGC targets scanned by MS1 and MS2 are 1×106 and 5×104, respectively, with longest injection times of MS1 and MS2 of 20 and 45MS, respectively; the removal of isotopes is disabled; the dynamic exclusion was set to 45 seconds.
Data analysis: the crosslinked peptides were identified using pLink software. pLink search parameters: parent ion mass tolerance 20ppm; fragment mass tolerance 20ppm; peptide length is at least 6 amino acids per chain, at most 60 amino acids; the minimum mass of each chain of peptide is 600 and the maximum mass of peptide is 6000Da, and the variable modification of cysteine is 57.02146; enzyme: trypsin; three cleavage sites deleted per chain. Protein sequences were downloaded from Uniprot.
The test results are shown in FIGS. 6-9. From the results of fig. 6 and 8, it can be seen that ePDES, ePDES both achieved cross-linking in vivo in living cells and successfully captured the TXN1 binding protein. In addition, as can be seen from fig. 7 and 9, ePDES, ePDES2 can effectively enrich the number of cross-linked peptides through click chemistry reaction and enrichment, and can further improve the resolution of protein mass spectrometry analysis, and identify low-expression proteins or transient interaction proteins which are not easy to capture.
The content fully proves that the small molecular chemical cross-linking agent provided by the invention can effectively capture direct interaction protein through chemical cross-linking, can effectively enrich cross-linked peptide fragments, has excellent technical effects, and provides an effective tool for researching the functional properties of the protein.
The applicant states that the present invention is illustrated by the above examples as well as the preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced in dependence on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (10)

1. The small molecule chemical crosslinking agent is characterized by having the following structure:
Wherein R is selected from any one of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 amido, and substituted or unsubstituted C6-C12 aryl;
the substituted substituent is selected from halogen, hydroxy, cyano, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.
2. The small molecule chemical crosslinker of claim 1, wherein R is selected from hydrogen, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 amide groups.
3. The small molecule chemical crosslinking agent of claim 1, wherein the small molecule chemical crosslinking agent is selected from any one of the following structures:
4. A small molecule chemical cross-linking agent as claimed in claim 3, wherein the small molecule chemical cross-linking agent is selected from any one of the following structures:
5. a method of preparing a small molecule chemical cross-linker according to any one of claims 1-4, characterized in that the method of preparation comprises the steps of:
condensing phenylenediamine compounds with 2-chloroethane sulfonyl chloride to obtain the micromolecular chemical cross-linking agent;
The reaction route is as follows:
wherein R has the same definition as any one of claims 1 to 4.
6. The process of claim 5, wherein the reaction is carried out in the presence of a base comprising any one or a combination of at least two of 4-dimethylaminopyridine, pyridine, triethylamine, diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, potassium carbonate or cesium carbonate.
7. The process of claim 5, wherein the temperature of the reaction is 20-30 ℃.
8. The method according to claim 5, wherein the reaction time is 10 to 18 hours.
9. Use of a small molecule chemical cross-linker according to any one of claims 1-4 in a method of non-disease diagnosis and treatment in studying enzyme and substrate interactions.
10. Use of the small molecule chemical cross-linker of any one of claims 1-4 in a method of non-disease diagnosis and treatment in protein analysis.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10325987A (en) * 1997-03-26 1998-12-08 Konica Corp Hardening agent, and silver halide photographic sensitive material and its image forming method by using the same
WO2001028537A2 (en) * 1999-10-15 2001-04-26 Arrow Therapeutics Limited Bissulfonamide derivatives as enzyme inhibitors
CN107721975A (en) * 2017-11-13 2018-02-23 上海应用技术大学 BRD4 micromolecular inhibitors, synthetic method and its application with antitumor activity
CN112694499A (en) * 2020-12-14 2021-04-23 上海科技大学 Crosslinking agent, preparation thereof and application thereof in mass spectrum crosslinking technology

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10325987A (en) * 1997-03-26 1998-12-08 Konica Corp Hardening agent, and silver halide photographic sensitive material and its image forming method by using the same
WO2001028537A2 (en) * 1999-10-15 2001-04-26 Arrow Therapeutics Limited Bissulfonamide derivatives as enzyme inhibitors
CN107721975A (en) * 2017-11-13 2018-02-23 上海应用技术大学 BRD4 micromolecular inhibitors, synthetic method and its application with antitumor activity
CN112694499A (en) * 2020-12-14 2021-04-23 上海科技大学 Crosslinking agent, preparation thereof and application thereof in mass spectrum crosslinking technology

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Solubility parameters of poly(ethylene sulfonamide)s by the use of group contribution technique;Ahmad Husain et al;《Farbe + Lack 》;第86卷(第9期);797-798 *

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