CN-121991128-A - Photoelectrochemistry-based phosphoric acid enrichment cross-linking agent and preparation method and application thereof

Abstract

The invention discloses a photoelectrochemistry-based phosphoric acid enrichment cross-linking agent, a preparation method and application thereof, and belongs to the technical field of protein structure analysis, wherein the cross-linking agent is 1, 3-bis (2- (1-methyl-3, 5-dioxo-1, 2, 4-triazolidine-4-yl) ethoxy) propane-2-yl dihydrogen phosphate, contains two identical reaction groups, namely a methyl urea azole group and a phosphoric acid group, as an affinity enrichment tag, and the preparation method comprises the synthesis of a methyl urea azole precursor, the synthesis of an OMs precursor, the synthesis of a compound 2 and the like, and tyrosine and histidine residues can be selectively targeted by using the cross-linking agent through controllable electrochemical click reaction and photocatalytic reaction. The preparation method has the advantages of simple synthesis steps, readily available raw materials, low price and environmental protection, and the crosslinking reaction can be completed under the condition of approaching physiology (pH 7.4), the prepared stable and water-soluble connecting agent has an innovative modularized skeleton, acts on various amino acids (such as tyrosine and histidine), and results show that the connection based on pBMT provides accurate and rich information, thereby being beneficial to protein structure analysis.

Inventors

• WEI ZHONGLIN
• ZHANG YANXIN
• CAO JUNGANG
• LIAO WEIWEI
• DUAN HAIFENG
• LIN YINGJIE

Assignees

• 吉林大学

Dates

Publication Date

20260508

Application Date

20260202

Claims (3)

1. 1. A photoelectrochemistry-based phosphoric acid enrichment cross-linking agent is 1, 3-bis (2- (1-methyl-3, 5-dioxo-1, 2, 4-triazolidine-4-yl) ethoxy) propane-2-yl dihydrogen phosphate, which is marked as pBMT, and is characterized by containing two identical reaction groups, namely methyl urea azole groups and one phosphoric acid group as an affinity enrichment label, wherein the reagent can realize targeted cross-linking of tyrosine residues under electrochemical conditions and histidine residues under photochemical conditions, and has the structural formula: 。
2. 2. a method of preparing the photoelectrochemical based phosphoric acid enriched cross-linking agent of claim 1, having the steps of: step one, synthesizing a methyl urea azole precursor: Adding methyl hydrazine sulfate into anhydrous dichloromethane to prepare methyl hydrazine sulfate solution, then adding triethylamine dropwise into the methyl hydrazine sulfate solution, stirring at room temperature for 20 minutes to neutralize sulfuric acid in the solution, then adding ethyl chloroformate dropwise into the solution under the condition of-60 oC, fully stirring, finally stirring the suspension at room temperature for 5 hours, washing twice with water and 5% NaHCO 3 aqueous solution respectively to remove salt and other water-soluble impurities in the system, collecting an organic phase, adding anhydrous Na 2 SO 4 for drying, and evaporating the solvent under reduced pressure to obtain a yellow oily product; Melting diphenyl carbonate in a pressure-resistant pipe at 80 ℃, then adding the yellow oily product of the previous step, and purifying the product by column chromatography for 3 hours to obtain colorless oily methyl urea azole precursor, wherein the diphenyl carbonate is a compound of 2=1:2 according to the molar ratio; step two, synthesizing OMs precursor: The flask was placed under an atmosphere of N 2 by three vacuums and a cycle of N 2 , and charged with alcohol dissolved in anhydrous dichloromethane. The solution was cooled to 0 o C, dried Et 3 N was added, then methanesulfonyl chloride was added, the reaction mixture was stirred at 0 ℃ for 3 hours, quenched by the addition of saturated aqueous NH 4 Cl to room temperature, the aqueous phase was extracted with Et 2 O, the combined organic extracts were washed with aqueous 1M, dried over MgSO 4 and concentrated to give the product Oms precursor as a colourless oil, molar ratio, alcohol: et 3 N: methanesulfonyl chloride = 1:2.1:1.4; Step three, synthesizing a compound 2: Adding NaH into a flask under N 2 , adding anhydrous DMF solution of a compound 1 at room temperature, stirring for 1 hour, adding anhydrous DMF solution of methanesulfonate, stirring for 17 hours, quenching the reaction by slowly adding saturated aqueous solution, washing with NH 4 Cl aqueous solution, extracting the aqueous phase with Et 2 O to obtain a compound 2; step four, synthesizing a compound 3: Adding the compound 2 into a dry dichloromethane solution, adding trifluoroacetic acid, stirring for 3 hours at room temperature, evaporating, mixing with dichloromethane, evaporating to obtain free acid, acidifying the water phase with 1N hydrochloric acid to pH=1, extracting with ethyl acetate for 5 times, drying the organic layer with MgSO 4 , and concentrating in vacuum to obtain a crude product of the compound 3, wherein the crude product can enter the next step without further purification; step five, synthesizing a compound 4: Compound 3 was added to anhydrous CH 3 OH, then triethylamine was added dropwise to the above solution at room temperature and mixed for 10 minutes, then this mixed solution was reacted with a methyl urea precursor in a pressure-resistant tube at 80 ℃ for 3 hours, and the product was purified by column chromatography to give compound 4 as a white solid, in a molar ratio of compound 3:et 3 N: methyl urea precursor=1:3:2; step six, synthesizing a compound 5: Adding an absolute ethanol solution of potassium hydroxide into a round-bottom flask, then adding a compound 4, carrying out reflux reaction for 12 hours at 78 ℃, and after the temperature of the reaction solution is reduced to room temperature, acidifying to pH 2.0 by using a hydrochloric acid solution, evaporating the solvent under reduced pressure, redissolving by using methanol, filtering out the precipitate and concentrating the solution in vacuum to obtain a white solid compound 5; step seven, synthesizing a compound 6: Pd/C and tetrahydrofuran are added into a flask filled with a compound 5 as a solvent, the suspension is placed under an atmosphere of H 2 and stirred for 24 hours at room temperature, the suspension is filtered by diatomite, the filtrate is concentrated, and the crude product is purified by flash chromatography to obtain a colorless oily product compound 6, wherein the molar ratio of the compound 5 to Pd/C=1 to 1; step eight, synthesis of a compound 7: Compound 6, phosphoric acid and tributylamine are added into DMF in turn, heating is carried out for 6 hours under azeotropic reflux condition, after the reaction mixture is cooled to ambient temperature, 1N MeONa MeOH solution is added, the reaction mixture is stirred for 30 minutes, after solvent evaporation, the residue is washed with methanol, after methanol evaporation, the solid residue is dissolved in water, finally freeze drying treatment is carried out, thus obtaining white product compound 7, pBMT, compound 6: phosphoric acid: tributylamine=1:1:1 according to molar ratio.
3. 3. Use of a photoelectrochemical based phosphoric acid enriched cross-linker as claimed in claim 1, wherein tyrosine and histidine residues are selectively targeted with said cross-linker by controlled electrochemical click reactions and photo-catalytic reactions, the specific steps being: (1) Electrochemical crosslinking reaction, namely, performing electric click chemical crosslinking in a three-electrode system, namely, a saturated calomel reference electrode (SCE), a graphite working electrode and a platinum wire auxiliary electrode, dissolving 10 mg pBMT in PB buffer solution of 1mL, adding 0.1 mM angiotensin II and 1mM pBMT into 3mL PB buffer solution according to the molar ratio of 1:10, applying voltage of 0.60V to the mixed system by using the three-electrode system, fully stirring and reacting for 4 hours at room temperature, and adding 0.01 mM r-hGH protein, 0.01 mM GST protein and 1mM pBMT into 3mL PB buffer solution according to the molar ratio of 1:100, respectively, so as to perform electric click chemical crosslinking; (2) Preparing 10mM MES and acetonitrile solution as buffer solution for photocatalysis crosslinking reaction, adding 0.1 mM angiotensin II and 1mM pBMT into 3 mL MES buffer solution in a molar ratio of 1:10, adding 1mM serving as photocatalyst Rose bengal into the mixed solution, irradiating the mixed system with white light at room temperature, fully stirring for 11 hours, respectively adding 0.01 mM r-hGH protein, 0.01 mM GST protein and 1mM pBMT into 3 mL MES buffer solution in a molar ratio of 1:100, and adding 1mM Rose bengal into the mixed solution for photocatalysis crosslinking reaction; (3) Enzyme digestion reaction, namely, separately carrying out electrochemical crosslinking, photochemical crosslinking and continuously carrying out two crosslinking on r-hGH protein and GST protein, respectively dissolving the r-hGH protein and the GST protein in a buffer solution of Tris-HCl, wherein the final concentration of the protein reaches 0.5 mg.mL -1 , incubating for 4: 4h at 37 ℃ for enzymolysis, and finally adding 1% formic acid aqueous solution for quenching reaction; (4) Enrichment of crosslinked peptides the digested peptides were then subjected to the Zip Tip method by centrifuging the trypsin digested solution, removing Tris-HCl buffer, dissolving in 10% TFA, then fixing the phosphopeptides to a 10. Mu.L Zip Tip pipette Tip equilibrated with TFA/MeOH eluent in advance, performing 10-15 aspirate-drain cycles on the whole sample, then draining into the waste solution, repeating five times to ensure adequate removal of salts and detergents, finally aspirating 5. Mu.L NH 4 OH/MeOH eluent into the Zip Tip, eluting two to three times, and then performing LC-MS mass spectrometry on the sample; (5) The mass spectrum test comprises the steps of analyzing a crosslinked product under electrochemical and photochemical conditions by using a liquid chromatograph and a mass spectrometer, carrying out liquid phase separation by using a 300SB-C18 reverse phase column before mass spectrum analysis, wherein the MS 2 spectrogram is generated by collision induced dissociation, the energy is 15 eV, analyzing the crosslinked protease cut product by using the liquid chromatograph and the mass spectrometer, carrying out crosslinked product separation by using a C18 reverse phase chromatographic column before mass spectrum analysis, operating the mass spectrometer by using a data dependency acquisition mode, wherein the MS scanning range is m/z 200-2000, the temperature of the liquid chromatographic column is kept at 60 ℃, the resolution of MS is 120000, the AGC target is set to be standard, the maximum is 50 MS, the resolution of MS 2 is 120000, the AGC target is set to be standard, the maximum IT is 118-MS, the isolation window is 1.2 m/s, the dynamic exclusion is set to be 7-s, and when the electrospray ionization source is operated, the intrathecal gas flow rate is 40L.min -1 , the auxiliary gas flow rate is 10L. -1 , the spray voltage is 348, and the auxiliary gas heating temperature is 325 ℃ of the capillary tube is kept at 30 ℃ when the temperature is kept at 350 ℃; (6) Mass spectrum data analysis, namely judging a crosslinking site through the mass difference of the peptide fragments after crosslinking enzyme digestion compared with the unmodified peptide fragments; (7) And (3) determining three-dimensional structure information of the protein, namely analyzing and sorting the obtained mass spectrum data, and respectively calculating the interval length of the cross-linking agent and the C alpha-C alpha Euclidean distance of tyrosine and histidine in the protein by using Gaussian View 6 software and PyMOL 2.3 software to obtain the three-dimensional structure information of the protein.

Description

Photoelectrochemistry-based phosphoric acid enrichment cross-linking agent and preparation method and application thereof Technical Field The invention belongs to the technical field of protein structure analysis. In particular to synthesis of a phosphoric acid-enriched selective cross-linking agent targeting tyrosine and histidine, and identification and research of polypeptide protein structure based on mass spectrum. Background Chemical cross-linked mass spectrometry (XL-MS) is a powerful and versatile technique that has proven to be very effective in mapping protein-protein interactions (PPI) both in vitro and in vivo (anal. Bioanal. Chem. 2010, 397, 3433-3440). XL-MS is unique in that it is able to capture interactions directly from the natural environment and deduce endogenous PPI networks in high throughput without cell engineering (Anal. Chem. 2018, 90,144) compared to other methods. Identification of cross-linked peptides allows simultaneous determination of PPI and its interaction site, revealing structural details of the protein that directly interact with the protein and provide residue-level resolution. In addition, crosslinking also provides distance constraints to facilitate overall structural analysis of large protein complexes by optimizing existing structures and/or assisting in de novo structural modeling (nat. Struct. Mol. Biol. 2018, 25, 1000-1008). It uses chemical agents to study the structure of proteins and complexes formed thereby. The reagents used are typically small bifunctional chemicals that can covalently link amino acids in close proximity to each other. Most commonly, efficient NHS chemistry is used to capture the side chains of lysines in proteins. The reactive groups are linked by a cross-linking arm, so that the cross-linking agent acts as a distance constraint between the captured amino acids. After crosslinking while the protein remains in its native state, the protein sample is typically alkylated, reduced, and eventually digested into peptide fragments by a protease (typically trypsin). Subsequently, all linear peptides and cross-linked peptide mixtures were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) identification. After detection, the cross-linked peptides provide important distance information about the tertiary or quaternary structure of the protein in the form of an inner chain (two peptides from the same protein) or an outer chain (two peptides from different proteins). By these measurements, the target cross-linked peptide is completely overwhelmed in both quantity and abundance by the non-cross-linked linear peptide. With the research of XL-MS, significant progress has been made in identifying and improving the accuracy of low abundance cross-linked peptides. Researchers have focused on developing cross-linking agents, protein digestion and cross-linked peptide enrichment strategies, and data analysis tools, where selective enrichment of cross-linked peptides is particularly critical. Early strategies relied on gel filtration chromatography (SEC) (Science 2012, 337, 1348-1352), strong cation exchange chromatography (SCX) (anal. Chim. Acta 2021, 1179, 338838), and high pH reversed phase fractionation (anal. Chem 2022, 94, 7551-7558) to reduce sample complexity. Enrichment crosslinking agents such as BDP-NHP(J. Proteome Res. 2013, 12, 1569–1579),BDRG(Mol. Cell. Proteomics 2012, 11, M111.008318),Leiker bAL1/2(Elife 2016, 5, e12509), and CBDPS (mol. Cell. Proteomics 2011, 10, M110.001420), direct introduction of biotin, one-step enrichment by the biotin-streptavidin system (BAS). Furthermore, the adverse effects of biotin on crosslinking were alleviated by the "alkynyl-azido click chemistry" method (e.g., leiker, cliXlink (ChemBioChem 2020, 21, 103-107), and Alkyne-A-DSBSO (Proc. Natl. Acad. Sci. USA 2021, 118, e 2023360118)). Although biotin is widely used as an affinity tag for enrichment of cross-linked peptides, the larger size of biotin tags can hinder cross-linking efficiency due to steric effects, thus resulting in fewer cross-linked peptide pairs being identifiable. In addition, the development of phosphoproteomics has driven the development of phosphate-based cross-linking agents (e.g., ,PhoX(ACSCent. Sci. 2019, 5, 1514–1522), tBu-PhoX（TBDSPP）(Angew. Chem. Int. Ed. 2022, 61, e202113937)）, but enrichment of phosphorylated peptide fragments also faces a number of technical challenges, such as low abundance of phosphorylated proteins and large dynamic range, relatively low ionization efficiency of phosphorylated peptide fragments, difficulty in determining the specific position of the phosphate modification group, etc. in current phosphoproteomics research, enrichment of phosphorylated peptide fragments in samples prior to mass spectrometry is often required to eliminate the identification inhibition of phosphorylated peptide fragments by non-modified peptide fragments. Tyrosine residues are often present in the protein-protein i