Synthesis of mass spectrum probe capable of enzyme digestion and application of mass spectrum probe in metabonomics

Abstract

The invention relates to synthesis of an enzyme-cleavable mass spectrum probe and application of the enzyme-cleavable mass spectrum probe in metabonomics analysis. The mass spectrum probe capable of being subjected to enzyme digestion is synthesized, and the probe comprises an active reaction group, a polypeptide chain with an enzyme digestion site and a silica gel carrier. The reaction group can be covalently bound with a target metabolite, the silica gel carrier enriches the target metabolite through centrifugation so as to eliminate the interference of a matrix, and the target metabolite is released from the silica gel carrier through a restriction enzyme cutting site on a polypeptide chain, so that the metabolite derivative with the TMT reaction group is obtained. The derivative can be subjected to TMT labeling, and multi-channel rapid relative quantification is realized. According to the method, multiple target metabolites can be derived and enriched at the same time, the chromatographic retention of the target polar metabolites can be improved, the target metabolites can be purified and enriched to enhance the sensitivity and selectivity of mass spectrometry, multi-channel simultaneous quantitative detection is achieved, and the efficiency and accuracy of metabonomics research are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of mass spectrometry probe preparation, and relates to the synthesis of an enzyme-cuttable chemically selective mass spectrometry probe and its application in metabolomics analysis. Background Art

[0002] Omics technologies represented by proteomics and metabolomics are powerful means to study disease pathogenesis, biomarkers and drug action mechanisms. The key purpose of omics technologies is to quickly discover differential substance groups in disease states or before and after drug intervention. The emergence of proteomics technologies based on isobaric isotope (iTRAQ or TMT) labeling of amino or carboxyl groups of peptides provides a pioneering method for the rapid discovery of differential substance groups. In this technology, isobaric isotope labeling reagents are composed of reporter groups, balance groups and reactive groups. Different isotopes are distributed in the balance group and the reporter group, but their total mass remains unchanged. Therefore, the molecular ion peaks of peptides labeled with different isobaric isotope reagents in MS (mass spectrometry) are the same, and during collision-induced dissociation (CID) or high-energy collision dissociation (HCD), the reporter group region is cut in the linker region to produce low-mass reporter ions with different mass numbers, which are used to distinguish which sample the target compound comes from, and to achieve relative quantification between different samples based on the relative abundance of different reporter ions. By labeling different samples with different isotopes of the same weight and then mixing them into one sample for LC-MS (liquid chromatography-mass spectrometry) analysis, multi-channel quantitative analysis can be achieved, that is, relative quantification of multiple samples at the same time, so that differential proteins or peptides in multiple samples can be quickly discovered, greatly promoting its application in biomedicine.

[0003] At present, metabolomics technology mainly discovers differential metabolites through targeted metabolomics and non-targeted metabolomics. The former requires complex methodological investigations for absolute quantitative analysis of metabolites, such as linearity, precision, repeatability, recovery rate and matrix effect; the latter analyzes the relative content of all detectable metabolites without bias, but requires complex data analysis and normalization processing. Different data analysis methods (such as different normalization methods) and different parameter settings will cause huge differences in results, which has a great impact on the reliability of the results. The above problems will not occur in the omics technology based on isobaric isotopes (iTRAQ or TMT) labeling. However, due to the large number of metabolites and large structural differences in metabolomics, it has always been a technical difficulty to achieve multi-channel quantitative analysis of metabolomics. Therefore, a multi-channel simultaneous quantitative detection method for metabolites of different structural types is proposed to improve the efficiency and accuracy of metabolomics research and promote the further development and improvement of metabolomics. Summary of the invention

[0004] The present invention proposes a chemically selective mass spectrometric probe that can be used to derivatize and enrich different target metabolites, and establishes a multi-channel relative quantitative analysis method for metabolites of different structural types through LC-MS / MS with the assistance of TMT labeling.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention synthesizes an enzyme-cleavable chemically selective mass spectrometry probe, which can simultaneously derive and enrich multiple target metabolites, and expose active reaction sites (TMT reaction groups) through enzyme cleavage reactions, so that the derivatives can be TMT-labeled, thereby achieving multi-channel rapid relative quantification.

[0007] The specific steps include:

[0008] First, compound 1 with an azide group that can simultaneously capture the target metabolite is synthesized, specifically: the compound containing the reactive group X and the carboxyl group is dissolved in acetonitrile, and hex-5-yn-1-amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and N-hydroxy-7-azabenzotriazole (HOAT) are added. After fully dissolved, the reaction is stirred at room temperature. The reaction process is monitored by thin layer chromatography. After the reaction is complete, ethyl acetate is added and extracted three times. The combined organic phase is washed with 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution and saturated brine in turn. The organic phase is dried by adding an appropriate amount of anhydrous sodium sulfate. Filter by suction to remove the agglomerated sodium sulfate, and the filtrate is concentrated under reduced pressure by a rotary evaporator to obtain a crude product, which is purified by column chromatography. The reaction formula is:

[0009]

[0010] Then, the amino silica gel was chemically modified by using succinic anhydride to modify the amino silica gel into carboxyl silica gel CSG, and a polypeptide silica gel PCSG with a specific enzyme cleavage site was bonded to the surface of the carboxyl silica gel. The polypeptide silica gel had an azide group. The previously synthesized compound 1 was connected through a click reaction to expose the active reaction site, and a mass spectrometry probe (ECCMSprobe) was synthesized to achieve the capture and release of metabolites.

[0011] Furthermore, the amino silica gel is chemically modified by using succinic anhydride to modify the amino silica gel into carboxyl silica gel, specifically:

[0012] Weigh 1g of succinic anhydride, fully dissolve it in 5mL N,N-dimethylformamide (DMF), add 500mg of amino silica gel, stir at 30℃ for 24h, and the speed of the magnetic stirrer is 800rpm to evenly suspend the silica gel in DMF. After the reaction is completed, centrifuge at 12000rpm for 10min, discard the supernatant, add 1mL DMF and ultrasonicate for 5min, centrifuge to remove the solvent, repeat three times to wash away the excess succinic anhydride, and dry to obtain carboxyl silica gel CSG.

[0013] Reaction equation:

[0014]

[0015] Furthermore, PCSG, a polypeptide silica gel with a specific enzyme cleavage site, is bonded to the surface of the carboxyl silica gel, specifically:

[0016] Weigh 50mg EDCI, 60mg N-hydroxysuccinimide (NHS) and dissolve in 1mL DMF, add 50mg carboxyl silica gel, place on a vertical flip instrument and react at room temperature for 12h, centrifuge at 12000rpm for 10min, and discard the supernatant. Add 1mL DMF to clean the silica gel carrier, centrifuge to remove excess EDCI and NHS, and repeat three times. Take 3mg of Peptide-N3 with the sequence of GLGSFGG{Lys-N3} synthesized by GenScript Biotech, dissolve it in PBS buffer solution; add silica gel activated by EDCI and NHS, and flip vertically to suspend the silica gel. Monitor the concentration of Peptide-N3 in the supernatant by liquid phase until Peptide-N3 no longer decreases and the reaction is over. Add 1mL phosphate buffer (PBS) and ultrasonically clean for five minutes, centrifuge and discard the supernatant, repeat three times; obtain polypeptide silica gel PCSG.

[0017] Reaction equation:

[0018]

[0019] Furthermore, a mass spectrometry probe ECCMS probe was synthesized by click reaction, specifically:

[0020] Prepare 100mM tris(3-hydroxypropyltriazolemethyl)amine THPTA solution, 20mM CuSO4·5H2O solution, and 300mM sodium ascorbate solution with water as solvent for standby use. Weigh 20mg PCSG, add 200μL DMF, vortex for 2min to fully suspend the silica gel, add THPTA solution, CuSO4·5H2O solution, sodium ascorbate solution in sequence, and finally add 1mg compound 1, make up the solution to 400μL, shake the reaction at room temperature, and detect the concentration of compound 1 in the supernatant by liquid phase until the peak area stops decreasing, and the reaction is over. Add 1mL DMF to wash, remove excess copper ions and ligands, and mass spectrometry probe Boc-PCSG with protective groups.

[0021] Reaction equation:

[0022]

[0023] Add 200 μL methanol to the Boc-PCSG synthesized in the previous step, vortex for 2 minutes to fully suspend the carrier in the liquid, add hydrochloric acid / dioxane solution with a volume ratio of 1:1 to methanol, shake at 50°C for 2 hours, centrifuge at 12000rpm to precipitate silica gel at the bottom of a 1.5mL centrifuge tube, and remove the supernatant. Add 1mL methanol, vortex for 2 minutes to fully suspend the carrier, then ultrasonicate for 5 minutes, centrifuge and discard the supernatant, remove excess hydrochloric acid, and repeat this step until the pH of the supernatant is 6.8-7.0. Volatilize the remaining methanol in a vacuum concentrator to obtain an enzyme-cuttable chemically selective mass spectrometry probe ECCMS probe.

[0024] The enzyme-cleavable chemically selective mass spectrometry probe prepared by the above method can derive and enrich a variety of metabolites, and through the TMT reaction group, the derivatives can be TMT-labeled to achieve multi-channel rapid relative quantification of metabolites of different structural types.

[0025] The specific steps for rapid multi-channel quantitative analysis of metabolites are as follows:

[0026] Step 1: extract the metabolites to be detected; the metabolites are usually extracted from cells, plasma, urine, and feces.

[0027] Step 2: reacting the metabolites to be detected with the mass spectrometry probe: reacting the extracted metabolites with the mass spectrometry probe for 2 hours;

[0028] Reaction conditions: 100 μL of metabolite standard mixed solution, 2 mM EDCI solution, 1% pyridine, and 1 mM mass spectrometry probe (calculated based on a hydrazine concentration of 0.03 μmol / mg) were added sequentially into a 1.5 mL centrifuge tube, vortexed and reacted at 40° C. for 2 h.

[0029] Step 3: After the reaction is completed, centrifuge (12000 rpm for 10 min) to precipitate the mass spectrometer probe, and discard the supernatant. Use H2O ultrasonic cleaning for 5 min, centrifuge and discard the supernatant, and repeat the cleaning three times.

[0030] Step 4: Take the mass spectrometry probe precipitate in step 3, add 20 μL of freshly prepared chymotrypsin solution with a concentration of 1 mg / mL, and digest at 30°C overnight.

[0031] Step 5: Add acetonitrile (ACN) to the solution treated in step 4 until ACN>95% to denature and precipitate the chymotrypsin, centrifuge to obtain the supernatant, and evaporate the solvent using a vacuum concentrator to obtain the derived metabolite.

[0032] Step 6: TMT labeling of the derived metabolites;

[0033] Step 8: LC-MS (liquid chromatography-mass spectrometry) detection and analysis.

[0034] Since the polarity of the metabolites after derivatization is relatively similar, optimization of chromatographic conditions including mobile phase, elution gradient, etc. is beneficial to obtain a longer retention time and better separation.

[0035] The liquid phase conditions used were as follows: mobile phase (A) 0.1% FA + 20 mM ammonium formate, (B) ACN, 0.0-8.0 min, 5%-18% B, 8.0-13.0 min, 18%-23% B, 13.0-14.0 min, 23%-30% B, 14.0-15.0 min, 30%-95% B, 15.0-18.0 min, 95% B, 18.0-18.1 min, 95%-5% B, 18.1-25 min, 5% B. The flow rate was 0.2 mL / min, the column temperature was 40°C, and the chromatographic column was ACQ BEH Shield RP18 (1.7μm, 2.1mm×100mm).

[0036] The present invention synthesizes a new mass spectrometry probe, which includes an active reactive group, a polypeptide chain with an enzyme cleavage site, and a silica gel carrier. The reactive group can covalently bind to the target metabolite, and the silica gel carrier can enrich the target metabolite by simple centrifugation to eliminate the interference of the matrix, and release it from the silica gel carrier through the enzyme cleavage site on the polypeptide chain to obtain a metabolite derivative with a TMT reactive group, and then TMT labeling can be used to achieve multi-channel rapid relative quantification. Not only can the chromatographic retention of the target polar metabolite be improved, but the target metabolite can also be purified and enriched to enhance the sensitivity and selectivity of mass spectrometry analysis, and multi-channel simultaneous quantitative detection can be achieved, which greatly improves the efficiency and accuracy of metabolomics research, and provides new ideas and solutions for the multi-channel simultaneous quantitative detection of various structural sub-metabolomes in future metabolomics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the preparation and application of a mass spectrometry probe based on enzyme cleavage chemistry selection in an example of the present invention;

[0038] FIG2 is a mass spectrum and a nuclear magnetic resonance spectrum of compound 1 in an example of the present invention (A) a high-resolution mass spectrum of compound 1; (B) a hydrogen nuclear magnetic resonance spectrum of compound 1; (C) a carbon nuclear magnetic resonance spectrum of compound 1;

[0039] Figure 3 The infrared spectra of five materials in the probe synthesis process in the example of the present invention;

[0040] Figure 4 The X-ray photoelectron spectroscopy (XPS) diagram of five materials in the probe synthesis process in the example of the present invention;

[0041] Figure 5 High-resolution XPS graphs of C1s, N1s and O1s of five materials during the probe synthesis process in the example of the present invention;

[0042] Figure 6 The structure of the TMT reagent in the example of the present invention is a functional region of the TMT reagent structure, including the sites of MS / MS fragments generated by high energy collision dissociation (HCD) and electron transfer dissociation (ETD); and TMT 10 -127N reagent and TMT 10 -Structure and isotopic position of 130N reagent (*);

[0043] Figure 7 is the relative content of each metabolite in the extract of human prostate cancer cells before and after intervention with elemene in the example of the present invention;

[0044] Figure 8 is the relative content of each metabolite in the plasma extract of prostate cancer patients in the examples of the present invention;

[0045] Fig. 9 is the absolute content of each metabolite in the human prostate cancer cell extract before and after elemene intervention in the example of the present invention;

[0046] Fig.10 LC-MS extracted ion chromatograms after different concentrations of metabolite standards were labeled with TMT reagent in the examples of the present invention (1: 3PG, 2: GAP, 3: DHAP, 4: ISOCIT, 5: CIT, 6: ACT, 7: MAL, 8: FUM, 9. α-KG, 10: PYR);

[0047] Fig.11 The figure is a comparison of the peak areas detected by LC-MS after the metabolite standards of different concentrations were labeled with TMT reagent in the examples of the present invention. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0049] In this embodiment, the multi-channel rapid quantitative analysis of metabolites extracted from cells is taken as an example, and the present invention is further described in detail in conjunction with the accompanying drawings:

[0050] like Figure 1 As shown in the figure, a multi-channel rapid quantitative analysis method for cell / plasma metabolites is first synthesized, and a chemically selective mass spectrometry probe that can be cleaved by enzymes is then synthesized; then the cell / plasma metabolite extract and the probe are reacted for 2 hours, and EDCI and pyridine are added as coupling agents and catalysts for the reaction. After the reaction is completed, the supernatant is discarded, and the excess catalyst and unreacted substrate are washed away with H2O. Chymotrypsin solution is added for enzymatic cleavage at 30°C overnight, excess acetonitrile ACN is added to precipitate the enzyme protein, centrifuged and evaporated, TMT labeling reagent is added and incubated at room temperature for 1 hour, 5% hydroxylamine solution is added to terminate the reaction, water is added to dilute until ACN <5% in the solution, and C18 SPE cartridges are used for desalting. Finally, the metabolites are relatively quantified by LC-MS.

[0051] The specific steps include:

[0052] Step 1: Synthesize a chemically selective mass spectrometry probe that can be cleaved by enzymes. The specific steps are as follows:

[0053] First, compound 1 was synthesized. In this example, compound 1 that simultaneously captured carbonyl, carboxyl and phosphate group metabolites was used as an example: 4-(2-N-tert-butyloxycarbonylhydrazine)benzoic acid (252.2 mg, 1 mmol, 1.0 equivalent) was dissolved in acetonitrile (10 mL), and hex-5-yn-1-amine (97.1 mg, 1 mmol, 1.0 equivalent), EDCI (287.5 mg, 1.5 mmol, 1.5 equivalent), and HOAT (204.2 mg, 1.5 mmol, 1.5 equivalent) were added. After fully dissolved, the mixture was stirred at room temperature for 24 hours. The reaction process was monitored by thin layer chromatography. After the reaction was complete, ethyl acetate (3×5 mL) was added for extraction three times, and the combined organic phase was washed with 0.1 M HCl solution (2×5 mL), 0.1 M NaOH solution (2×5 mL) and saturated brine (2×5 mL) in turn. The organic phase was dried by adding an appropriate amount of anhydrous sodium sulfate. The filtrate was filtered to remove the agglomerated sodium sulfate, and the filtrate was concentrated under reduced pressure using a rotary evaporator to obtain a crude product, which was purified by column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain 188.3 mg of a light yellow solid. Compound 1 was obtained with a yield of 56.89%. The reaction equation is:

[0054]

[0055] In this example, the following experiment was performed to verify the obtained compound 1:

[0056] Figure 2 (A) is the high-resolution mass spectrum of compound 1, the molecular formula is C 18 H 25 N3O3, molecular weight is 331.1869. Quasi-molecular ion peak [M+H] was detected in the positive ion mode of mass spectrometry + and [2M+H] + .

[0057] Figure 2(B) is the 1 H-NHR spectrum, HPLC detection purity was 95.78%, and the solvent was deuterated DMSO (DMSO-d6). 1 H NMR (500MHz, DMSO-d6) δ8.86(s,1H),8.10(t,J=5.7Hz,1H),7.99(s,1H),7.65(d,J=8.5Hz,2H),6.63(d,J=7.9Hz,2H),3.2 2(q,J=6.7Hz,2H),2.75(t,J=2.6Hz,1H),2.18(td,J=7.0,2.7Hz,2H),1.61–1.54(m,2H),1.51–1.44(m,2H),1.42(s,9H).

[0058] Figure 2(C) is the 13C-NHR spectrum, HPLC detection purity is 97.65%, the solvent is deuterated DMSO (DMSO-d6), and the multiple peaks of the solvent deuterated DMSO are at 39.6ppm. 13 C NMR (126MHz, DMSO-d6) δ166.43,156.37,152.46,128.81,124.60,110.87,84.92,79.47,71.71,38.83,28.95,28.57,25.97,17.90.

[0059] According to Figures 2(A) to (C), it can be concluded that compound 1 in this example was successfully synthesized.

[0060] Then, carboxyl silica gel (CSG) was synthesized by weighing 1g of succinic anhydride, fully dissolving it in 5mL DMF, adding 500mg of amino silica gel (ASG), stirring at 30°C for 24h, and the speed of the magnetic stirrer was 800rpm, so that the silica gel was evenly suspended in DMF. After the reaction was completed, centrifugation was performed at 12000rpm for 10min, the supernatant was discarded, 1mL of DMF was added for ultrasonication for 5min, and the solvent was removed by centrifugation. The excess succinic anhydride was washed off three times, and then dried to obtain carboxyl silica gel.

[0061] Reaction equation:

[0062]

[0063] Synthetic peptide silica gel (PCSG): Weigh 50mg EDCI, 60mg NHS dissolved in 1mL DMF, add 50mg CSG, place on a vertical flip instrument to react at room temperature for 12h, centrifuge at 12000rpm for 10 minutes, and discard the supernatant. Add 1mL DMF to wash the silica gel carrier, centrifuge to remove excess EDCI and NHS, and repeat three times. Take 3mg Peptide-N3 (synthesized by GenScript Biotech, sequence GLGSFGG{Lys-N3}) and dissolve it in PBS buffer solution, add silica gel activated by EDCI / NHS, and flip vertically to suspend the silica gel. LC monitors the concentration of Peptide-N3 in the supernatant until Peptide-N3 no longer decreases and the reaction is over. Add 1mL PBS buffer for ultrasonic cleaning for five minutes, centrifuge and discard the supernatant, repeat three times. The obtained peptide silica gel (PCSG) is placed in PBS buffer and stored at 4°C.

[0064] Reaction equation:

[0065]

[0066] Synthesis of mass spectrometry probe with protective group (Boc-PCSG) by click reaction:

[0067] Prepare 100mM tris(3-hydroxypropyltriazolemethyl)amine (THPTA) solution, 20mM CuSO4·5H2O solution, and 300mM sodium ascorbate solution with water as solvent for standby use. Weigh 20mg of silica gel synthesized in the previous step, add 200μL DMF, vortex for 2min to fully suspend the silica gel, add THPTA solution, CuSO4·5H2O solution, sodium ascorbate solution in sequence, and finally add 1mg of compound 1 synthesized in advance, make up the solution to 400μL, shake the reaction at room temperature, and detect the concentration of compound 1 in the supernatant by LC until the peak area stops decreasing, and the reaction is over. Add 1mL DMF to wash the silica gel carrier, remove excess copper ions and ligands, and obtain a mass spectrometry probe with tert-butyloxycarbonyl protection.

[0068] Reaction equation:

[0069]

[0070] The protective group of Boc-PCSG was removed with hydrochloric acid / dioxane solution to expose the active reaction site and synthesize the mass spectrometry probe (ECCMS probe):

[0071] Add 200 μL methanol to the Boc-PCSG synthesized in the previous step, vortex for 2 minutes to fully suspend the carrier in the liquid, add hydrochloric acid / dioxane solution with a volume ratio of 1:1 to methanol, shake at 50°C for 2 hours, centrifuge at 12000rpm to precipitate silica gel at the bottom of a 1.5mL centrifuge tube, and remove the supernatant. Add 1mL methanol, vortex for 2 minutes to fully suspend the carrier, then ultrasonicate for 5 minutes, centrifuge and discard the supernatant, remove excess hydrochloric acid, and repeat this step until the pH of the supernatant is 6.8-7.0. Volatilize the remaining methanol in a vacuum concentrator to obtain a chemically selective mass spectrometry probe that can be cleaved by enzymes.

[0072] Reaction equation:

[0073]

[0074] The following infrared spectroscopy and X-ray photoelectron spectroscopy were performed on the five materials (a) ASG, (b) CSG, (c) PCSG, (d) Boc-PCSG, and (e) ECCMS probe prepared in this example using silica gel as a carrier:

[0075] FT-IR (Fourier transform infrared spectroscopy) results are as follows Figure 3 As shown. At 804cm -1 Nearby is the Si-O-Si symmetric stretching vibration peak, 3434 cm -1 There is an amino absorption peak at 2949.0 cm -1 The stretching vibration peak of alkane CH is 1632.8 cm-1 The out-of-plane vibration peak of NH at 1727.1cm -1 A new peak is generated at 1561.1cm, which is the absorption peak of carboxyl C=O. -1 The vibration peak of amide is at 1641.1cm -1 Nearby is the C=O absorption of amide, indicating the successful synthesis of carboxyl silica gel. -1 The newly appeared absorption peak at 2100 cm-1 is the asymmetric stretching vibration peak of the azido group, indicating that the azido peptide has been successfully modified onto the surface of CSG. -1 The azide peak at 1556.9 cm -1 Nearby is the bending vibration peak of NH in the triazole ring, indicating that the click reaction is complete. Material (e) has a peak at 3500-3300 cm -1 The vibration absorption peak of the amino group appeared at , indicating that the tert-butyloxycarbonyl group was successfully removed and the ECCMS probe was successfully synthesized. The characteristic functional group information displayed by Fourier transform infrared spectroscopy was consistent with the synthetic route designed in this experiment, indicating that the mass spectrometry probe was successfully prepared. Afterwards, the elemental chemical state of the material surface was analyzed by XPS.

[0076] Figure 4 The XPS spectra of five materials with silica gel as the carrier during the reaction process. All five materials contain C, N, O, and Si elements. Table 1 shows the proportions of the three elements C, N, and O in each group of materials. Compared with ASG, the proportion of O elements on the surface of CSG increased from 23.18% to 60.77%, indicating that the amino silica gel was successfully connected to the carboxyl group. The proportion of N elements in PCSG increased from 2.39% to 3.16%, indicating that the azidated polypeptide was fixed on the silica gel surface, and the proportion of C elements in Boc-PCSG increased, indicating that compound 1 and the azidated polypeptide were successfully clicked. The proportion of O elements in the ECCMS probe decreased from 54.64% to 14.40%, indicating that the tert-butyloxycarbonyl group was successfully removed. In summary, the chemically selective mass spectrometry probe with silica gel as the carrier prepared by the method described in the present invention was successfully synthesized.

[0077] Table 1 Statistics of element content of materials

[0078]

[0079]

[0080] like Figure 5 As shown, high-resolution XPS spectra of C1s, N1s, and O1s are displayed to show the differences in binding energies.

[0081] The C1s binding energy of ASG is at 288.24, 285.66, and 284.10 eV, which are attributed to CN, CO, and CC, respectively. The C1s binding energy of CSG is at 289.03, 285.66, and 284.61 eV, which are attributed to C=O, CO, and CC, respectively, indicating that the surface silica gel is bound to carboxyl groups. The C1s binding energy of PCSG is at 289.78, 288.24, 286.46, and 284.61 eV, which are attributed to C=O, CN, CO, and CC, respectively, indicating that the azidated polypeptide is successfully bonded to the silica gel surface. The C 1s binding energy of Boc-PCSG is at 288.91, 287.53, 285.66, and 284.63 eV, which are attributed to C=O, CN, CO, and CC, respectively. The C1s binding energies of the ECCMS probe were at 286.20, 285.30, and 284.60 eV, which were attributed to CN, CO, and CC, respectively, indicating that the tert-butyloxycarbonyl group was successfully removed.

[0082] The N1s binding energies of ASG and CSG are at around 400.85 and 399.35 eV, which are attributed to CN and NH, respectively. The N1s binding energies of PCSG, Boc-PCSG, and ECCMS probe are at around 402.11, 401.06, and 400.04 eV, which are attributed to triazole ring N, CN, and NH, respectively. The O1s binding energies of ASG are at around 534.04, 533.06, and 532.38 eV, which are attributed to OH, Si-O, and CO, respectively.

[0083] The O1s binding energies of CSG, PCSG, Boc-PCSG and ECCMS probe are around 533.14, 532.45 and 531.39 eV, which are attributed to Si-O, CO and C=O.

[0084] In summary, the XPS characterization results are consistent with the Fourier transform infrared spectrometer (FT-IR) characterization results, which fully demonstrates that this experiment bonded the azidated peptide to the surface of the silica gel material and successfully completed the construction of a chemically selective mass spectrometry probe with silica gel as the carrier through a click reaction.

[0085] Step 2: Extraction of cell metabolites: In this example, IC 50 The specific process is as follows: After cell passage, the cells were incubated in a carbon dioxide incubator for 24 hours until the cells adhered to the wall. The cell samples were divided into two groups, a control group and an elemene intervention group, with n=3 samples in each group. The old culture medium was discarded, 10 mL of culture medium was added to the control group, and IC 5010mL culture medium with elemene concentration was cultured in an incubator for 24h. The cell culture dish was placed on ice, the culture medium was aspirated with a pipette, and the cells were washed 2-3 times with pre-cooled PBS buffer (3mL each time). 1mL 50% methanol solution (pre-cooled at -20℃) was added, and the cells were gently scraped off with a cell scraper and collected into a 1.5mL centrifuge tube. Ultrasonication was performed on ice for 1min (pause every 30s), and then 225μL of chloroform (pre-cooled at -20℃ in advance) was added to the centrifuge tube and vortexed for 10min. Then 75μL of cold water (pre-cooled at 4℃) was added to the mixture and vortexed again for 2min. The extraction mixture was placed in a -20℃ refrigerator and incubated for 20min. The metabolite extract was centrifuged at 10000rpm and 4℃ for 15min. The extract after centrifugation was divided into three layers, and the supernatant was transferred to a new 1.5mL centrifuge tube and dried by a vacuum concentrator for standby use.

[0086] Extraction of plasma metabolites: Before extracting metabolites, the plasma samples were frozen and thawed. Immediately after the plasma was thawed, 20 μL was mixed with 80 μL methanol, 40 μL chloroform, and 80 μL water in sequence. The mixture was vigorously vortexed for 1 min after adding each extraction reagent. Thereafter, the mixture was incubated at -20°C for 20 minutes. Then, the mixture was centrifuged at 13,000 rpm for 15 minutes at 4°C. Finally, the supernatant was transferred to a new 1.5 mL centrifuge tube and dried for later use.

[0087] Step 3: Cell metabolites and plasma metabolites were reacted with mass spectrometry probes under the action of coupling agent EDCI and catalyst pyridine at 40°C for 2h. Take the metabolites, add 100μL of 50% acetonitrile (ACN) aqueous solution, sonicate for 5min to fully dissolve the metabolites, centrifuge at 12000rpm for 10min, and take the supernatant. Add 1mM mass spectrometry probe (calculated based on the concentration of hydrazine groups connected to the carrier), EDCI (final concentration of 2mM), 1% pyridine, and oscillate at 40°C for 2h.

[0088] Step 4: After the reaction is completed, centrifuge at 12000 rpm for 10 min to precipitate the mass spectrometer probe, discard the supernatant, add 400 μL H2O and sonicate for 5 min, centrifuge again and discard the supernatant, and repeat three times to remove excess catalyst.

[0089] Step 5: Take the mass spectrometry probe precipitate in step 3, add chymotrypsin solution, and digest at 30°C overnight.

[0090] Weigh 1.0 mg of chymotrypsin and add 1 mL of H2O to fully dissolve it to prepare a chymotrypsin solution. Resuspend the cleaned mass spectrometer probe in 90 μL of H2O, add 10 μL of freshly prepared chymotrypsin solution, and shake in a mixer at 30°C overnight for enzyme digestion.

[0091] Step 6: Add acetonitrile (ACN) until ACN>95%, precipitate the protein, evaporate the supernatant, and obtain the derivatized metabolite.

[0092] After the reaction, 400 μL ACN was added to precipitate chymotrypsin, the supernatant was collected by centrifugation, the liquid was evaporated with a vacuum concentrator, 20 μL 50% ACN was added for re-dissolution, and the supernatant was collected by centrifugation at 12000 rpm for 15 min.

[0093] Step 7: TMT labeling of the derived metabolites.

[0094] The structure of TMT reagent is as follows Figure 6 As shown, it consists of an amine-reactive NHS ester group, a spacer arm, and a mass reporter group. 13 C. 15 N isotopes provide different reporter ions; as shown in Table 2, the TMT used in this example 10 -127N reagent and TMT 10 The -130N reagent generates reporter groups at m / z 127 and m / z 130, which can be used to distinguish different samples.

[0095] Table 2 TMT reagent types and reporter ions for each group of metabolite derivatives

[0096]

[0097] Add 50 μL of 200 mM HEPES buffer to the dried digested products and shake them to fully dissolve. Add 41 μL of ACN to each 0.8 mg of TMT labeling reagent to dissolve, let it stand for 5 minutes, then vortex, and add 20 μL of TMT labeling solvent to the digested products (41 μL per 100 μg of product). After shaking at room temperature for 1 hour, add 8 μL of 5% hydroxylamine solution. Shake the reaction at room temperature for 15 minutes and terminate the reaction. Dilute the sample with water until ACN <5% in the solution, and use a C18 SPE cartridge for desalting.

[0098] Step 8: LC-MS detection and analysis, quantitative analysis of target metabolite derivatives:

[0099] Using the Tuning Mode of the mass spectrometer for analysis, a full scan mass spectrum of the secondary ion can be obtained, a secondary ion with a higher response can be selected as the qualitative ion, and the DP value and CE value can be optimized. A new MRM analysis method is established and the mobile phase elution gradient is optimized. In this embodiment, the liquid phase conditions finally determined are as follows: mobile phase (A) 0.1% FA + 20mM ammonium formate, (B) ACN, 0.0-8.0min, 5%-20%B, 8.0-13.0min, 20%-35%B, 13.0-14.0min, 35%-40%B, 14.0-15.0min, 40%-95%B, 15.0-18.0min, 95%B, 18.0-18.1min, 95%-5%B, 18.1-25min, 5%B. The flow rate is 0.2mL / min, the column temperature is 40°C, and the chromatographic column is ACQUITY BEH Shield RP18 (1.7μm, 2.1mm×100mm).

[0100] The protein concentration of cells in the two groups (control group and elemene group) was calculated by BCA quantitative method. The metabolite concentrations of the two groups were normalized by protein concentration, and the changes in the content of each metabolite in the two groups were compared. The results are as follows Figure 7 As shown. It can be seen that the 10 quantitative metabolites all changed significantly. Compared with before administration, the concentration of glyceraldehyde 3-phosphate (GAP) in the glycolysis pathway increased, the concentration of 3-phosphoglycerate (3PG) downstream decreased, and the concentration of dihydroxyacetone phosphate (DHAP) that was mutually converted with GAP also decreased (P<0.01). The concentration of pyruvate (PYR) downstream of the glycolysis pathway increased significantly. In the tricarboxylic acid cycle, the metabolites citric acid (CIT), isocitrate (ISOCIT), α-ketoglutarate (α-KG) and malic acid (MAL) all increased significantly after administration, and only the concentration of fumaric acid (FUM) decreased.

[0101] Plasma samples from three prostate cancer patients (P1, P2, P3) and three healthy volunteers (H1, H2, H3) were selected. Metabolites in the plasma of prostate cancer patients and healthy individuals were quantitatively analyzed using the ECCMS probe combined with TMT labeling (MMCPT) method. Figure 8 As shown in the data, the concentrations of dihydroxyacetone phosphate (DHAP), fumaric acid (FUM), glyceraldehyde 3-phosphate (GAP), 3-phosphoglyceric acid (3PG), isocitrate (ISOCIT), and malic acid (MAL) in the plasma of prostate cancer patients were higher, with ratios greater than 1.0, while the concentrations of pyruvate (PYR), cis-aconitic acid (ACT), α-ketoglutaric acid (α-KG), and citric acid (CIT) were lower, with ratios less than 1.0.

[0102] Based on the above results, the combination of a novel ECCMS probe, TMT labeling, and LC-MS / MS detection allowed for robust relative quantification of glucose metabolites in various sample types, including standards, cell samples, and human plasma samples.

[0103] The following experiments were performed to quantitatively analyze and verify the target metabolite derivatives:

[0104] 1. Absolute quantitative method:

[0105] The mass spectrometry fragmentation rules of the metabolite standards after derivatization with mass spectrometry probes were studied. According to the optimized derivatization conditions, 13 metabolite standards were derivatized to obtain 13 single standards. TM 5500LC-MS / MS) was used to detect the sample. First, the Q1 scan was performed using the tuning mode of the mass spectrometer. After obtaining the parent ion, the DP parameters were optimized. The ProductIon scan was performed at the optimal DP value to obtain a secondary spectrum, and the fragments of the daughter ions were attributed to determine the compound structure, and the MRM daughter-mother ion pairs that met the qualitative and quantitative requirements were selected. The CE value of each ion pair was optimized by MRM to obtain the best mass spectrometry capture behavior. The results are shown in Table 3.

[0106] Table 3 LC-MS / MS MRM detection ion pairs of each metabolite derivative and optimized mass spectrometry parameters and retention time

[0107]

[0108]

[0109] Absolute quantitative analysis of metabolites in human prostate cancer cells after elemene intervention was performed. First, a series of metabolite standard mixtures with different concentrations (4.00, 2.00, 0.50, 0.10, and 0.02 μM) were derivatized and analyzed by LC-MS to develop calibration curves for each metabolite. The results are shown in Table 4. R 2 All of them were greater than 0.99, indicating that the calibration curves of these 13 metabolites had good linearity and the content of each metabolite in the sample could be calculated based on the calibration curves.

[0110] Table 4 Calibration curves of 13 metabolite derivatives

[0111]

[0112] The content of each metabolite was expressed in ng / μg protein after normalization based on protein concentration. 50The changes of cell metabolites in the drug concentration elemene intervention group were as follows Fig. 9 As shown. It can be found that after 24 hours of elemene treatment of cells, except for the metabolites D-fructose-6-phosphate disodium F6P, GAP, and 3PG, the glycolysis-related metabolites of the cells changed significantly. Compared with the control group without medication, the contents of D-glucose-6-phosphate disodium G6P, D-fructose-1,6-diphosphate trisodium salt FBP, and DHAP in the elemene group cells were significantly decreased (P<0.01), while the content of PYR located downstream of the glycolysis pathway was significantly increased (P<0.01). The intermediate metabolites produced by the TCA cycle play an important role in regulating cancer metabolic reprogramming. After elemene treatment, CIT, ISOCIT, α-KG, and MAL in prostate cancer cells were significantly increased (P<0.01), which was consistent with the relative quantitative results.

[0113] 2. Relative quantification of standards at different concentrations

[0114] Two groups of standard solutions with different concentrations (10 μM and 5 μM) were derivatized and TMT labeled. The retention time of each group of metabolites was determined after LC-MS analysis. The results are shown in Table 3. Metabolites G6P, F6P and FBP did not obtain good peak shapes and the response decreased after TMT labeling. This may be because these three metabolites have high polarity and cannot be retained on the C18 column when desalted by SPE column. The chromatograms of the remaining groups of metabolite derivatives are shown in Table 3. Fig.10 As shown, the peak area comparison is Fig.11 As shown, it can be seen that the peak area ratios of the Standard-10μM group and the Standard-5μM group are both close to 2:1, which is similar to the concentration ratio of the added standard mixture, indicating that the relative quantitative analysis results using TMT labeling reagent are reliable.

[0115] 3. Quantification of plasma samples by hydrophilic interaction chromatography (HILIC-MS / MS)

[0116] Metabolites were directly analyzed by HILIC-MS / MS, and their absolute concentrations were determined using calibration curves. The concentrations of metabolites in plasma of prostate cancer patients and healthy individuals were as follows: Figure 8 As shown in Figure 4, the HILIC-MS / MS method failed to detect four metabolites (3PG, CIT, ISOCIT and MAL), while the multi-channel metabolomics method developed in the present invention could detect these four metabolites with good selectivity and sensitivity.

[0117] The above description only points out the preferred implementation and operation scheme of the present invention; for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an enzyme-cleavable mass spectrometry probe, characterized in that: The specific steps include: First, a compound 1 having an azide group and capable of simultaneously capturing target metabolites is synthesized, specifically: a compound containing a reactive group X and a carboxyl group is dissolved in acetonitrile, and hex-5-yn-1-amine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI, and N-hydroxy-7-azabenzotriazole HOAT are added, and the mixture is stirred at room temperature after being fully dissolved; the reaction process is monitored by thin layer chromatography, and ethyl acetate is added for extraction three times after the reaction is complete, and the combined organic phase is washed with 0.1M hydrochloric acid solution, 0.1M sodium hydroxide solution, and saturated brine in sequence; an appropriate amount of anhydrous sodium sulfate is added to the organic phase for drying; suction filtration is performed to remove agglomerated sodium sulfate, and the filtrate is concentrated under reduced pressure by a rotary evaporator to obtain a crude product, which is purified by column chromatography; the reaction formula is: Then, the amino silica gel was chemically modified by using succinic anhydride to modify the amino silica gel into carboxyl silica gel, and a polypeptide with a specific enzyme cleavage site was bonded to the surface of the carboxyl silica gel. The polypeptide also had an azide group. The previously synthesized compound 1 was connected through a click reaction to expose the active reaction site, and a mass spectrometry probe ECCMS probe was synthesized to achieve the capture and release of metabolites.

2. The method for preparing the mass spectrometry probe cleavable by enzyme according to claim 1, characterized in that: The amino silica gel is chemically modified by using succinic anhydride to modify the amino silica gel into carboxyl silica gel, specifically: Weigh 1g of succinic anhydride and fully dissolve it in 5mL of N,N-dimethylformamide DMF, add 500mg of amino silica gel, stir at 30℃ for 24h, and the speed of the magnetic stirrer is 800rpm to make the silica gel evenly suspended in DMF; after the reaction is completed, centrifuge at 12000rpm for 10min, discard the supernatant, add 1mL of DMF and ultrasonicate for 5min, centrifuge to remove the solvent, repeat three times to wash away the excess succinic anhydride, and dry to obtain carboxyl silica gel CSG; Reaction equation:

3. The method for preparing the mass spectrometry probe cleavable by enzyme according to claim 1, characterized in that: The peptide with specific enzyme cleavage sites is bonded to the surface of carboxyl silica gel, specifically: Synthetic peptide silica gel PCSG: Weigh 50mg EDCI, 60mg N-hydroxysuccinimide NHS and dissolve them in 1mL DMF, add 50mg carboxyl silica gel, place on a vertical flip instrument to react at room temperature for 12h, centrifuge at 12000rpm for 10min, and discard the supernatant; add 1mL DMF to clean the silica gel carrier, centrifuge to remove excess EDCI and NHS, and repeat three times; take 3mg of Peptide-N3 with the sequence of GLGSFGG{Lys-N3} synthesized by GenScript Biotech, and dissolve it in PBS buffer solution; add silica gel activated by EDCI and NHS, and flip vertically to suspend the silica gel; monitor the concentration of Peptide-N3 in the supernatant by liquid phase until Peptide-N3 no longer decreases and the reaction is over; add 1mL phosphate buffer PBS and ultrasonically clean for five minutes, centrifuge and discard the supernatant, and repeat three times; obtain Reaction equation:

4. The method for preparing the mass spectrometry probe cleavable by enzyme according to claim 1, characterized in that: The mass spectrometry probe ECCMS probe was synthesized by click reaction, specifically: Use water as solvent to prepare 100mM tris(3-hydroxypropyltriazolemethyl)amine THPTA solution, 20mM CuSO4·5H2O solution, and 300mM sodium ascorbate solution for standby use; weigh 20mg PCSG, add 200μL DMF, vortex for 2min to fully suspend the silica gel, add THPTA solution, CuSO4·5H2O solution, sodium ascorbate solution in sequence, and finally add 1mg compound 1, make up the solution to 400μL, shake the reaction at room temperature, and detect the concentration of compound 1 in the supernatant by liquid chromatography until the peak area stops decreasing, and the reaction is completed; add 1mL DMF for washing to remove excess copper ions and ligands, and the mass spectrometry probe Boc-PCSG with a protective group; Reaction equation: Add 200 μL of methanol to the Boc-PCSG synthesized in the previous step, vortex for 2 minutes to fully suspend the carrier in the liquid, add a hydrochloric acid / dioxane solution with a volume ratio of 1:1 to methanol, oscillate at 50°C for 2 hours, centrifuge at 12000 rpm to precipitate silica gel at the bottom of a 1.5 mL centrifuge tube, and remove the supernatant; add 1 mL of methanol, vortex for 2 minutes to fully suspend the carrier, then ultrasonicate for 5 minutes, centrifuge and discard the supernatant, remove excess hydrochloric acid, and repeat this step until the pH of the supernatant is 6.8-7.0; evaporate the remaining methanol in a vacuum concentrator to obtain an enzyme-cleavable chemically selective mass spectrometry probe.

5. The mass spectrometry probe cleavable by enzymes prepared by the method of claim 1, characterized in that: It can derive and enrich a variety of metabolites, and through the TMT reaction group, the derivatives can be TMT-labeled to achieve multi-channel rapid relative quantification of metabolites of different structural types.

6. A method for rapid multi-channel quantitative analysis of metabolites using an enzyme-cleavable mass spectrometry probe prepared by the method of claim 1, characterized in that: The specific steps include: Step 1: extracting the metabolites to be detected; Step 2: react the extracted metabolites with the mass spectrometry probe for 2 h; Step 3: After the reaction is completed, centrifuge to precipitate the mass spectrometer probe and discard the supernatant; use H2O ultrasonic to clean the mass spectrometer probe for 5 minutes, centrifuge and discard the supernatant, and repeat the cleaning three times; Step 4: Take the mass spectrometry probe precipitate in step 3, add 20 μL of freshly prepared chymotrypsin solution with a concentration of 1 mg / mL, and digest at 30°C overnight; Step 5: Add acetonitrile (ACN) to the solution treated in step 4 until ACN>95% to denature and precipitate the chymotrypsin, centrifuge to obtain the supernatant, and evaporate the solvent using a vacuum concentrator to obtain the derived metabolite; Step 6: TMT labeling of the derived metabolites; Step seven: Liquid chromatography-mass spectrometry LC-MS detection and analysis.

7. The method for rapid multi-channel quantitative analysis of metabolites according to claim 6, characterized in that: The metabolites are extracted from cells, plasma, urine and feces.

8. The method for rapid multi-channel quantitative analysis of metabolites according to claim 6, characterized in that: Since the polarity of the metabolites after derivatization is relatively similar, the chromatographic conditions were optimized and the liquid phase conditions used were as follows: mobile phase (A) 0.1% FA + 20mM ammonium formate, (B) ACN, 0.0-8.0min, 5%-18%B, 8.0-13.0min, 18%-23%B, 13.0-14.0min, 23%-30%B, 14.0-15.0min, 30%-95%B, 15.0-18.0min, 95%B, 18.0-18.1min, 95%-5%B, 18.1-25min, 5%B; flow rate was 0.2mL / min, column temperature was 40°C, and the chromatographic column was ACQ BEH Shield RP18, 1.7μm, 2.1mm×100mm.

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