WO2015167216A1 - Matériau de marqueur de masse photoclivable et son utilisation - Google Patents

Matériau de marqueur de masse photoclivable et son utilisation Download PDF

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WO2015167216A1
WO2015167216A1 PCT/KR2015/004242 KR2015004242W WO2015167216A1 WO 2015167216 A1 WO2015167216 A1 WO 2015167216A1 KR 2015004242 W KR2015004242 W KR 2015004242W WO 2015167216 A1 WO2015167216 A1 WO 2015167216A1
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antigen
maldi
mmol
antibody
dichloromethane
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Korean (ko)
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문봉진
오한빈
강나나
전애란
박계신
박형순
방주용
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다이아텍코리아 주식회사
서강대학교 산학협력단
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Publication of WO2015167216A1 publication Critical patent/WO2015167216A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/02—Iron compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/26—Mass spectrometers or separator tubes

Definitions

  • the present invention relates to a photodegradable mass labeling substance and its use, and more particularly, to a photodegradable mass labeling substance capable of easily photolysing to release a cation labeled with a specific mass and a matrix-free condition using the substance.
  • So-called "soft ionization” mass spectroscopy methods including matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI), allow for complete ionization, detection and mass measurement of macromolecules whose mass exceeds 300 kDa [ See Fenn et al., Science 246: 64-71 (1989); Karas and Hillenkamp, Anal. Chem. 60: 2299-3001 (1988).
  • MALDI-MS requires incorporation into the matrix of the macromolecules to be analyzed and has been performed on polypeptides and nucleic acids mixed in a solid (ie crystalline) matrix.
  • a laser is used to exfoliate the biopolymer / matrix mixture that crystallizes on the probe tip, which affects the desorption and ionization of the biopolymer.
  • MALDI-MS uses organic acids or glycerol such as hydrated water (i.e. ice) benzoic acid as a matrix, and the complexity of the matrix impedes the analysis of trace polypeptides, and when using hydrated water as a matrix, MALDI-MS is used as a matrix.
  • the protein Before performing, the protein must first be lyophilized or air dried. See Berkenkamp et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7003-7007.
  • MALDI-TOF matrix-assisted laser desorption / ionization-time-of-flight
  • the current MALDI-TOF method is not enough to obtain information on the amount as well as the presence or absence of a biomarker. Therefore, it is absolutely necessary to develop a MALDI-TOF high-sensitivity quantitative method to overcome this problem, and furthermore, to develop a mass spectrometry method capable of amplifying signals such as quantitative analysis of trace biomolecules even if the matrix-assisted method is not used.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a photodegradable mass labeling material which can be measured in matrix-free conditions, unlike conventional MALDI-TOF MS.
  • the present invention to achieve the above object
  • R1 and R2 are H, alkyl, alkenyl, and Carney day, alkoxy, or aryl group
  • R3 is R 3 is a reactor that can react with the amine group of a protein or non-protein molecule
  • the reactor is N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, benzotriazol-1-yloxyl ester, pentahalobenzyl ester and 4- It is preferably selected from the group consisting of nitrophenyl esters, but is not limited thereto.
  • the material is preferably a compound of any one of the following formulas (2) to (4);
  • the yellow circle portion is the mass changer portion
  • the yellow circle portion is the mass changer portion
  • R1 and R2 are H, alkyl, alkenyl, alkaniyl, alkoxy, or an aryl group
  • R ' is N.
  • n is 0 Is an integer of 20, but is not limited thereto.
  • the material is preferably a compound of Formula 5, but is not limited thereto;
  • n is preferably 0 to 20, but is not limited thereto.
  • the present invention also provides a substance comprising an antibody or an amine group conjugated with the labeling substance of the present invention.
  • the present invention is to treat the antigen-labeled sample on a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody. And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
  • LBI-TOF MS laser desorption ionization time-of-flight mass spectrometry
  • the present invention is to treat a sample containing a different type of antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface to which the capture antibody can be bound to selectively select between the antigen and the capture antibody.
  • An antigen antibody complex is generated, and the complex is treated with a detection antibody labeled with the photodegradable mass label of the present invention, so that only when the antigen to be detected is present in the antigen, the capture antibody does not bind to another part of the antigen to which the capture antibody does not bind.
  • the plate is subjected to laser desorption ionization time-of flight mass spectrometry (LDI-TOF MS) without a matrix to provide a method for quantitative detection of multiple antigens.
  • LTI-TOF MS laser desorption ionization time-of flight mass spectrometry
  • the present invention is to treat the sample containing the antigen in a maldi (MALDI) plate treated with a capture antibody or a Maldi (MALDI) plate including a surface capable of binding the capture antibody to generate an antigen antibody complex between the antigen and the capture antibody And treating the detection antibody labeled with the photodegradable mass labeling substance of the present invention to the complex to bind to another portion of the antigen to which the capture antibody does not bind only when the detection antibody is present in the antigen to be detected. It provides a method for quantitative analysis of antigen by performing laser desorption ionization time-of-flight mass spectrometry (LDI-TOF MS) without matrix.
  • LBI-TOF MS laser desorption ionization time-of-flight mass spectrometry
  • An object of the present invention is photolysis capable of releasing a specific mass labeled cation by easily photolysing under a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF MS) under irradiation with a 355 nm laser.
  • MALDI-TOF MS matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer
  • a MALDI plate treated with a capture antibody (or a primary antibody) and a detection antibody (or a secondary antibody) labeled with a photodegradable mass labeling agent are prepared, respectively.
  • the biomarkers are sprayed onto the prepared MALDI plate, only the antigen to be detected is selectively bound to the capture antibody immobilized by the antigen-antibody reaction, and when the labeled antibody is processed again, the detection antibody is like a sandwich. Bind to the antigen.
  • the labeled detection antibody is immobilized on the MALDI plate only when there is a biomarker (antigen) to be detected in the sample.
  • the mass-labeling material is photolyzed to generate cations, thereby analyzing the presence of antigen.
  • the biggest advantage of this method is that there is no signal contamination due to the non-specific antigen-antibody binding which is always present in the sandwich type detection method using antigen-antibody binding. Even if unwanted biomaterials are captured, their mass values do not appear at all because the matrix is not used. In this case, the measured signal intensity is proportional to the amount of labeling material, so that quantitative analysis of captured biomarkers is possible, and even if the labeling materials having different structures (mass values) are labeled with different antibodies, they can be used at once. Can be used to detect multiple markers because it releases cations of different mass values independently without affecting.
  • the new photodegradable mass labeling substance should basically have a structure capable of producing stable cations by light.
  • a reactor capable of binding the antibody and a mass changer capable of varying the mass for detecting multiple markers should be provided at the same time.
  • they must be materials that can be synthesized in a relatively simple and high yield protocol.
  • Ferrocene a lead compound used in the present invention, has been studied for a long time that the ⁇ -carbon cation of this ligand has a very stable metallocene structure. Based on this fact, the inventors devised ferrocene derivatives as shown in FIG. 3.
  • This derivative can be decomposed into stable carbon cations and sulfur anions while the CS bond at the ligand ⁇ position is unevenly decomposed by a 355 nm laser.
  • the mass labeling material of the present invention can be used for the multiplexing of multiple markers (FIG. 7).
  • the present inventors synthesized ferrocene derivatives having three different mass change groups as shown in FIG. 8 and compared their matrix-less LDI-TOF MS results to find the most suitable structure for the purpose.
  • Suzuki cross-coupling reaction of an intermediate made through a boronation reaction between a 9-BBN (9-borabicyclo [3.3.1] nonane) and a hydrocarbon compound having a terminal double (C C) as shown in FIG. 9.
  • the three compounds have two carbon chains of mass changer. Irrespective of the structure difference of the mass changer, matrix-less LDI-TOF MS was measured after mixing 6a-c in the same number of moles to verify whether the matrix-less LDI-TOF MS detection intensity was the same.
  • Conjugation of a novel mass labeling substance to a detection antibody requires a reactor capable of pairing with an amine group of a protein in high yield.
  • the most widely used of the N - hydroxy mugwort god imide ester (N -hydroxysuccinimide ester, NHS ester) It is known that this reactor reacts very effectively with the amine groups of a protein.
  • N - hydroxy mugwort god imide N -hydroxysuccinimide, NHS
  • N, N'- dicyclohexyl carbodiimide N, N '-dicyclohexylcarbodiimide, DCC
  • ferrocene derivatives 6-carboxylic acid and thereby the reaction of the terminal NHS ester is introduced at the site in high yield.
  • novel photodegradable mass labeling materials 17a, 17b, and 17c as shown in FIG. 14 were synthesized, and the compound was actually conjugated to a detection antibody and applied to detection of a biomarker.
  • the photodegradable mass labeling substances were dissolved in tetrahydrofuran (1.0 ⁇ 10 ⁇ 6 ⁇ 1.0 ⁇ 10 ⁇ 15 ) at various concentrations, then 1 ⁇ l was taken at each spot on the plate and exposed to air at room temperature to dry the solvent.
  • the dried samples on the plates were analyzed with a MALDI-TOF mass spectrometer (matrix assisted laser desorption time-of-flight mass spectrometer, Autoflex Speed series, BrukerDaltonics, Leipzig, Germany). All spectra were measured in cationic reflecton mode using lasers of 335 nm wavelength of varying intensity in the range of 10% to 70%.
  • the mass range was set at 0-800 Da, after which data analysis was performed using the flexAnalysis program.
  • the photodegradable mass labeling material of the present invention can be used for detection and quantitative analysis of biomarkers, and can be used for simultaneous detection of multiple markers.
  • FIG. 1 is a schematic view of the principle of operation of the photodegradable mass labeling material to be developed in the present invention
  • FIG. 3 is a diagram showing a photolysis reaction of a ferrocene mass labeling material
  • FIG. 7 is a schematic diagram of a principle of simultaneous detection of multiple markers using photodegradable mass labeling substances
  • 11 to 13 are matrix-less LDI-TOF MS detection limit measurement spectra of ferrocene derivatives 6a-c (laser power: 70%): detection signal intensity comparison spectra according to the absolute moles of molecules placed on a plate;
  • 15 is a diagram showing the results of LDI experiments in a matrix free state of the BSA conjugated tags
  • 16 is a verification diagram of tag signal ratio change according to protein mixing ratio
  • Figure 17 is a tag signal detection in the antibody system conjugated with leptin conjugated with a tag.
  • 18 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
  • 19 is a diagram showing a method for synthesizing derivatives of photodegradable labeling substances of the present invention.
  • Ferrocene carboxyaldehyde (0.100 g, 0.467 mmol) was dissolved in ethanol (8 mL), and sodium borohydride (0.090 g, 2.4 mmol) was slowly added slowly at 0 ° C in small portions. The reaction mixture was stirred at room temperature for 3 hours. Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.087 g, 2.2 mmol) was slowly added in small portions at 0 ° C.
  • the reaction mixture was stirred at room temperature for 3 hours.
  • Water (3 mL) and dichloromethane (10 mL) were added sequentially to terminate the reaction.
  • the organic layer was separated and the remaining water layer was extracted three times with dichloromethane (15 mL x 3).
  • the combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.080 g, 0.28 mmol) was dissolved in ethanol (5 mL), and sodium borohydride (0.052 g, 1.4 mmol) was slowly added in small portions at 0 ° C.
  • the reaction mixture was stirred at room temperature for 3 hours.
  • the reaction was terminated by adding water (2 mL) and dichloromethane (8 mL) in this order.
  • the organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • the combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Acetyl ferrocene ( 1b , 0.100 g, 0.438 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 1.1 mL, 1.8 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at ⁇ 78 ° C. for 11 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL) and cooled to -78 ° C, and then methyllithium solution (1.67 M diethyl ether solution, 0.46 mL, 0.77 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at ⁇ 78 ° C. for 2.5 hours, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Benzoylferrocene ( 1c , 0.100 g, 0.345 mmol) was dissolved in anhydrous tetrahydrofuran (3.5 mL), cooled to -78 ° C, and the phenyllithium solution (1.35 M dinormalbutylether solution, 0.51 mL, 0.69 mmol) was added to a nitrogen atmosphere. Slowly put in. After stirring at -78 ° C for 20 minutes, water (2 mL) and dichloromethane (8 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenemethanol ( 2a , 0.020 g, 0.093 mmol) is dissolved in dichloromethane (0.4 mL), and thiophenol (0.031 g, 0.28 mmol) is added thereto. To this solution was added an aqueous solution of fluoroboric acid (48 wt%, 0.034 mL, 0.18 mmol). The reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (phenyl) methanol ( 2c , 0.030 g, 0.10 mmol) is dissolved in dichloromethane (1.0 mL), and then thiophenol (0.012 g, 0.11 mmol) is added thereto.
  • dichloromethane 1.0 mL
  • thiophenol 0.012 g, 0.11 mmol
  • the reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyldiphenylmethanol ( 2f, 0.042 g, 0.11 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.029 g, 0.26 mmol) is added.
  • thiophenol 0.029 g, 0.26 mmol
  • the reaction mixture is stirred at room temperature for 5 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (5 mL), and diluted with dichloromethane (10 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocene (0.500 g, 2.69 mmol) was dissolved in anhydrous dichloromethane (5 mL), and then paraanisoyl chloride ( p- anisoyl chloride, 0.504 g, 2.96 mmol) was added to the solution.
  • Aluminum chloride (0.394 g, 2.96 mmol) was slowly added in small portions at 0 ° C. The reaction mixture was stirred at room temperature for 11 hours, and then ice water (5 mL) and dichloromethane (10 mL) were added sequentially. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (20 mL x 3).
  • Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.152 g, 0.475 mmol) was dissolved in anhydrous dichloromethane (1.5 mL), cooled to -78 ° C, and boron tribromide (0.054 mL, 0.57 mmol) was added slowly under nitrogen atmosphere. . The temperature of the reaction mixture was slowly raised to room temperature and stirred for 8 hours. The reaction mixture was poured into iced water (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (4-hydroxylphenyl) methanone ( 5 , 0.094 g, 0.24 mmol) was dissolved in ethanol (1 mL), and sodium borohydride (0.027 g, 0.71 mmol) was slowly added slowly at 0 ° C. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3). The combined organic layers were washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate was added and filtered under reduced pressure.
  • Ferrocenyl (4-methoylphenyl) methanone ( 4 , 0.060 g, 0.19 mmol) was dissolved in a mixture of ethanol (0.5 mL) / tetrahydrofuran (0.5 mL), followed by a small amount of sodium borohydride (0.035 g, 0.94 mmol) at 0 ° C. Slowly put in. The reaction mixture was stirred at room temperature for 3 hours. Water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3).
  • Ferrocenyl (4-methoxyphenyl) methanol ( 7b , 0.059 g, 0.18 mmol) is dissolved in dichloromethane (0.8 mL) and thiophenol (0.022 g, 0.20 mmol) is added.
  • thiophenol 0.022 g, 0.20 mmol
  • the reaction mixture is stirred at room temperature for 3 minutes, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL). The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (7 mL x 3).
  • Ferrocenyl (4-iodophenyl) methanone 9 , 0.100 g, 0.240 mmol
  • tetrakis (triphenylphosphine) palladium 0.028 g, 0.024 mmol
  • copper iodide I
  • 0.009 g, 0.05 mmol It was dissolved in furan (1 mL).
  • Triethylamine (0.27 mL, 1.9 mmol) was added to this solution in a nitrogen atmosphere. After removing the oxygen inside by blowing argon gas into the solution for 20 minutes, 1-decane (1-decyne, 0.037 g, 0.26 mmol) was put in an argon atmosphere, and stirred at room temperature for 12 hours.
  • Ferrocenyl (4-hexylphenyl) methanone ( 14a , 0.040 g, 0.11 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.09 mL, 0.1 mmol) was slowly added in a nitrogen atmosphere. After stirring at ⁇ 78 ° C. for 1 minute, water (1 mL) and dichloromethane (3 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (5 mL x 3).
  • Ferrocenyl (4-octylphenyl) methanone ( 14b , 0.121 g, 0.301 mmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and cooled to -78 ° C, followed by phenyllithium solution (1.35 M dinormalbutylether solution, 0.22 mL, 0.30 mmol) was slowly added in a nitrogen atmosphere. After stirring at ⁇ 78 ° C. for 1 minute, water (2 mL) and dichloromethane (5 mL) were added sequentially to terminate the reaction. The organic layer was separated and the remaining water layer was extracted three times with dichloromethane (10 mL x 3).
  • Ferrocenyl (4-hexylphenyl) (phenyl) methanol 15a , 0.048 g, 0.11 mmol was dissolved in dichloromethane (1 mL), and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.012 g, 0.12 mmol) was added thereto.
  • an aqueous solution of fluoroboric acid 48 wt%, 0.025 mg, 0.14 mmol.
  • the reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL).
  • Ferrocenyl (4-octylphenyl) (phenyl) methanol 15b , 0.138 g, 0.287 mmol was dissolved in dichloromethane (1 mL) and 3-mercaptopropionic acid (3-mercaptopropionic acid, 0.037 g, 0.35 mmol) was added thereto.
  • an aqueous solution of fluoroboric acid 48 wt%, 0.074 mg, 0.40 mmol.
  • the reaction mixture is stirred at room temperature for 3 hours, then poured into saturated aqueous sodium hydrogen carbonate solution (3 mL), and diluted with dichloromethane (5 mL).
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.014 g, 0.14 mmol
  • anhydrous dichloromethane 0.3 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C.
  • the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure.
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol
  • anhydrous dichloromethane 0.4 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C.
  • the solid that had settled by precipitation was washed with cold dichloromethane under reduced pressure.
  • N, N'-dicyclohexylcarbodiimide N, N'-dicyclohexylcarbodiimide 0.028 g, 0.14 mmol
  • anhydrous dichloromethane 0.4 mL
  • the temperature of the final reaction mixture was slowly raised to room temperature and stirred for 8 hours.
  • the reaction mixture was cooled to 0 ° C., and the solids which settled by precipitation were washed with cold dichloromethane under reduced pressure.
  • NHS-Tag was prepared by dissolving 50 times the amount of BSA mole number in 100% DMSO (50nmoles).
  • Conjugation reaction was carried out in 50mM MES, pH 6, 10% Acetonitrile, the final volume of the reaction was 80uL, 1hr reaction at room temperature. After conjugation, Bio-Spin (Bio Rad) P-30 was buffered with water, and the remaining free NHS-Tag was removed. Dry in vacuo and concentrate to 50 uL. (MA) LDI-MS without matrix was confirmed that the tagging on the BSA.
  • Table 1 Tag1 Tag2 Tag3 Tag4 Remarks BSA 1 nmole 7 uL 7 uL 7 uL 7 uL 10ug / uL stock in MES MES 62.2 uL 61.8 uL 61.6 uL 61.5uL 50 mM, pH 6 ACN 8uL 8uL 8uL 8uL 10% ACN Tag 50nmoles 2.8uL 3.2 uL 3.4 uL 3.5 uL 10ug / uL stock in DMSO Total Vol. 80 uL 80 uL 80 uL 80 uL 80 uL 80 uL
  • the AB Scix 4800 instrument and ASTA micro focus 384 well plate were used to determine if the tag was conjugated.
  • Tag4 1 1 10: 1 1:10 Tag2 Tag4 Tag2 Tag4 Tag2 Tag4 m / z 435.29 491.37 435.27 491.35 435.25 491.32 Height 2149.64 683.92 2508.49 252.18 411.24 2454.64 Area 112.5 39.16 147.33 13.14 22.88 130.52 Ratio 2.87 11.21 5.7
  • Antibody-antigen system condition experiment to detect antigens with conjugated tag

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Abstract

La présente invention concerne un matériau de marqueur de masse photoclivable et une utilisation de celui-ci. Le matériau de marqueur de masse photoclivable de la présente invention peut être utilisé pour la détection et l'analyse quantitative d'un biomarqueur, et peut être utilisé pour la détection simultanée de marqueurs multiples.
PCT/KR2015/004242 2014-04-28 2015-04-28 Matériau de marqueur de masse photoclivable et son utilisation WO2015167216A1 (fr)

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KR1020140050964A KR101583811B1 (ko) 2014-04-28 2014-04-28 광분해성 질량 표지 물질 및 그 용도
KR10-2014-0050964 2014-04-28

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