CN117457312A - Magnetic molecular probe for specifically capturing metabolites from complex sample, and preparation method and application thereof - Google Patents
Magnetic molecular probe for specifically capturing metabolites from complex sample, and preparation method and application thereof Download PDFInfo
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Classifications
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- 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
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/42—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of organic or organo-metallic materials, e.g. graphene
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
- H01F41/02—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for manufacturing cores, coils, or magnets
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Manufacturing & Machinery (AREA)
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Abstract
The invention discloses a magnetic molecular probe for specifically capturing metabolites from complex samples, which combines a magnetic separation technology with a chemical derivatization technology of amino, carboxyl, sulfhydryl, hydroxyl, phosphoric acid and aldehyde ketone compounds, and is covalently bound on a magnetic solid phase material to be used as the molecular probe for specifically capturing the compounds in the complex samples. The probe can selectively break disulfide bonds, dissociate captured compounds from magnetic materials into solutions, and can be used for subsequent experimental analysis after magnetic separation and centrifugation. The probe has the label for improving the ionization efficiency of the mass spectrum, and can obviously improve the detection sensitivity. The probe has an isotope labeling label, and can realize identification and full quantitative analysis of the metabolites in complex samples under the condition of lacking a standard substance. The magnetic molecular probe provides a new technical method for realizing high-flux, multi-sub-metabolism group, high-sensitivity and full-quantitative metabolite analysis.
Description
Technical Field
The invention relates to the technical field of metabolite analysis, in particular to a magnetic molecular probe for specifically capturing metabolites from complex samples, and a preparation method and application thereof.
Background
Small molecules of the metabolite are the material basis upon which organisms survive, and cellular metabolism is increasingly thought to be a key driver of a range of common diseases. Metabolites play an important role in the biosynthesis of cells, cell growth, signal transduction and immunomodulation processes. Metabolomics is a recent discipline developed following genomics and proteomics, and the study of the metabolome at a given moment in time of the collection of all metabolites in the cell. The aim of the research is to quantitatively analyze the content of all metabolites in a biological system, and establish the connection between the level of the metabolites and abnormal physiological states, thereby being used for diagnosing and treating diseases.
Common metabolites include amino, carboxyl, sulfhydryl, hydroxyl, phosphoric acid and aldehyde ketone compounds, which have different physical and chemical properties, can be used as a material base to participate in the construction of living bodies, can also participate in the transduction of signal paths, influence the physiological states of the living bodies, and are currently becoming important points of metabolite analysis. Conventional analysis techniques for small molecules of metabolites include Nuclear Magnetic Resonance (NMR), ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS), allowing for the parallel determination of hundreds of metabolites. Among them, mass Spectrometry (MS) is a major technique for studying complex metabolite mixtures in human samples due to its excellent detection range and sensitivity.
The components in the biological sample are often complex, and besides target metabolite molecules, a large amount of inorganic salts, organic salts, phospholipids, proteins and the like are contained to seriously interfere the test result, so that the sample is required to be pretreated to remove or weaken the interference, and the result with high stability and good repeatability is obtained. Although derivatization reagents are used for derivatizing amino compounds at present to reduce the polarity of the compounds, improve the separation effect of chromatographic columns and improve the detection sensitivity. However, there is a problem in that derivatization does not remove a large amount of matrix, and excessive derivatization reagents can interfere with detection, contaminate and clog chromatographic columns, reducing instrument life.
The derivatizing agent is immobilized on a solid phase material, and the coupled small molecule metabolites can be purified by simple separation, separation of impurities, and subsequent washing after selective coupling of the metabolites. The target metabolite is then released under mild conditions for subsequent detection. The magnetic material can simplify the separation process and improve the separation efficiency. By this method, analysis of the sub-metabolome can be achieved.
Because Beijing interference exists in the mass spectrum detection process, the method is very important for peak extraction, qualitative and quantitative analysis of small molecules of the metabolite. In peak extraction and qualitative analysis, the analysis is generally confirmed by retention time, accurate molecular weight and mass spectrum fragmentation rules of the standard substances. In terms of quantification, analysis is often performed by standard curves established by standards. However, biological samples are often complex, and the ionization efficiency of the target metabolite is disturbed by the components in the biological samples, so that the ionization response of the actual samples deviates from the standard curve, and the actual content of the target metabolite in the biological samples is difficult to obtain. The standard curve established in the real sample can greatly improve the quantitative accuracy of the target molecules. The standard variety restricts the accurate quantification of most metabolites in biological samples, and a novel accurate relative quantification technical method for the metabolites in the biological samples is required to be established.
Therefore, it is a technical problem that needs to be solved by those skilled in the art to provide a method capable of extracting, qualitatively and accurately quantifying metabolite molecules in a real sample.
Disclosure of Invention
In view of the above, the invention provides a probe for selectively capturing metabolites from complex samples, a preparation method and application thereof, and the probe can improve the detection sensitivity and fully quantify the target metabolites.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a magnetic molecular probe for specifically capturing metabolites from complex samples, which has a structure shown in a general formula (I):
wherein, the black round dots in the general formula (I) are magnetic cores with magnetic response; x is an ion response improving label, Y is an isotope label, and Z is a targeting coupling group; the side chains contain disulfide bonds as orthogonal cleavage groups;
the positions of X and Y may be interchanged, either only one is present, or neither X nor Y is present;
the metabolite is any one or more of amino compound, carboxyl compound, sulfhydryl compound, hydroxyl compound, phosphoric acid compound and aldehyde ketone compound.
Further, the magnetic core material with magnetic response is iron, cobalt, nickel, ferroferric oxide or a coating material containing the material.
Still further, the coating of the coating material is any one of aminated silica, polystyrene, polyacrylic acid and polyurea.
Preferably, the magnetic core material may be any material with magnetic response, and may be selected from iron powder, ferroferric oxide, nickel powder, cobalt powder and combinations thereof, and may also be a combination of the magnetic core and other materials, such as silicon dioxide coated ferroferric oxide, silicon dioxide amino coated ferroferric oxide, silicon dioxide hydroxyl coated ferroferric oxide and silicon dioxide carboxyl coated ferroferric oxide. The surface of the magnetic core contains amino and substituted amino for subsequent derivatization.
More preferably, the core is ferroferric oxide (FSP, fe) coated with polyurea amino groups 3 O 4 @SiO 2 -Polymers-NH 2 )。
The beneficial effect of adopting above-mentioned further scheme lies in: the magnetic bead coated with the polymer has larger specific surface area, higher load and higher efficiency.
Further, the connecting chain X contains a pyridine ring.
Further, X is any one of the following structures:
wherein R is H, CH 3 、CH 2 CH 3 、CH 2 CH 2 One of OH; m is any one of 0,1,2,3,4,5,6,7,8 and 9; n is any one of 0,1,2,3,4,5,6,7,8 and 9;
preferably, X is any one of the following structures:
the beneficial effect of adopting above-mentioned further scheme lies in: the scheme can obviously improve the ionization efficiency in the detection process of the probe mass spectrum.
Further, the linker Y contains glycine or isotopically labeled glycine.
Further, Y has the following general structure:
wherein o is any one of 0,1,2,3,4,5,6,7,8 and 9; the atomic composition in the tag may be 12 C、 13 C、 14 N、 15 N、 16 O、 18 O、H、D。
Preferably, the linker Y is an isotopically labeled tag, which is any one of the following structures:
the beneficial effect of adopting above-mentioned further scheme lies in: isotope labeling is introduced, so that the qualitative and quantitative determination of the metabolites can be assisted.
Further, the linking chain Z is a metabolite targeting coupling group, which is any one of the following structures:
wherein p is one of 1,2,3,4,5,6,7,8, 9.
Preferably, Z is any one of the following structures:
the beneficial effect of adopting above-mentioned further scheme lies in: the above scheme can realize selective coupling of different types of target metabolites.
Further, the magnetic molecular probe for specifically capturing the metabolite from the complex sample can be selected from the following structures
The invention also provides a preparation method of the magnetic molecular probe for specifically capturing the metabolite from the complex sample, which comprises the following steps:
1) Ferroferric oxide @ silicon dioxide magnetic beads (Fe 3 O 4 @SiO 2 FS) preparation:
adding ethanol into ferroferric oxide powder, dispersing with ultrasound, mechanically stirring in water bath, and adding NH dropwise 3 ·H 2 O, dropwise adding tetraethyl orthosilicate, and after the reaction, magnetically separating, washing and drying to obtain gray brown powder Fe 3 O 4 -SiO 2 I.e., FS;
2) Ferroferric oxide @ silica magnetic bead-polymer (Fe 3 O 4 @SiO 2 -Polymers, FSP) preparation:
suspending the FS powder in DMF, then adding the DMF powder into an EP tube, and removing the solvent for later use; adding EDCI and NHS into a centrifuge tube, adding MES solution, adding the mixture into the centrifuge tube into an EP tube containing FS after complete dissolution, stirring for reaction, removing solvent by magnetic separation after the reaction is finished, and washing residues to obtain Fe 3 O 4 -SiO 2 Polymers, i.e. FSP;
3) Ferroferric oxide @ silicon dioxide magnetic bead-polymer-amino (Fe 3 O 4 @SiO 2 -Polymers-NH 2 ,FSP-NH 2 ) Is prepared from the following steps:
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mixing 1, 3-diaminopropane with MES, adding into FSP for reaction, removing solvent by magnetic separation after reaction, washing residues with DMF, water and DMSO in sequence to obtain Fe 3 O 4 -SiO 2 -Polymers-NH 2 I.e. FSP-NH 2 ；
4)Fe 3 O 4 -SiO 2 -Polymers-NH-CS-NH 2 (FSP-NH-CS-NH 2 ) Is prepared from the following steps:
Fmoc-CS was dissolved in dimethyl sulfoxide, HATU and DIPEA were added, and the above mixture was added to FSP-NH prepared in step 3) 2 After the reaction, the solvent was removed by magnetic separation, and the residue was washed with DMSO, and DMF and DBU were then added to obtain Fe after the reaction 3 O 4 -SiO 2 -Polymers-NH-CS-NH 2 I.e. FSP-NH-CS-NH 2 ；
5) Synthesis of 4- ((6- (((9H-fluoren-9-yl) method) carbonyl) amino) -1-oxo-1- ((pyridin-3-ylmethyl) amino) hexan-2-yl) amino) -4-oxobutanoc-id:
dissolving N-Boc-N' -Fmoc-lysine in DMF, adding HATU, DIPEA and benzyl amine pyridine, diluting with ethyl acetate after the reaction is completed, washing with water, removing solvent to obtain solid, adding dichloromethane into the obtained solid, uniformly mixing, adding methanol for dissolution, then adding dioxane hydrochloride for reaction, and removing solvent after the reaction is completed; adding 200mL of acetonitrile into the reaction product to be fully dissolved, then adding 100mL of acetonitrile solution containing 1.0eq of succinic anhydride, then adding DIPEA until the reaction system is slightly alkaline, removing the solvent after the reaction is finished, and washing to obtainTo 7.34g of product; adding 1.60g succinic anhydride, 3.34mL triethylamine and 30mL methanol and 50mL acetonitrile into 7.34g succinic anhydride, dissolving, reacting to generate white solid, filtering and collecting the solid, washing with acetonitrile to obtain target product, named PyNH 2 ；
6)FSP-Py-Gly-NH 2 Synthesis of magnetic beads:
will PyNH 2 Dissolving HATU in DMSO, mixing, and adding DIPEA and FSP-NH-CS-NH 2 After the reaction is finished, washing, adding DMF and DBU into the obtained product, removing Fmoc, and after the reaction is finished, washing; then Fmoc-Gly, HATU and DIPEA are dissolved in DMSO, added into the reaction product, washed after the reaction is completed, added with DBU to remove Fmoc, and FSP-Py-Gly-NH is obtained 2 Magnetic bead (FSP-NH) 2 -CS-Py-Gly-NH 2 Magnetic beads);
7) Synthesis of magnetic molecular probes for specific capture of metabolites from complex samples: taking FSP-Py-Gly-NH 2 The magnetic beads further react to obtain the magnetic molecular probe for specifically capturing the metabolite from the complex sample.
Further, the mass of the ferroferric oxide and NH in the step 1) 3 ·H 2 The volume ratio of O to tetraethyl orthosilicate is 4g:10mL:10mL;
the water bath temperature is 40 ℃;
the beneficial effect of adopting above-mentioned further scheme lies in: by adopting the scheme, the Fe can be treated 3 O 4 -SiO 2 Is a high-efficiency synthesis of (2).
Further, the ratio of the molar mass of EDCI to the molar mass of NHS to the volume of MES solution in step 2) was 125mmol:125mmol:5mL;
the concentration of the MES solution is 100mM, and the solvent is tween with the mass concentration of 0.5%;
the ratio of the mass of FS powder to the volume of the mixture was 2mg:3mL;
stirring speed is 1800rpm, and stirring time is 2h;
the beneficial effect of adopting above-mentioned further scheme lies in: the scheme is favorable for realizing the efficient encapsulation of the polymer on the magnetic beads.
Further, the volume ratio of 1, 3-diaminopropane to MES in step 3) is 1:9.
The beneficial effect of adopting above-mentioned further scheme lies in: can realize the efficient amination of the polymer on the surface of the magnetic bead.
Further, the Fmoc-CS mass to HATU mass to DIPEA volume ratio in step 4) was 10mg:12mg: 100. Mu.L;
the volume ratio of DMF to DBU is 1:1;
the beneficial effect of adopting above-mentioned further scheme lies in: efficient removal of Fmoc protecting groups can be achieved.
Further, the equivalent ratio of N-Boc-N' -Fmoc-lysine to HATU to DIPEA to benzylamine pyridine in step 5) is 1:1.2:2:1.1;
the volume ratio of the dichloromethane to the methanol to the dioxane hydrochloride is 4:1:1;
the beneficial effect of adopting above-mentioned further scheme lies in: can realize the efficient synthesis of N-Boc-N' -Fmoc-lysine and the Boc removal protection.
Further, in step 6), pyNH 2 The equivalent ratio of HATU to DIPEA is 1:2:3;
the volume ratio of DMF to DBU is 1:1;
the equivalent ratio of Fmoc-Gly to HATU to DIPEA is 1:1.2:3.
The beneficial effect of adopting above-mentioned further scheme lies in: fmoc-Gly can be efficiently coupled on the magnetic beads.
Further, the synthesis of the magnetic molecular probe in the step 7) is any one of the following steps:
7-1)FSP-NH 2 -synthesis of CS-Py-Gly-NCS:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Weighing 100mg of 4-carboxyphenyl isothiocyanate, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH 2 The CS-Py-Gly-NCS probe is a magnetic molecular probe for specifically capturing metabolites from complex samples;
7-2)FSP-NH 2 -synthesis of CS-Py-Gly-COOH:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Magnetic beads, weighing 1.0g of succinic anhydride, adding 100mg of DMAP, and reacting for 1h to obtain FSP-NH 2 -CS-Py-Gly-COOH probe, i.e. a magnetic molecular probe that specifically captures metabolites from complex samples;
7-3)FSP-NH 2 -synthesis of CS-Py-Gly-MAL:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Magnetic beads, weighing 86.8mg of 2-maleimidoacetic acid, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH 2 The CS-Py-Gly-MAL probe is a magnetic molecular probe for specifically capturing metabolites from complex samples;
7-4)FSP-NH-CS-PY-Gly-NHNH 2 is synthesized by the following steps:
300mg FSP-NH-CS-PY-Gly-NH was taken 2 Weighing 142mg Fmoc-hydrazine, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH-CS-PY-Gly-NHNH 2 The probe is a magnetic molecular probe for specifically capturing metabolites from complex samples.
The invention also provides a scheme for preparing magnetic molecular probes for specifically capturing metabolites from complex samples by other magnetic beads, which is any one of the following methods:
preparation of FSP-NH-CS-Gly-NH 2 ：
2.4g of magnetic beads were taken, fmoc-Gly (1.0 g,3.36mmol,1.0 eq) and HATU (1.53 g,4.03mmol,1.2 eq) were added, after complete dissolution, DIPEA (1.30 g,1.76mL,10.08mmol,3.0 eq) was added. Post-washing, adding DBU, removing Fmoc to obtain FSP-NH-CS-Gly-NH 2 And (3) a probe.
Preparation of FSP-NH-CS-Gly-COOH:
300mg of magnetic beads are weighed, 1.0g of succinic anhydride is weighed, 100mg of DMAP is added, and the mixture is reacted for 1h.
Preparation of FSP-NH-CS-Gly-NCS:
NCS (100 mg,0.56mmol,1.0 eq) was weighed out, HATU (255 mg,0.67mmol,1.2 eq) was added, DIPEA (0.293 mL,1.68mmol,3.0 eq) was dissolved in DMSO and reacted at room temperature for 30min to give FSP-NH-CS-Gly-COOH probe.
Preparation of FSP-NH-CS-Gly-MAL:
300mg of magnetic beads were weighed, 2-maleimidoacetic acid (86.8 mg,0.56mmol,1.0 eq) was added to HATU (255 mg,0.67mmol,1.2 eq), DIPEA (0.293 mL,1.68mmol,3.0 eq) was dissolved in DMSO, and reacted at room temperature for 30min to obtain FSP-NH-CS-Gly-MAL probe.
Preparation of FSP-NH-CS-Gly-NHNH 2 ：
300mg of magnetic beads were weighed, fmoc-hydrazine (142 mg,0.56mmol,1.0 eq) was added to HATU (255 mg,0.67mmol,1.2 eq), DIPEA (0.293 mL,1.68mmol,3.0 eq) was dissolved in DMSO and reacted at room temperature for 30min to give FSP-NH-CS-Gly-NHNH 2 And (3) a probe.
The invention also provides an application method of the magnetic molecular probe for specifically capturing the metabolite from the complex sample, which comprises the following steps:
1) Taking 0.1-10mg of the material, adding 0.2-1mL of dimethyl sulfoxide, 0.3mL of serum and 0.001-0.1mL of triethylamine, oscillating for 0.1-5h at the temperature of 0-50 ℃, removing the solvent by magnetic separation after the reaction is finished, washing with phosphate buffer solution, washing with water, washing with a mixed solution of acetonitrile and water, and replacing a centrifuge tube for later use;
2) Capturing the obtained compound to break bonds:
suspending the magnetic material prepared in the step 1) in 0.1-1mL of acetonitrile or methanol water solution, adding 0.01mL of dithiothreitol, oscillating to break bonds for 1-24h, magnetically separating after breaking bonds, sucking the solution, diluting by 5 times, centrifuging, and using for subsequent mass spectrum detection and analysis.
The invention also provides a qualitative method of the material for specifically capturing the ammonia compound from the complex sample, which comprises the following steps:
will be 15 N、 14 N、 13 And C, mixing the marked biological samples in an equal ratio, and determining a target molecular peak and eliminating the interference of a mixed peak through the ratio of the accurate molecular weight and the intensity of the three.
The invention also provides an absolute quantification method of the material for specifically capturing the ammonia compound from the complex sample, which comprises the following steps:
by combining 15 N-labeled amino standard with constant concentration is used as an internal standard and is matched with 14 Mixing N-labeled gradient amino standards, mass spectrometric detection, establishing concentration (C 14 N Ratio of X) to intensity (I) 15 N /I 14 N Marks of Y)The quasi-curve can realize absolute quantification of target molecules.
The invention also provides a relative quantification method of the total target molecules of the material for specifically capturing the ammonia compound from the complex sample, which comprises the following steps:
by passing through 15 N marked unknown concentration biological sample is used as internal standard and is graded 14 Mixing N-labeled biological samples, detecting by mass spectrum, and establishing concentration ratio (C 15 N /C 14 N Ratio of X) to intensity (I) 15 N /I 14 N The standard curve of Y) can realize the relative quantification of target molecules of biological samples.
The beneficial effect of adopting above-mentioned further scheme lies in: the method is simpler and more convenient to operate, is not limited by a large instrument (such as a high-speed centrifugal machine), realizes thorough separation of materials and solutions, and is more beneficial to follow-up experiments.
The invention has the beneficial effects that: the invention provides a brand-new molecular probe, a preparation method and an application method of the probe for selectively capturing metabolic compounds in complex samples. The magnetic molecular probe is formed by immobilizing amino, carboxyl, isothiocyanate, maleimide, hydrazide groups and analogues thereof on a magnetic material, and is used for selectively coupling carboxyl, phosphoric acid, amino, hydroxyl, sulfhydryl and aldehyde ketone compounds, and other impurities can be removed by a simple magnetic separation and washing method. In addition, disulfide bonds are contained in the connecting chain, and the compound is stable in the amino capturing and washing processes, and can be selectively broken in the presence of reducing agents such as dithiothreitol and the like, so that the captured target compound can be broken off from the magnetic material. The bond-breaking solution can be directly used for subsequent experimental analysis after simple magnetic separation.
Compared with the traditional analysis method, the method provided by the invention has the advantages of mild enrichment condition, low-cost and easily obtained materials, easy preparation, high capture specificity and remarkable effect. The method provided by the invention can remove a large amount of matrix interferences such as phospholipid, various salts and the like, is favorable for subsequent mass spectrometry, and can realize confirmation, absolute quantification and relative quantification of target molecules in complex biological samples by taking the glycine isotope labeled metabolite as an internal standard. The method has important significance for the discovery of new metabolic compounds, biomarker screening research, early warning of serious diseases, disease development, prognosis evaluation and the like.
Drawings
FIG. 1 is a scanning electron micrograph of a magnetic molecular probe prepared in example 1 of the present invention;
FIG. 2 is a diagram of the leucine mass spectrometry analysis in example 2 of the present invention;
FIG. 3 is a standard curve for absolute quantification of proline in example 3 of the present invention.
FIG. 4 is a graph showing the relative quantification of glycine in example 4 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the probe for specifically capturing the ammonia compound from the complex sample comprises the following steps:
1) Ferroferric oxide @ silicon dioxide magnetic beads (Fe 3 O 4 @SiO 2 FS) preparation:
adding 4.0g of ferroferric oxide into 250mL of ethanol, dispersing for 10min by ultrasonic, placing into a water bath at 40 ℃ for mechanical stirring, and then adding NH dropwise 3 ·H 2 O (mass concentration 25%,10 mL), tetraethyl orthosilicate (TEOS, 10 mL) is added dropwise, after the reaction, 5.80g of gray brown powder Fe is obtained by magnetic separation, washing and drying at 50 DEG C 3 O 4 -SiO 2 I.e., FS;
2) Ferroferric oxide @ silica magnetic bead-polymer (Fe 3 O 4 @SiO 2 -Polymers, FSP) preparation:
FS (2 mg) powder was suspended in DMF (1.00 mL) and then added to the EP tube and the solvent was removed for use; EDCI (2.40 g,125.00 mmol), NHS (1.44 g,125.00 mmol) were added to a 15mL centrifuge tube, and 5.00mM MES solution (100 mM, solvent 0.5% Tween, 4 ℃ C.) was added, the mixture (0.30 mL) in the centrifuge tube was added to the FS-containing EP tube after vigorous stirring to complete dissolution, at 1,800rpm for 2h, after the reaction was completed, the solvent was removed by magnetic separation, washed twice with DMF, repeated 8 times, and the resulting Fe was combined 3 O 4 -SiO 2 Polymers, i.e. FSP;
3) Ferric oxide @ silica magnetic bead-polymer-amino (Fe 3 O 4 @SiO 2 -Polymers-NH 2 ,FSP-NH 2 ) Is prepared from the following steps:
1, 3-diaminopropane (1.00 mL) was mixed with MES (9.00 mL) and added to FSP for reaction, after the reaction was completed, the solvent was removed by magnetic separation, and the residue was washed with DMF, water and DMSO in this order to obtain Fe 3 O 4 -SiO 2 -Polymers-NH 2 I.e. FSP-NH 2 ；
4)Fe 3 O 4 -SiO 2 -Polymers-NH-CS-NH 2 (FSP-NH-CS-NH 2 ) Is prepared from the following steps:
Fmoc-CS (10.00 mg) was dissolved in dimethyl sulfoxide (10.00 mL), HATU (12.00 mg) and DIPEA (100. Mu.L) were added, and the above mixture was then added toFSP-NH prepared in step 3) 2 After the completion of the reaction, the solvent was removed by magnetic separation, and the residue was washed 3 times with DMSO, and then DMF (5.00 mL) and DBU (5.00 mL) were added, and Fe was obtained after the completion of the reaction 3 O 4 -SiO 2 -Polymers-NH-CS-NH 2 I.e. FSP-NH-CS-NH 2 ；
5) Ferroferric oxide @ silicon dioxide magnetic bead-polymer-amino-cystamine-glycine-amino (FSP-NH-CS-Gly +. 15 N-Gly/ 13 C-Gly-NH 2 ) Is prepared from the following steps:
2.2g of magnetic beads were taken and Fmoc-Gly/Fmoc-Henoc was added 15 N-Gly/Fmoc- 13 C-Gly (1.0 g,3.36mmol,1.0 eq), HATU (1.53 g,4.03mmol,1.2 eq) was dissolved thoroughly and DIPEA (1.30 g,1.76mL,10.08mmol,3.0 eq) was added. Washing after the reaction is completed, adding DBU, removing Fmoc to obtain FSP-NH-CS-Gly +. 15 N-Gly/ 13 C-Gly-NH 2 。
6) Ferroferric oxide @ silicon dioxide magnetic bead-polymer-amino-cystamine-amino-glycine-isothiocyanate (FSP-NH-CS-Gly +. 15 N-Gly/ 13 C-Gly-NCS) preparation:
300mg of magnetic beads were taken. 4-carboxyphenyl isothiocyanate (100 mg,0.56mmol,1.0 eq) was weighed, HATU (255 mg,0.67mmol,1.2 eq) was added, DIPEA (0.293 mL,1.68mmol,3.0 eq) was dissolved in DMSO and the reaction was continued at room temperature for 30min. Washing after the reaction is finished to obtain FSP-NH-CS-Gly +. 15 N-Gly/ 13 C-Gly-NCS。
Example 2 covalent capture of amino compounds in serum by magnetic probes:
1) FSP-NH-CS-Gly-NCS prepared in example 1 was taken 15 N-Gly-NCS and FSP-NH-CS- 13 C-Gly-NCS 4mg each was added to two EP tubes, followed by 1.0mL of dimethyl sulfoxide and 0.0 mL of dimethyl sulfoxide, respectively3mL of serum was added with 0.05mL of triethylamine and shaken at 25℃for 3 hours. After the reaction is completed, the solvent is removed by magnetic separation, and the centrifuge tube is replaced for standby after washing by phosphate buffer solution, water washing and acetonitrile and water mixed solution in sequence.
2) Capturing the obtained amino compound to break bonds:
the magnetic material of step 1) was suspended in 0.5mL of an aqueous solution (50%) of methanol, and 0.01mL of dithiothreitol (0.1M) was added thereto to cleave the bond for 10 minutes with shaking. After the bond breaking is completed, the magnetic separation is carried out, 200 mu L of each sucking solution is uniformly mixed, diluted by 5 times and centrifuged for subsequent mass spectrum detection and analysis.
Wherein part of the mass spectrum results are shown in figure 2. (Gly-NCS-LEU, theory 425.13227, actual 425.13233; 15 N-Gly-NCS-LEU, theory 426.12931, actual 426.12937, 13 C-Gly-NCS-LEU, theory 426.13563, actual 426.13559. ) Leucine can be determined as a target molecule, and not as a hybrid peak, by the ratio of the precise molecular weight and the intensity of the three peaks.
Example 3 absolute quantification of amino compounds in serum by magnetic probes:
1) FSP-NH-CS-Gly-NCS prepared in example 1 was taken 15 N-Gly-NCS 4mg each was added to six EP tubes. A mixed amino acid standard was prepared at a concentration of 1mM, and then 1.0mL of dimethyl sulfoxide and 0.03mL of the mixed standard solution were added, respectively, and 0.05mL of triethylamine was added thereto, followed by shaking at 25℃for 3 hours. After the reaction is completed, the solvent is removed by magnetic separation, and the centrifuge tube is replaced for standby after washing by phosphate buffer solution, water washing and acetonitrile and water mixed solution in sequence.
2) The magnetic material of step 1) was suspended in 0.5mL of an aqueous solution (50%) of methanol, and 0.01mL of dithiothreitol (0.1M) was added thereto to cleave the bond for 10 minutes with shaking. After the bond breaking is completed, magnetically separating, and respectively combining the solutions after the bond breaking. Respectively suck up 14 N-labeled solutions 100, 80, 60, 40, 20, 10, 5, 2, 1. Mu.L were added to the EP tube. Suction is carried out 15 50. Mu.L of N-labeled solution was added to the above EP tube, and an aqueous solution (50%) of methanol was added to a constant volume of 0.5mL, and the mixture was uniformly mixed, centrifuged, mass-detected, and the concentration (C) 14 N Ratio of X) to intensity (I) 15 N /I 14 N Standard curve of Y). The standard curve for proline is shown in figure 3.
Example 4 relative quantification of amino compounds in serum by magnetic probes:
1) FSP-NH-CS-Gly-NCS prepared in example 1 was taken 15 N-Gly-NCS (3X 4 mg) was added to each of the six EP tubes. 1.0mL of dimethyl sulfoxide and 0.03mL of serum were then added, respectively, and 0.05mL of triethylamine was added thereto, followed by shaking at 25℃for 3 hours. After the reaction is completed, the solvent is removed by magnetic separation, and the centrifuge tube is replaced for standby after washing by phosphate buffer solution, water washing and acetonitrile and water mixed solution in sequence.
2) The magnetic material of step 1) was suspended in 0.5mL of an aqueous solution (50%) of methanol, and 0.01mL of dithiothreitol (0.1M) was added thereto to cleave the bond for 10 minutes with shaking. After the bond breaking is completed, magnetically separating, and respectively combining the solutions after the bond breaking. 400. Mu.L, 300. Mu.L, 200. Mu.L, 100. Mu.L, 50. Mu.L, 30. Mu.L, 10. Mu.L, 5. Mu.L of the non-isotopically labeled solution were aspirated, respectively, and added to the EP tube. Suction is carried out 15 100 μl of N-labeled solution was added to the above EP tube, and an aqueous solution of methanol (50%) was added to a constant volume of 0.5mL, and the mixture was uniformly mixed, centrifuged, mass-detected, and a concentration ratio (C) was established 15 N /C 14 N Ratio of X) to intensity (I) 15 N /I 14 N Standard curve of Y). The standard curve of glycine is shown in FIG. 4. By adding the same 15 An N-labeled internal standard sample can achieve total relative quantification of all biological samples.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (13)
1. A magnetic molecular probe for specifically capturing metabolites from complex samples, which is characterized by having a structure shown in a general formula (I):
wherein, the black round dots in the general formula (I) are magnetic cores with magnetic response; x is a label for improving ionization efficiency of mass spectrum, Y is an isotope label, and Z is a targeting coupling group; the side chains contain disulfide bonds as orthogonal cleavage groups;
the metabolite is any one or more of amino compound, carboxyl compound, sulfhydryl compound, hydroxyl compound, phosphoric acid compound and aldehyde ketone compound.
2. The magnetic molecular probe for specifically capturing metabolites from complex samples according to claim 1, wherein the magnetic core material with magnetic response is iron, cobalt, nickel, ferroferric oxide or a coating material containing the same.
3. The magnetic molecular probe for specifically capturing metabolites from complex samples according to claim 2, wherein the coating material is any one of aminated silica, polystyrene, polyacrylic acid and polyurea.
4. A magnetic molecular probe for specifically capturing metabolites from complex samples according to claim 1, wherein said connecting strand X contains a pyridine ring.
5. A magnetic molecular probe for specifically capturing metabolites from complex samples according to claim 1, characterized in that said linker chain Y contains glycine or isotopically labeled glycine.
6. A method for preparing a magnetic molecular probe for specifically capturing metabolites from complex samples according to any one of claims 1 to 5, comprising the steps of:
1) Adding ethanol into ferroferric oxide, and adding ethanol into the ferroferric oxideDispersing with sound, mechanically stirring in water bath, and adding NH dropwise 3 ·H 2 O, dropwise adding tetraethyl orthosilicate, and after the reaction, magnetically separating, washing and drying to obtain gray brown powder Fe 3 O 4 -SiO 2 I.e., FS;
2) Suspending the FS powder in DMF, then adding the DMF powder into an EP tube, and removing the solvent for later use; adding EDCI and NHS into a centrifuge tube, adding MES solution, adding the mixture into the centrifuge tube into an EP tube containing FS after complete dissolution, stirring for reaction, removing solvent by magnetic separation after the reaction is finished, and washing residues to obtain Fe 3 O 4 -SiO 2 Polymers, i.e. FSP;
3) Mixing 1, 3-diaminopropane with MES, adding into FSP for reaction, removing solvent by magnetic separation after reaction, washing residues with DMF, water and DMSO in sequence to obtain Fe 3 O 4 -SiO 2 -Polymers-NH 2 I.e. FSP-NH 2；
4) Fmoc-CS was dissolved in dimethyl sulfoxide, HATU and DIPEA were added, and the above mixture was added to FSP-NH prepared in step 3) 2 After the reaction, the solvent was removed by magnetic separation, and the residue was washed with DMSO, and DMF and DBU were then added to obtain Fe after the reaction 3 O 4 -SiO 2 -Polymers-NH-CS-NH 2 I.e. FSP-NH-CS-NH 2 ；
5) Synthesis of 4- ((6- (((9H-fluoren-9-yl) method) carbonyl) amino) -1-oxo-1- ((pyridin-3-ylmethyl) amino) hexan-2-yl) amino) -4-oxobutanoc-id:
dissolving N-Boc-N' -Fmoc-lysine in DMF, adding HATU, DIPEA and benzyl amine pyridine, diluting with ethyl acetate after the reaction is completed, washing with water, removing solvent to obtain solid, adding dichloromethane into the obtained solid, uniformly mixing, adding methanol for dissolution, then adding dioxane hydrochloride for reaction, and removing solvent after the reaction is completed; adding 200mL of acetonitrile into the reaction product to be fully dissolved, then adding 100mL of acetonitrile solution containing 1.0eq of succinic anhydride, then adding DIPEA until the reaction system is slightly alkaline, removing the solvent after the reaction is finished, and washing to obtain 7.34g of product; 7.34g of the product was taken and addedAdding 1.60g succinic anhydride, 3.34mL triethylamine, adding 30mL methanol and 50mL acetonitrile for dissolution, reacting to generate white solid, filtering and collecting the solid, washing the solid with acetonitrile to obtain a target product, namely PyNH 2 ；
6)FSP-Py-Gly-NH 2 Synthesis of magnetic beads:
will PyNH 2 Dissolving HATU in DMSO, mixing, and adding DIPEA and FSP-NH-CS-NH 2 After the reaction is finished, washing, adding DMF and DBU into the obtained product, removing Fmoc, and after the reaction is finished, washing; then Fmoc-Gly, HATU and DIPEA are dissolved in DMSO, added into the reaction product, washed after the reaction is completed, added with DBU to remove Fmoc, and FSP-Py-Gly-NH is obtained 2 Magnetic beads;
7) Synthesis of magnetic molecular probes for specific capture of metabolites from complex samples: taking FSP-Py-Gly-NH 2 The magnetic beads further react to obtain the magnetic molecular probe for specifically capturing the metabolite from the complex sample.
7. The method for preparing a magnetic molecular probe for specifically capturing metabolites from a complex sample as set forth in claim 6, wherein the mass of said ferroferric oxide, NH in step 1), is as follows 3 ·H 2 The volume ratio of O to tetraethyl orthosilicate is 4g:10mL:10mL;
the water bath temperature is 40 ℃;
the ratio of the molar mass of EDCI to the molar mass of NHS to the volume of MES solution in step 2) was 125mmol:125mmol:5mL;
the concentration of the MES solution is 100mM, and the solvent is tween with the mass concentration of 0.5%;
the ratio of the mass of FS powder to the volume of the mixture was 2mg:3mL;
the stirring speed is 1800rpm, and the stirring time is 2h;
the volume ratio of the 1, 3-diaminopropane to the MES in the step 3) is 1:9.
8. The method of claim 6, wherein the ratio of the mass of Fmoc-CS to the mass of HATU to the volume of DIPEA in step 4) is 10mg:12mg: 100. Mu.L;
the volume ratio of DMF to DBU is 1:1;
the equivalent ratio of N-Boc-N' -Fmoc-lysine to HATU to DIPEA to benzylamine pyridine in step 5) is 1:1.2:2:1.1;
the volume ratio of the dichloromethane to the methanol to the dioxane hydrochloride is 4:1:1;
PyNH in step 6) 2 The equivalent ratio of HATU to DIPEA is 1:2:3;
the volume ratio of DMF to DBU is 1:1;
the equivalent ratio of Fmoc-Gly to HATU to DIPEA is 1:1.2:3.
9. The method for preparing a magnetic molecular probe for specifically capturing metabolites from a complex sample according to claim 6, wherein the synthesis of the magnetic molecular probe in step 7) is any one of the following steps:
7-1)FSP-NH 2 -synthesis of CS-Py-Gly-NCS:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Weighing 100mg of 4-carboxyphenyl isothiocyanate, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH 2 The CS-Py-Gly-NCS probe is a magnetic molecular probe for specifically capturing metabolites from complex samples;
7-2)FSP-NH 2 -synthesis of CS-Py-Gly-COOH:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Magnetic beads, weighing 1.0g of succinic anhydride, adding 100mg of DMAP, and reacting for 1h to obtain FSP-NH 2 -CS-Py-Gly-COOH probe, i.e. a magnetic molecular probe that specifically captures metabolites from complex samples;
7-3)FSP-NH 2 -synthesis of CS-Py-Gly-MAL:
300mg FSP-NH-CS-Py-Gly-NH was taken 2 Magnetic beads, weighing 86.8mg of 2-maleimidoacetic acid, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH 2 The CS-Py-Gly-MAL probe is used for specifically capturing metabolites from complex samplesA magnetic molecular probe;
7-4)FSP-NH-CS-PY-Gly-NHNH 2 is synthesized by the following steps:
300mg FSP-NH-CS-PY-Gly-NH was taken 2 Weighing 142mg Fmoc-hydrazine, adding 255mg HATU,0.293mL DIPEA, dissolving in DMSO, and reacting at room temperature for 30min to obtain FSP-NH-CS-PY-Gly-NHNH 2 The probe is a magnetic molecular probe for specifically capturing metabolites from complex samples.
10. An application method of a magnetic molecular probe for specifically capturing metabolites from complex samples is characterized by comprising the following steps:
1) Taking the material according to any one of claims 1-5, adding dimethyl sulfoxide, serum and triethylamine, oscillating, magnetically separating to remove solvent after the reaction is completed, washing with phosphate buffer solution, washing with water, washing with a mixed solution of acetonitrile and water, and replacing a centrifuge tube for later use;
2) Capturing the obtained compound to break bonds:
suspending the magnetic material prepared in the step 1) in acetonitrile or methanol water solution, adding dithiothreitol, oscillating to break bonds, magnetically separating after the break bonds are completed, sucking the solution, diluting, centrifuging, and using for subsequent mass spectrometry detection analysis.
11. Use of a magnetic molecular probe according to any one of claims 1-5 for specifically capturing metabolites from complex samples for the characterization of target compounds.
12. Use of a magnetic molecular probe for specific capture of metabolites from complex samples according to any one of claims 1-5 for absolute quantification of target compound molecules.
13. Use of a magnetic molecular probe according to any one of claims 1-5 for the specific capture of metabolites from complex samples for the relative quantification of molecules of all compounds of interest.
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