CN115124442B - Monosaccharide derivatization reagent, preparation method thereof and mass spectrometry imaging method of monosaccharide isomer - Google Patents
Monosaccharide derivatization reagent, preparation method thereof and mass spectrometry imaging method of monosaccharide isomer Download PDFInfo
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- 150000002772 monosaccharides Chemical class 0.000 title claims abstract description 61
- 238000001212 derivatisation Methods 0.000 title claims abstract description 56
- 238000004949 mass spectrometry Methods 0.000 title claims abstract description 37
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 32
- 238000003384 imaging method Methods 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 238000005507 spraying Methods 0.000 claims abstract description 44
- 238000004458 analytical method Methods 0.000 claims abstract description 17
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 claims abstract description 11
- 238000011065 in-situ storage Methods 0.000 claims abstract description 9
- 238000009826 distribution Methods 0.000 claims abstract description 8
- FLHXVKZDKJAVMB-UHFFFAOYSA-N 2-naphthalen-1-ylacetohydrazide Chemical group C1=CC=C2C(CC(=O)NN)=CC=CC2=C1 FLHXVKZDKJAVMB-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000011159 matrix material Substances 0.000 claims abstract description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 48
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 150000002500 ions Chemical class 0.000 claims description 15
- KQSABULTKYLFEV-UHFFFAOYSA-N naphthalene-1,5-diamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1N KQSABULTKYLFEV-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
- 238000001819 mass spectrum Methods 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 6
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 claims description 6
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical group OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 claims description 4
- 208000007976 Ketosis Diseases 0.000 claims description 3
- 150000001323 aldoses Chemical class 0.000 claims description 3
- 150000002584 ketoses Chemical group 0.000 claims description 3
- 238000001869 matrix assisted laser desorption--ionisation mass spectrum Methods 0.000 claims description 2
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000002243 precursor Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 238000004885 tandem mass spectrometry Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 36
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical group C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 26
- 239000012634 fragment Substances 0.000 description 16
- 241000220223 Fragaria Species 0.000 description 12
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 12
- 241000208843 Arctium Species 0.000 description 11
- 235000003130 Arctium lappa Nutrition 0.000 description 11
- 235000008078 Arctium minus Nutrition 0.000 description 11
- 244000000626 Daucus carota Species 0.000 description 11
- 235000002767 Daucus carota Nutrition 0.000 description 11
- 150000002402 hexoses Chemical class 0.000 description 10
- GZCGUPFRVQAUEE-UHFFFAOYSA-N 2,3,4,5,6-pentahydroxyhexanal Chemical compound OCC(O)C(O)C(O)C(O)C=O GZCGUPFRVQAUEE-UHFFFAOYSA-N 0.000 description 9
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N 3,4,5,6-tetrahydroxyoxane-2-carboxylic acid Chemical compound OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 9
- 235000016623 Fragaria vesca Nutrition 0.000 description 9
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 9
- 229930091371 Fructose Natural products 0.000 description 7
- 239000005715 Fructose Substances 0.000 description 7
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 7
- OFLXLNCGODUUOT-UHFFFAOYSA-N acetohydrazide Chemical compound C\C(O)=N\N OFLXLNCGODUUOT-UHFFFAOYSA-N 0.000 description 7
- 239000011521 glass Substances 0.000 description 7
- 239000005971 1-naphthylacetic acid Substances 0.000 description 6
- IIDAJRNSZSFFCB-UHFFFAOYSA-N 4-amino-5-methoxy-2-methylbenzenesulfonamide Chemical compound COC1=CC(S(N)(=O)=O)=C(C)C=C1N IIDAJRNSZSFFCB-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000000007 visual effect Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000009210 therapy by ultrasound Methods 0.000 description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000021012 strawberries Nutrition 0.000 description 3
- 229910001887 tin oxide Inorganic materials 0.000 description 3
- BJHIKXHVCXFQLS-UHFFFAOYSA-N 1,3,4,5,6-pentahydroxyhexan-2-one Chemical compound OCC(O)C(O)C(O)C(=O)CO BJHIKXHVCXFQLS-UHFFFAOYSA-N 0.000 description 2
- 150000001312 aldohexoses Chemical class 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- YSULOORXQBDPCU-UHFFFAOYSA-N 2-(trimethylazaniumyl)ethanehydrazonate;hydrochloride Chemical compound [Cl-].C[N+](C)(C)CC(=O)NN YSULOORXQBDPCU-UHFFFAOYSA-N 0.000 description 1
- NDXLVXDHVHWYFR-UHFFFAOYSA-N 2-pyridin-1-ium-1-ylacetohydrazide;chloride Chemical compound [Cl-].NNC(=O)C[N+]1=CC=CC=C1 NDXLVXDHVHWYFR-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- GZCGUPFRVQAUEE-VANKVMQKSA-N aldehydo-L-glucose Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-VANKVMQKSA-N 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C243/00—Compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
- C07C243/24—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids
- C07C243/26—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C243/30—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of an unsaturated carbon skeleton
- C07C243/32—Hydrazines having nitrogen atoms of hydrazine groups acylated by carboxylic acids with acylating carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C241/00—Preparation of compounds containing chains of nitrogen atoms singly-bound to each other, e.g. hydrazines, triazanes
- C07C241/04—Preparation of hydrazides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- 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
- G01N27/64—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 using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
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- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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Abstract
The invention discloses a monosaccharide derivatization reagent, a preparation method thereof and a mass spectrometry imaging method of monosaccharide isomers, wherein the derivatization reagent is sprayed on the surface of a tissue slice to carry out in-situ derivatization reaction to obtain different monosaccharide derivatization products; and spraying a matrix on the tissue slice after the derivatization reaction, and then carrying out MALDI mass spectrometry imaging analysis to obtain the spatial distribution characteristics of different monosaccharide isomers in the tissue slice. The monosaccharide derivatization reagent is 1-naphthalene acethydrazide, and the structural formula is shown in the formula (I):formula (I).
Description
Technical Field
The invention belongs to the technical field of mass spectrometry detection, and relates to a monosaccharide derivatization reagent, a preparation method thereof and a mass spectrometry imaging method of monosaccharide isomers, wherein the mass spectrometry imaging method can be applied to in-situ analysis of biological tissue monosaccharides.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Monosaccharides are the most basic form of carbohydrates present in the body and play an important role in many biological processes. Mass spectrometry has become a powerful tool for detecting monosaccharides in biological samples due to its high analysis speed and large throughput. However, due to the low natural abundance of monosaccharides, poor hydrophobicity, low ionization efficiency, highly sensitive detection of monosaccharides in biological tissues remains a significant challenge.
Chemical derivatization is an effective strategy to increase saccharide detection sensitivity during mass spectrometry. There are researchers that introduce permanent cationic groups on sugar molecules to improve the detection sensitivity of sugar molecules by using the Girard reagent P and the Girard reagent T as derivatization reagents. In addition, the mass spectrum ionization efficiency of the sugar molecules can be improved and the mass spectrum detection sensitivity of the sugar molecules can be enhanced by introducing a hydrophobic group at the reducing end of the sugar molecules. However, these methods are all performed by liquid-mass spectrometry (LC-MS), and liquid-mass spectrometry analysis necessitates tissue homogenization, which tends to result in loss of spatial information about saccharide molecules in biological tissues. MALDI mass spectrometry imaging techniques can perform visual analysis of molecules in tissue without destroying tissue spatial features. However, for monosaccharide isomers in biological tissues, since they have the same mass-to-charge ratio (m/z) value, it is difficult to directly analyze different monosaccharide isomers by using MALDI mass spectrometry imaging technology, and visual characterization of monosaccharide isomers in biological tissues cannot be achieved.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a monosaccharide derivatization reagent, a preparation method thereof and a mass spectrometry imaging method of monosaccharide isomers. The method is characterized in that a novel monosaccharide derivatization reagent is synthesized and sprayed on the surface of a tissue slice to carry out in-situ derivatization reaction, and the derivatized tissue slice sprayed matrix is further subjected to visual analysis by adopting a MALDI mass spectrometry imaging technology, so that in-situ visual characterization of monosaccharide isomers in biological tissues is realized.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a monosaccharide derivatization reagent, wherein the derivatization reagent is 1-naphthalene acethydrazide, and the structural formula of the derivatization reagent is shown as a formula (I):
in a second aspect, the invention provides a preparation method of the monosaccharide derivatization reagent, which comprises the following synthetic process routes:
in a third aspect, the invention provides a mass spectrometry imaging method of monosaccharide isomers, comprising the steps of:
spraying the derivatization reagent on the surface of a tissue slice to perform in-situ derivatization reaction to obtain different monosaccharide derivatization products;
and spraying a matrix on the tissue slice after the derivatization reaction, and then carrying out MALDI mass spectrometry imaging analysis to obtain the spatial distribution characteristics of different monosaccharide isomers in the tissue slice.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
(1) The derivatization reagent is 1-naphthalene acetyl hydrazine, can react with carbonyl or aldehyde groups in monosaccharide rapidly, has mild reaction conditions, is suitable for in-situ derivatization of tissue slice surfaces, can introduce naphthalene rings with larger hydrophobicity into sugar molecules by a chemical derivatization method, can obviously improve ionization efficiency of the sugar molecules, and can obviously improve mass spectrum detection sensitivity of the sugar molecules.
(2) By carrying out in-situ derivatization on monosaccharide molecules in the tissue slice and combining with MALDI imaging technology, in-situ visual analysis of various monosaccharide molecules in biological tissues can be realized.
By performing secondary mass spectrometry on the derivatized monosaccharide molecules, characteristic fragment ions of the ketose and aldose molecules can be obtained, and MALDI imaging of the characteristic fragment ions can characterize the spatial distribution characteristics of different monosaccharide isomers in tissue sections.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a reaction scheme of monosaccharides with 1-naphthalene acetohydrazide;
FIG. 2 is a secondary mass spectrum profile and fragment ion structure of the resulting derivative after reaction of different monosaccharide isomers with 1-naphthalenyl-acethydrazide.
FIG. 3 is a mass spectrometry imaging of the hexulose, aldohexose isomer in carrot tissue in example 2 of the invention.
FIG. 4 is a mass spectrum image of the hexulose, aldohexose isomer in strawberry tissue in example 3 of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Aiming at the description in the background art, as monosaccharide isomers in biological tissues have the same mass-to-charge ratio (m/z) value, different monosaccharide isomers are difficult to directly analyze by adopting a MALDI mass spectrometry imaging technology, and visual characterization of the monosaccharide isomers in the biological tissues cannot be realized. The invention provides a monosaccharide isomerism MALDI mass spectrum imaging method based on chemical derivatization.
In a first aspect, the invention provides a monosaccharide derivatization reagent, wherein the derivatization reagent is 1-naphthalene acethydrazide, and the structural formula of the derivatization reagent is shown as a formula (I):
in a second aspect, the invention provides a preparation method of the monosaccharide derivatization reagent, which comprises the following synthetic process routes:
in a third aspect, the invention provides a mass spectrometry imaging method of monosaccharide isomers, comprising the steps of:
spraying the derivatization reagent on the surface of a tissue slice to perform in-situ derivatization reaction to obtain different monosaccharide derivatization products;
and spraying a matrix on the tissue slice after the derivatization reaction, and then carrying out MALDI mass spectrometry imaging analysis to obtain the spatial distribution characteristics of different monosaccharide isomers in the tissue slice.
In some embodiments, the solvent for the derivatizing reagent is a mixed solution of acetonitrile and acetic acid, the volume ratio of acetonitrile to acetic acid is 1-9:1, preferably, the volume ratio of acetonitrile to acetic acid is 7:3. The volume ratio of acetonitrile to acetic acid may also be specifically: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1.
The acetonitrile is used for dissolving the derivatization reagent, and the acetic acid solution can provide an acidic environment to accelerate the derivatization reaction.
Preferably, the concentration of the derivatizing agent is 0.1-1.0mg/mL, as may be: 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1.0mg/mL, etc., preferably 0.5mg/mL.
Preferably, the spraying temperature of the derivatizing agent is 30-70 ℃, for example, the spraying temperature can be: 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃. The spraying temperature of the derivatizing agent is preferably 55 ℃. The derivatization reagent has better atomization effect at higher temperature and more uniform distribution on the surface of the tissue slice.
In some embodiments, the derivatizing agent is sprayed onto the tissue slice surface in an amount of 5-20ng/cm 2 For example, it may be: 5ng/cm 2 、6ng/cm 2 、7ng/cm 2 、8ng/cm 2 、9ng/cm 2 、10ng/cm 2 、11ng/cm 2 、12ng/cm 2 、13ng/cm 2 、14ng/cm 2 、15ng/cm 2 、16ng/cm 2 、17ng/cm 2 、18ng/cm 2 、19ng/cm 2 、20ng/cm 2 . The spraying amount of the derivatization reagent on the surface of the tissue slice is preferably 10ng/cm 2 。
Preferably, after the derivatization reagent is sprayed on the surface of the tissue slice, the tissue slice is incubated at constant temperature, the derivatization reaction is performed, and the incubation temperature is 40-70 ℃, for example, the following steps are adopted: 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃.
In some embodiments, the sprayed substrate is selected from 2, 5-dihydroxybenzoic acid, 1, 5-diaminonaphthalene, or α -cyano-4-hydroxycinnamic acid, preferably 1, 5-diaminonaphthalene.
In some embodiments, MALDI mass spectrometry is performed by scanning a tissue slice of the sprayed matrix using a MALDI-TOF/TOF mass spectrometer, and selecting an MS or MS/MS scanning mode.
Preferably, when the fragments are subjected to MALDI-MS/MS mass spectrometry imaging analysis, the CID energy is opened and the analysis window of the precursor ions is selected to be 0.6%.
Preferably, the monosaccharide isomers are ketoses and aldoses.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
Chemical derivatization mass spectrometry imaging analysis of different monosaccharide isomers on slides: the method comprises the following steps:
(1) Glucose (aldohexose) and fructose (ketohexose) controls, each precisely weighed 5.0mg, were placed in 10mL volumetric flasks and 10mL acetonitrile was added: acetic acid (70:30, v/v) solution, vortex mixing, ultrasonic treatment for 5 minutes, and obtaining glucose and fructose solution with concentration of 0.5 mg/mL;
(2) Using HTX TM-Splayer TM Spraying the glucose and fructose solutions of 0.5mg/mL onto the conductive glass slide with the spraying area of 1cm multiplied by 1cm and the spraying density of 10ng/cm 2 。
(3) Accurately weighing 5.0mg of 1-naphthalene acetohydrazide in a 10mL volumetric flask, adding 10mL acetonitrile: acetic acid (70:30, v/v) solution, vortex and mix well, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic acid hydrazide solution.
(4) Using HTX TM-Splayer TM Spraying the 1-naphthalene acethydrazide solution with the concentration of 10ng/cm on a glass slide with glucose and fructose by a spraying instrument, wherein the spraying density of the 1-naphthalene acethydrazide solution is 0.5mg/mL 2 。
(5) And (3) placing the indium tin oxide conductive glass slide sprayed with the monosaccharide and the 1-naphthalene acetic hydrazide in a constant temperature incubator, wherein the temperature of the incubator is set to be 60 ℃, and the incubation time is 2 hours.
(6) 25mg of 1, 5-diaminonaphthalene was precisely weighed into a 10mL volumetric flask, and 10mL of acetonitrile was added: the water (70:30, v/v) solution is mixed by vortex, and ultrasonic treatment is carried out for 10 minutes to obtain 2.5mg/mL 1, 5-diaminonaphthalene solution;
(7) Using HTX TM-Splayer TM Spraying 2.5mg/mL of 1, 5-diaminonaphthalene solution onto the derivatized indium tin oxide conductive glass slide, wherein the spraying speed of the solution is 75 mu L/min, and the spraying cycle times are 10 times;
(8) Secondary mass spectrometry of glucose and fructose after derivatization of indium tin oxide conductive slides was performed using a bruke rapidex MALDI-TOF/TOF mass spectrometer, and it was found that glucose (aldohexose) was able to generate characteristic fragment ions m/z 265.1 and fructose (ketohexose) was able to generate characteristic fragment ions m/z 295.1, see fig. 2.
(9) The mass spectrum imaging diagram of the characteristic fragment ions m/z 265.1 and m/z 295.1 of glucose (aldohexose) and fructose (ketohexose) on the indium tin oxide conductive glass slide is respectively targeted and extracted by the SCiLS Lab 2018b data processing software, and is shown in figure 3.
Example 2
Derivatization mass spectrometry imaging analysis of monosaccharides and monosaccharide isomers in carrots
(1) Taking fresh carrot, and making frozen slices with the thickness of 20 mu m;
(2) Frozen sections of carrot tissue were transferred to ITO-indium tin oxide conductive glass slides and dried in vacuo for 15 minutes.
(3) Accurately weighing 5.0mg of 1-naphthalene acetohydrazide in a 10mL volumetric flask, adding 10mL acetonitrile: acetic acid (70:30, v/v) solution, vortex and mix well, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic acid hydrazide solution.
(4) Using HTX TM-Splayer TM Spraying the 1-naphthalene acetic hydrazine solution with the concentration of 0.5mg/mL on the carrot slices by a spraying instrument, wherein the spraying density of the 1-naphthalene acetic hydrazine solution on the tissue surface is 10ng/cm 2 。
(5) The carrot tissue slices sprayed with the 1-naphthalene acetic acid hydrazide are placed in a constant temperature incubator, the temperature of the incubator is set to be 60 ℃, and the incubation time is 2 hours.
(6) 25mg of 1, 5-diaminonaphthalene was precisely weighed into a 10mL volumetric flask, and 10mL of acetonitrile was added: the water (70:30, v/v) solution is mixed by vortex, and ultrasonic treatment is carried out for 10 minutes to obtain 2.5mg/mL 1, 5-diaminonaphthalene solution;
(7) Using HTX TM-Splayer TM Spraying 2.5mg/mL of 1, 5-diaminonaphthalene solution onto the derivatized carrot slices by a spraying instrument, wherein the spraying rate of the solution is 75 mu L/min, and the spraying cycle times are 10 times;
(8) A Bruce Rapid flow MALDI-TOF/TOF mass spectrometer is used for carrying out secondary mass spectrometry on the carrot tissue sections, and the characteristic fragment ions m/z 265.1 of the hexose and the characteristic fragment ions m/z 295.1 of the hexose in the carrot are found, so that the hexose and the hexose are shown in the carrot.
(9) The distribution characteristics of aldohexose and ketohexose in carrot tissue sections were visualized by targeted extraction of aldohexose and ketohexose characteristic secondary fragment ions by means of SCiLS Lab 2018b data processing software, see fig. 3.
Example 3
Derivatized mass spectrometry imaging analysis of monosaccharides and monosaccharide isomers in strawberries
(1) Taking fresh strawberry, and making frozen sections of strawberry tissues with the thickness of 20 mu m;
(2) Frozen sections of strawberry tissue were transferred to ITO-indium tin oxide conductive slides and dried in vacuo for 15 minutes.
(3) Accurately weighing 5.0mg of 1-naphthalene acetohydrazide in a 10mL volumetric flask, adding 10mL acetonitrile: acetic acid (70:30, v/v) solution, vortex and mix well, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic acid hydrazide solution.
(4) Using HTX TM-Splayer TM Spraying 0.5mg/mL of 1-naphthalene acetic hydrazine solution on the strawberry slices by a spraying instrument, wherein the spraying density of the 1-naphthalene acetic hydrazine solution on the tissue surface is 10ng/cm 2 。
(5) And placing the strawberry tissue slices sprayed with the 1-naphthalene acetic acid hydrazide in a constant temperature incubator, wherein the temperature of the incubator is set to be 60 ℃, and the incubation time is set to be 2 hours.
(6) 25mg of 1, 5-diaminonaphthalene was precisely weighed into a 10mL volumetric flask, and 10mL of acetonitrile was added: the water (70:30, v/v) solution is mixed by vortex, and ultrasonic treatment is carried out for 10 minutes to obtain 2.5mg/mL 1, 5-diaminonaphthalene solution;
(7) Using HTX TM-Splayer TM Spraying 2.5mg/mL of 1, 5-diaminonaphthalene solution onto the derivatized strawberry slices by a spraying instrument, wherein the spraying rate of the solution is 75 mu L/min, and the spraying cycle times are 10 times;
(8) A Bruker Rapid flow MALDI-TOF/TOF mass spectrometer is used for carrying out secondary mass spectrometry on the strawberry tissue slices, and the characteristic fragment ions m/z 265.1 and m/z 295.1 of the hexose in the strawberries are found, so that the strawberries have the characteristic fragments of the hexose and the hexose.
(9) The distribution characteristics of aldohexose and ketohexose in strawberry tissue sections were visualized by targeted extraction of aldohexose and ketohexose characteristic secondary fragment ions by means of SCiLS Lab 2018b data processing software, see fig. 4.
Example 4
Mass spectrometry imaging analysis of derivatization of monosaccharides and monosaccharide isomers in burdock
(1) Taking fresh burdock, and preparing frozen slices of burdock tissues with the thickness of 20 mu m;
(2) Frozen sections of burdock tissue were transferred to an ITO-indium tin oxide conductive glass slide and dried in vacuo for 15 minutes.
(3) Accurately weighing 5.0mg of 1-naphthalene acetohydrazide in a 10mL volumetric flask, adding 10mL acetonitrile: acetic acid (70:30, v/v) solution, vortex and mix well, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic acid hydrazide solution.
(4) Using HTX TM-Splayer TM Spraying 0.5mg/mL of 1-naphthalene acetic hydrazine solution onto the burdock slices by a spraying instrument, wherein the spraying density of the 1-naphthalene acetic hydrazine solution on the tissue surface is 10ng/cm 2 。
(5) And (3) placing the burdock tissue slices sprayed with the 1-naphthalene acethydrazide in a constant-temperature incubator, wherein the temperature of the incubator is set to be 60 ℃, and the incubation time is set to be 2 hours.
(6) 25mg of 1, 5-diaminonaphthalene was precisely weighed into a 10mL volumetric flask, and 10mL of acetonitrile was added: the water (70:30, v/v) solution is mixed by vortex, and ultrasonic treatment is carried out for 10 minutes to obtain 2.5mg/mL 1, 5-diaminonaphthalene solution;
(7) Using HTX TM-Splayer TM Spraying 2.5mg/mL of 1, 5-diaminonaphthalene solution onto the derivatized burdock slices by a spraying instrument, wherein the spraying rate of the solution is 75 mu L/min, and the spraying cycle times are 10 times;
(8) A Bruker Rapid flow MALDI-TOF/TOF mass spectrometer is used for carrying out secondary mass spectrometry on the burdock tissue slices, and the characteristic fragment ions m/z 265.1 and m/z 295.1 of the hexose in the burdock are found, so that the burdock has the characteristic fragments of the hexose and the hexose.
(9) The distribution characteristics of aldohexose and ketohexose in burdock tissue sections were visualized by targeted extraction of aldohexose and ketohexose characteristic secondary fragment ions by means of SCiLS Lab 2018b data processing software.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A mass spectrometry imaging method of monosaccharide isomers, characterized by: the method comprises the following steps:
spraying a monosaccharide derivatization reagent onto the surface of a tissue slice to perform in-situ derivatization reaction to obtain different monosaccharide derivatization products;
spraying a matrix on the tissue slice after the derivatization reaction, and then carrying out MALDI mass spectrometry imaging analysis to obtain the spatial distribution characteristics of different monosaccharide isomers in the tissue slice;
the monosaccharide derivatization reagent is 1-naphthalene acethydrazide, and the structural formula is shown in the formula (I):
the monosaccharide isomer is ketose and aldose;
the solvent of the derivatization reagent is a mixed solution of acetonitrile and acetic acid, and the volume ratio of acetonitrile to acetic acid is 1-9:1; the concentration of the derivatization reagent is 0.1-1.0mg/mL;
the spraying temperature of the derivatization reagent is 30-70 ℃;
spraying a derivatization reagent on the surface of a tissue slice, and then incubating the tissue slice at a constant temperature, wherein the derivatization reaction is carried out at the incubation temperature of 40-70 ℃;
the sprayed substrate is selected from 2, 5-dihydroxybenzoic acid, 1, 5-diaminonaphthalene or alpha-nitrile-4-hydroxycinnamic acid.
2. The method of mass spectrometry imaging of monosaccharide isomers according to claim 1, wherein: the spraying amount of the derivatization reagent on the surface of the tissue slice is 5-20ng/cm 2 。
3. The method of mass spectrometry imaging of monosaccharide isomers according to claim 1, wherein: in MALDI mass spectrum imaging analysis, a MALDI-TOF/TOF mass spectrum imager is selected to scan and analyze the tissue slice sprayed with the matrix, and MS or MS/MS scanning mode is selected;
when the sections were subjected to MALDI-MS/MS mass spectrometry imaging analysis, the CID energy was turned on and the analysis window for the precursor ions was selected to be 0.6%.
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