CN115124442A - 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 65
- 238000001212 derivatisation Methods 0.000 title claims abstract description 60
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 37
- 238000003384 imaging method Methods 0.000 title claims abstract description 33
- 238000004949 mass spectrometry Methods 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 238000005507 spraying Methods 0.000 claims abstract description 36
- 238000004458 analytical method Methods 0.000 claims abstract description 15
- 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 13
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 claims abstract description 13
- 238000011065 in-situ storage Methods 0.000 claims abstract description 9
- 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
- 238000000034 method Methods 0.000 claims description 19
- 150000002500 ions Chemical class 0.000 claims description 16
- KQSABULTKYLFEV-UHFFFAOYSA-N naphthalene-1,5-diamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1N KQSABULTKYLFEV-UHFFFAOYSA-N 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 7
- 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 class 0.000 claims description 3
- 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 compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 28
- 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 15
- 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 15
- OFLXLNCGODUUOT-UHFFFAOYSA-N acetohydrazide Chemical compound C\C(O)=N\N OFLXLNCGODUUOT-UHFFFAOYSA-N 0.000 description 14
- 239000012634 fragment Substances 0.000 description 14
- 241000220223 Fragaria Species 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
- 235000016623 Fragaria vesca Nutrition 0.000 description 9
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 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
- 238000001819 mass spectrum Methods 0.000 description 7
- 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
- 238000001514 detection method Methods 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000000007 visual effect Effects 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
- 238000005516 engineering process Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 235000021012 strawberries Nutrition 0.000 description 3
- 229910001887 tin oxide Inorganic materials 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 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
- 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
- 230000000694 effects Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 238000011896 sensitive detection 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
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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
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- 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
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- 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
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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
- 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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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 for 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 with a structural formula shown in a 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.
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 found 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 fast analysis speed and large throughput. However, due to the low natural abundance, poor hydrophobicity and low ionization efficiency of monosaccharides, highly sensitive detection of monosaccharides in biological tissues remains a significant challenge.
In the process of mass spectrometric detection, chemical derivatization is an effective strategy for improving the detection sensitivity of saccharides. Some researchers use the Gilard reagent P and the Gilard reagent T as derivatization reagents, and introduce permanent cationic groups on sugar molecules to improve the detection sensitivity of the sugar molecules. In addition, the hydrophobic group is introduced into the reducing end of the sugar molecule, so that the ionization efficiency of the mass spectrum of the sugar molecule can be improved, and the mass spectrum detection sensitivity of the sugar molecule can also be enhanced. However, these methods are performed by liquid-mass spectrometry (LC-MS), and homogenization is necessary for LC-MS, which inevitably results in loss of spatial information of carbohydrate molecules in biological tissues. MALDI mass spectrometry imaging techniques allow visual analysis of molecules in tissue without destroying spatial characteristics of the tissue. However, for the monosaccharide isomers in the biological tissue, because 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 the monosaccharide isomers in the biological tissue cannot be realized.
Disclosure of Invention
Aiming at the defects 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. A novel monosaccharide derivatization reagent is synthesized and sprayed on the surface of a tissue slice for in-situ derivatization reaction, and the tissue slice sprayed matrix after derivatization 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 purpose, the invention is realized by the following technical scheme:
in a first aspect, the present invention provides a monosaccharide derivatization reagent, wherein the derivatization reagent is 1-naphthylacethydrazide having a structural formula shown in formula (I):
in a second aspect, the invention provides a preparation method of the monosaccharide derivatization reagent, and the synthetic process route is as follows:
in a third aspect, the present invention provides a method for mass spectrometry imaging of monosaccharide isomers, comprising the steps of:
spraying the derivatization reagent on the surface of the tissue slice for 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 of the embodiments of the invention described above are as follows:
(1) the derivatization reagent is 1-naphthylacethydrazide, the derivatization reagent can rapidly react with carbonyl or aldehyde groups in monosaccharide, the reaction condition is mild, the derivatization reagent is suitable for in-situ derivatization on the surface of a tissue slice, naphthalene rings with high hydrophobicity can be introduced into sugar molecules by a chemical derivatization method, the ionization efficiency of the sugar molecules can be obviously improved, and the mass spectrum detection sensitivity of the sugar molecules can be obviously improved.
(2) In-situ derivatization is carried out on monosaccharide molecules in a tissue slice, and in-situ visual analysis of various monosaccharide molecules in biological tissues can be realized by combining MALDI imaging technology.
By performing secondary mass spectrometry on the derivatized monosaccharide molecules, characteristic fragment ions of ketose and aldose molecules can be obtained, MALDI imaging is performed on the characteristic fragment ions, and spatial distribution characteristics of different monosaccharide isomers in a tissue slice can be characterized.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a reaction scheme of a monosaccharide with 1-naphthylacethydrazide;
FIG. 2 shows the secondary mass spectrum profile and fragment ion structure of the derivative generated by the reaction of different monosaccharide isomers with 1-naphthylacethydrazide.
FIG. 3 is a photograph of the mass spectrum of an isomer of ketohexose and aldohexose in carrot tissue in example 2 of the present invention.
FIG. 4 is a photograph of the mass spectrum of an isomer of ketohexose and aldohexose in strawberry tissue in example 3 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
In contrast to the description in the background art, because the monosaccharide isomers in the biological tissue 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 the visualized characterization of the monosaccharide isomers in the biological tissue cannot be realized. The invention provides a monosaccharide isomer MALDI mass spectrometry imaging method based on chemical derivatization.
In a first aspect, the present invention provides a monosaccharide derivatization reagent, wherein the derivatization reagent is 1-naphthylacethydrazide having a structural formula shown in formula (I):
in a second aspect, the invention provides a preparation method of the monosaccharide derivatization reagent, and the synthetic process route is as follows:
in a third aspect, the present invention provides a method for mass spectrometry imaging of monosaccharide isomers, comprising the steps of:
spraying the derivatization reagent on the surface of the tissue slice for 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 of the derivatization reagent is a mixed solution of acetonitrile and acetic acid, the volume ratio of acetonitrile to acetic acid is 1-9:1, and preferably, the volume ratio of acetonitrile to acetic acid is 7: 3. The volume ratio of the acetonitrile to the acetic acid can also be specifically as follows: 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 and accelerate the derivatization reaction.
Preferably, the concentration of the derivatizing agent is 0.1-1.0mg/mL, such as can 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 0.5 mg/mL.
Preferably, the spraying temperature of the derivatization reagent is 30-70 ℃, such as the following temperature: 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 atomization effect of the derivatization reagent is better at higher temperature, and the distribution on the surface of the tissue slice is more uniform.
In some embodiments, the amount of derivatizing agent sprayed onto the surface of the tissue slice is between 5 and 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 a constant temperature, and the derivatization reaction is performed, wherein the incubation temperature is 40-70 ℃, and the incubation temperature can be as follows: 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 the matrix-coated tissue section using a MALDI-TOF/TOF mass spectrometer in a MS or MS/MS scanning mode.
Preferably, when MALDI-MS/MS mass spectrometry analysis is performed on the slices, the CID energy is opened and the analysis window for 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 thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Performing chemical derivatization mass spectrometry imaging analysis on different monosaccharide isomers on a glass slide: the method comprises the following steps:
(1) glucose (aldohexose) and fructose (ketohexose) controls, each 5.0mg, were precisely weighed into 10mL volumetric flasks, 10mL acetonitrile: mixing acetic acid (70:30, v/v) solution, vortex mixing, and ultrasonic treating for 5 min to obtain glucose and fructose solution with concentration of 0.5 mg/mL;
(2) using HTX TM-Sprayer TM Spraying 0.5mg/mL glucose and fructose solution on ITO conductive glass slide respectively with spraying area of 1cm × 1cm and spraying density of 10ng/cm 2 。
(3) 1-naphthalene acetic hydrazide 5.0mg was precisely weighed into a 10mL volumetric flask, 10mL acetonitrile was added: acetic acid (70:30, v/v) solution, vortex and mix evenly, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic hydrazide solution.
(4) Using HTX TM-Sprayer TM Spraying instrument, spraying 0.5mg/mL 1-naphthalene acethydrazide solution onto glass slide containing glucose and fructose, wherein the spraying density of the 1-naphthalene acethydrazide solution is 10ng/cm 2 。
(5) And (3) placing the indium tin oxide conductive glass slide sprayed with monosaccharide and 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 h.
(6) Precisely weigh 25mg of 1, 5-diaminonaphthalene into a 10mL volumetric flask, add 10mL of acetonitrile: vortex and mix the water (70:30, v/v) solution evenly, and obtain 2.5mg/mL 1, 5-diaminonaphthalene solution by ultrasonic treatment for 10 minutes;
(7) using HTX TM-Sprayer TM A spraying instrument, wherein 2.5mg/mL of 1, 5-diaminonaphthalene solution is sprayed on the derivatized indium tin oxide conductive glass slide, the spraying rate of the solution is 75 muL/min, and the spraying cycle times are 10 times;
(8) secondary mass spectrometry of glucose and fructose derivatised on an indium tin oxide conducting slide using a Brooks Rapiflex MALDI-TOF/TOF type mass spectrometer found that glucose (aldohexose) produced the characteristic fragment ion m/z 265.1 and fructose (ketohexose) produced the characteristic fragment ion m/z 295.1, see FIG. 2.
(9) The imaging of mass spectra of characteristic fragment ions m/z 265.1, m/z 295.1 of glucose (aldohexose), fructose (ketohexose) on indium tin oxide conducting glass slides were targeted for extraction by the SCiLS Lab 2018b data processing software, respectively, see fig. 3.
Example 2
Derivatization mass spectrometry imaging analysis of monosaccharide and monosaccharide isomer in carrot
(1) Taking fresh carrots, and making frozen slices with the thickness of 20 mu m;
(2) frozen carrot tissue sections were transferred to ITO-indium tin oxide conductive slides and dried for 15 minutes in vacuo.
(3) 1-naphthalene acetic hydrazide 5.0mg was precisely weighed into a 10mL volumetric flask, 10mL acetonitrile was added: acetic acid (70:30, v/v) solution, vortex and mix evenly, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic hydrazide solution.
(4) Using HTX TM-Sprayer TM A spraying instrument, wherein 0.5mg/mL of 1-naphthalene acetic hydrazide solution is sprayed on the carrot slices, and the spraying density of the 1-naphthalene acetic hydrazide solution on the tissue surface is 10ng/cm 2 。
(5) Putting the carrot tissue slices sprayed with 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 h.
(6) Precisely weigh 25mg of 1, 5-diaminonaphthalene into a 10mL volumetric flask, add 10mL of acetonitrile: vortex and mix the water (70:30, v/v) solution evenly, and obtain 2.5mg/mL 1, 5-diaminonaphthalene solution by ultrasonic treatment for 10 minutes;
(7) using HTX TM-Sprayer TM A spraying instrument, wherein 2.5mg/mL of 1, 5-diaminonaphthalene solution is sprayed on the derivatized carrot slices, the spraying rate of the solution is 75 muL/min, and the spraying cycle times are 10 times;
(8) secondary mass spectrometry of carrot tissue sections by using a Bruker Rapiflex MALDI-TOF/TOF type mass spectrometer found that carrot has both the characteristic fragment ion m/z 265.1 of aldohexose and the characteristic fragment ion m/z 295.1 of ketohexose, indicating that carrot has both aldohexose and ketohexose.
(9) The distribution characteristics of aldohexose and ketohexose in carrot tissue sections were visualized by targeted extraction of their characteristic secondary fragment ions with the help of the SCiLS Lab 2018b data processing software, see fig. 3.
Example 3
Derivatization mass spectrometry imaging analysis of monosaccharide and monosaccharide isomer in strawberry
(1) Taking fresh strawberries, and making frozen sections of strawberry tissues with the thickness of 20 mu m;
(2) the frozen sections of strawberry tissue were transferred to ITO-indium tin oxide conductive slides and dried for 15 minutes in vacuum.
(3) 1-naphthylacethydrazide of 5.0mg was precisely weighed into a 10mL volumetric flask, 10mL acetonitrile was added: acetic acid (70:30, v/v) solution, vortex and mix evenly, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic hydrazide solution.
(4) Using HTX TM-Sprayer TM A spraying instrument, wherein 0.5mg/mL of 1-naphthylacethydrazide solution is sprayed on the strawberry slices, and the spraying density of the 1-naphthylacethydrazide solution on the tissue surface is 10ng/cm 2 。
(5) Placing the strawberry tissue slices sprayed with the 1-naphthylacethydrazide in a constant-temperature incubator, wherein the temperature of the incubator is set to be 60 ℃, and the incubation time is 2 h.
(6) Precisely weigh 25mg of 1, 5-diaminonaphthalene into a 10mL volumetric flask, add 10mL of acetonitrile: vortex and mix the water (70:30, v/v) solution evenly, and obtain 2.5mg/mL 1, 5-diaminonaphthalene solution by ultrasonic treatment for 10 minutes;
(7) using HTX TM-Sprayer TM A spraying instrument, wherein 2.5mg/mL of 1, 5-diaminonaphthalene solution is sprayed on the derivatized strawberry slices, the solution spraying rate is 75 muL/min, and the spraying cycle times are 10 times;
(8) secondary mass spectrometry of strawberry tissue sections using a mass spectrometer of the Bruker Rapiflex MALDI-TOF/TOF type revealed that strawberries contained both the characteristic fragment ion m/z 265.1 for aldohexose and the characteristic fragment ion m/z 295.1 for ketohexose, indicating that strawberries contained both aldohexose and ketohexose.
(9) The distribution characteristics of aldohexose and ketohexose in strawberry tissue sections were visualized by targeted extraction of their characteristic secondary fragment ions with the help of the SCiLS Lab 2018b data processing software, see fig. 4.
Example 4
Derivatization mass spectrometry imaging analysis of monosaccharide and monosaccharide isomer in burdock
(1) Taking fresh burdock, and making frozen burdock tissue slices with the thickness of 20 mu m;
(2) frozen burdock tissue sections were transferred to ITO-indium tin oxide conductive slides and dried for 15 minutes in vacuum.
(3) 1-naphthalene acetic hydrazide 5.0mg was precisely weighed into a 10mL volumetric flask, 10mL acetonitrile was added: acetic acid (70:30, v/v) solution, vortex and mix evenly, ultrasonic for 10 minutes, get 0.5mg/mL 1-naphthalene acetic hydrazide solution.
(4) Using HTX TM-Sprayer TM A spraying instrument, wherein 0.5mg/mL of 1-naphthalene acetic hydrazide solution is sprayed on the burdock slices, and the spraying density of the 1-naphthalene acetic hydrazide solution on the tissue surface is 10ng/cm 2 。
(5) Placing the burdock tissue slices sprayed with 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) Precisely weigh 25mg of 1, 5-diaminonaphthalene into a 10mL volumetric flask, add 10mL of acetonitrile: vortex and mix the water (70:30, v/v) solution evenly, and obtain 2.5mg/mL 1, 5-diaminonaphthalene solution by ultrasonic treatment for 10 minutes;
(7) using HTX TM-Sprayer TM A spraying instrument, wherein 2.5mg/mL of 1, 5-diaminonaphthalene solution is sprayed on the derivatized burdock slices, the spraying speed of the solution is 75 mu L/min, and the spraying cycle times are 10;
(8) secondary mass spectrometry analysis is carried out on burdock tissue sections by using a Bruker Rapiflex MALDI-TOF/TOF type mass spectrometer, and the Burdock is found to have characteristic fragment ions m/z 265.1 of aldohexose and m/z 295.1 of ketohexose, which indicates that the Burdock has both aldohexose and ketohexose.
(9) The distribution characteristics of aldohexose and ketohexose in burdock tissue sections are visualized by targeted extraction of aldohexose and ketohexose characteristic secondary fragment ions with the help of the SCiLS Lab 2018b data processing software.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A monosaccharide derivatization reagent, comprising: the derivatization reagent is 1-naphthalene acethydrazide with a structural formula shown in a formula (I):
2. a method of preparing a monosaccharide derivatizing reagent according to claim 1, wherein: the synthetic process route is as follows:
3. a method of mass spectrometric imaging of a monosaccharide isomer, characterized by: the method comprises the following steps:
spraying the derivatization reagent on the surface of the tissue slice for 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.
4. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: the solvent of the derivatization reagent is a mixed solution of acetonitrile and acetic acid, and the volume ratio of the acetonitrile to the acetic acid is 1-9: 1;
the concentration of the derivatization reagent is 0.1-1.0 mg/mL.
5. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: the spraying temperature of the derivatization reagent is 30-70 ℃.
6. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: the spraying amount of the derivatization reagent on the surface of the tissue slice is 5-20ng/cm 2 。
7. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: after the derivatization reagent is sprayed on the surface of the tissue slice, the tissue slice is incubated at a constant temperature, and the derivatization reaction is carried out, wherein the incubation temperature is 40-70 ℃.
8. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: the sprayed substrate is selected from 2, 5-dihydroxy benzoic acid, 1, 5-diaminonaphthalene or alpha-nitrile-4-hydroxycinnamic acid.
9. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: in MALDI mass spectrometry, MALDI-TOF/TOF type mass spectrometry imaging instrument is selected to scan and analyze the tissue section coated with the matrix, and MS or MS/MS scanning mode is selected;
when MALDI-MS/MS mass spectrometry imaging analysis is performed on the section, CID energy is opened, and the analysis window of precursor ions is selected to be 0.6%.
10. The method of mass spectrometric imaging of monosaccharide isomers according to claim 3, characterized in that: the monosaccharide isomers are ketose and aldose.
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