CN112162028A - Mass spectrum imaging method for vitamin C in strawberry tissue - Google Patents
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Abstract
The invention belongs to the technical field of mass spectrometry imaging, and particularly relates to a mass spectrometry imaging method for vitamin C in strawberry tissues. The invention provides a mass spectrometry imaging method for vitamin C in strawberry tissues, which comprises the steps of slicing strawberry fruits, preparing a coating matrix of the strawberry fruits and preparing a mass spectrometry image. The invention can effectively avoid the interference of the substrate to the target object by limiting the type of the substrate and the coating method. The mass spectrometry imaging method provided by the invention is rapid, simple and convenient, does not need a complex pretreatment technology, can observe a high-resolution morphological image, can identify and visually analyze the specific molecules, and provides a brand new method for researching the spatial distribution of vitamin C on strawberry fruits.
Description
Technical Field
The invention belongs to the technical field of mass spectrometry imaging, and particularly relates to a mass spectrometry imaging method for vitamin C in strawberry tissues.
Background
Strawberries (Fragaria x ananassa Duch.) are important horticultural crops all over the world, and because the strawberries are sour and sweet in fruit, rich in fragrance and rich in various nutrients such as carbohydrates, polyphenols, vitamins and the like, the strawberries play an important role in the production and consumption of fruits and vegetables in China. Vitamin C, a water-soluble vitamin, is highly effective in oxidation resistance and is also known as ascorbic acid because it is susceptible to scurvy when lacking in human body. The content of vitamin C in the strawberries is 40-90 mg/kg, which is higher than that of other common fruits such as apples.
An imaging mass spectrum microscope (iMScope TRIO) is matched with a high-resolution optical microscope at the front end, and a matrix-assisted laser desorption ionization source, an ion trap and a flight mass spectrum tandem mass spectrum (MALDI-IT-TOF-MS) are adopted for the rear-end mass spectrum, so that the optical microscope and a mass spectrometer are integrated into a whole, a high-resolution morphological image can be observed, and multi-stage identification and visual distribution analysis can be carried out on specific molecules. Meanwhile, matrix-assisted laser desorption ionization is an emerging technology for rapidly detecting unknown substances at high flux, and the technology can effectively detect the accurate position of the unknown substances in a tissue sample through the ionization of biomolecules.
At present, a specific matrix is required to be adopted for assisting laser desorption ionization in an imaging mass spectrum technology, but the commonly used auxiliary matrix in the prior art is easy to generate interference on a target object.
Disclosure of Invention
In order to solve the above problems, the present invention provides a mass spectrometry imaging method for vitamin C in strawberry tissue. The mass spectrometry imaging method provided by the invention can effectively eliminate the interference of the matrix on the target object by using a proper matrix and a specific matrix coating method, has the characteristics of rapidness and directness, and provides a powerful basis for researching the spatial distribution of the vitamin C.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a mass spectrometry imaging method for vitamin C in strawberry tissues, which comprises the following steps:
(1) freezing strawberry fruits, slicing to obtain strawberry tissue slices, adhering the strawberry tissue slices to a conductive glass slide, and drying to obtain dried strawberry tissue slices;
(2) coating a matrix for assisting laser desorption ionization on the dried strawberry tissue slices to obtain matrix-coated strawberry tissue slices; the substrate comprises 9-aminoacridine; the method of coating a substrate comprises: sublimating 9-aminoacridine on the strawberry tissue slices, and spraying 1mL of methanol aqueous solution with the volume percentage of 30% methanol on the strawberry tissue slices after sublimation is finished;
(3) and ionizing the strawberry tissue sections coated with the matrix in a mass spectrometer, and processing by data processing software to obtain a mass spectrum image of the vitamin C on the strawberry tissue sections.
Preferably, the freezing method in the step (1) comprises freezing using liquid nitrogen.
Preferably, the step (1) further comprises thawing before slicing, wherein the thawing temperature is-18 to-20 ℃, and the time is 3-5 min.
Preferably, the drying method in the step (1) comprises vacuum drying, and the drying time is 3-5 min.
Preferably, the thickness of the tissue slice of the strawberry in the step (1) is 90-100 μm.
Preferably, the ionization operation in the step (3) includes sequentially performing scanning in a primary mass spectrum negative ion mode and secondary mass spectrum scanning on the vitamin C parent ions.
Preferably, the mass-to-charge ratio range of scanning in the primary mass spectrum negative ion mode is 50-300, the laser energy is 75, the diameter of a laser spot is 25 microns, and the ion mass-to-charge ratio is 170.02; in the secondary mass spectrum scanning, the mass-to-charge ratio of precursor ions is 170.02, scanning is performed in a negative ion mode, the scanning mass-to-charge ratio range is 50-300, the laser energy is 75, the diameter of a laser spot is 25 mu m, and the mass-to-charge ratio of sub-ions is 115.00.
Preferably, the data processing software in step (3) includes Imaging MS Solution software.
The invention provides a mass spectrometry imaging method for vitamin C in strawberry tissues, which comprises the steps of slicing strawberry fruits, preparing a coating matrix of the strawberry fruits and preparing a mass spectrometry image. The invention can effectively avoid the interference of the substrate to the target object by limiting the type of the substrate and the coating method. The mass spectrometry imaging method provided by the invention is rapid, simple and convenient, does not need complex pretreatment, can observe a high-resolution morphological image, can identify and visually analyze specific molecules, and provides a brand new method for researching the spatial distribution of vitamin C on strawberry fruits. According to the embodiment, the mass spectrum imaging method provided by the invention can effectively avoid interference, and the imaging is clear and visual.
Drawings
FIG. 1 is a mass spectrum image of strawberries from example 1 at day 5 of plucking;
FIG. 2 is a mass spectrum image of strawberries from example 1 at day 5 of plucking;
FIG. 3 is a mass spectrum image of strawberries from example 1 at 6 hours of harvest;
FIG. 4 is a mass spectrum image of strawberries from example 1 at 6 hours of harvest;
FIG. 5 is a mass spectrum image of strawberries from example 1 at 6 hours of harvest;
FIG. 6 is a mass spectral image at 6 hours of picking of the raspberries of example 1;
FIG. 7 is a mass spectrum obtained by detecting a vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser desorption after spraying a 9AA matrix in comparative example 1;
FIG. 8 is a mass spectrum obtained by detecting a vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser desorption after spraying a 5-DAN matrix in comparative example 1;
FIG. 9 is an image of a vitamin C mass spectrum obtained by analyzing strawberry slices with a laser desorption ionization time-of-flight mass spectrometer after spraying 9AA matrix in comparative example 1;
FIG. 10 is an image of a vitamin C mass spectrum obtained by analyzing strawberry slices with a laser-resolved ionization time-of-flight mass spectrometer after spraying a 5-DAN matrix in comparative example 1;
FIG. 11 is a mass spectrum obtained by detecting a vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser desorption after spraying a DHB matrix in comparative example 1;
FIG. 12 is a mass spectrum image of vitamin C in a strawberry sample detected by a laser desorption ionization time-of-flight mass spectrometer in a spray coating manner in comparative example 2;
FIG. 13 is a mass spectrum image of vitamin C in a strawberry sample detected by a laser desorption ionization time-of-flight mass spectrometer in a sublimation coating manner in comparative example 2;
FIG. 14 is a mass spectrometry imaging chart of detecting vitamin C in a strawberry sample by using an ionization time-of-flight mass spectrometer after laser resolution in the coating mode of sublimating and spraying methanol in comparative example 3;
FIG. 15 is a mass spectrometry image of detecting vitamin C in a strawberry sample by using laser desorption followed by ionization time-of-flight mass spectrometry in the coating mode of sublimation in comparative example 3;
FIG. 16 is a histogram comparing response values obtained by spraying methanol of different concentrations and detecting a vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser desorption in comparative example 4;
FIG. 17 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 10% in comparative example 4;
FIG. 18 is a mass spectrum obtained by mass spectrometry of vitamin C standard substance in comparative example 4, when the volume percentage concentration of methanol in the methanol aqueous solution is 20%;
FIG. 19 is a mass spectrum of vitamin C standard substance detected by a mass spectrometer in example 1 when the volume percentage concentration of methanol in the methanol aqueous solution is 30%;
FIG. 20 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 40% in comparative example 4;
FIG. 21 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 50% in comparative example 4;
FIG. 22 is a mass spectrum obtained by mass spectrometry of vitamin C standard substance in comparative example 4, when the volume percentage concentration of methanol in the methanol aqueous solution is 60%;
FIG. 23 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 70% in comparative example 4;
FIG. 24 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 80% in comparative example 4;
FIG. 25 is a mass spectrum obtained by detecting a vitamin C standard substance by a mass spectrometer when the volume percentage concentration of methanol in the methanol aqueous solution is 90% in comparative example 4;
FIG. 26 is a mass spectrum of the volume percentage concentration of methanol in the aqueous methanol solution of comparative example 4.
Detailed Description
The invention provides a mass spectrometry imaging method for vitamin C in strawberry tissues, which comprises the following steps:
(1) freezing strawberry fruits, slicing to obtain strawberry tissue slices, adhering the strawberry tissue slices to a conductive glass slide, and drying to obtain dried strawberry tissue slices;
(2) coating a matrix for assisting laser desorption ionization on the dried strawberry tissue slices to obtain matrix-coated strawberry tissue slices; the substrate comprises 9-aminoacridine; the method of coating a substrate comprises: sublimating 9-aminoacridine on the strawberry tissue slices, and spraying 1mL of methanol solution with the volume percentage of 30% on the strawberry tissue slices after sublimation is finished;
(3) and ionizing the strawberry tissue sections coated with the matrix in a mass spectrometer, and processing by data processing software to obtain a mass spectrum image of the vitamin C on the strawberry tissue sections.
The mass spectrometry imaging method provided by the invention can be used for rapidly and directly preparing the mass spectrometry image of the strawberry vitamin C, can effectively avoid the interference of the common matrix in the prior art to the mass spectrometry image, provides a simple sample pretreatment process, can highly reduce the state of the vitamin C in the strawberry, is beneficial to the confirmation of mass spectrometry conditions, and also can provide a foundation for the development of mass spectrometry imaging of plant tissues in the future.
Freezing strawberry fruits, slicing to obtain strawberry tissue slices, adhering the strawberry tissue slices on a conductive glass slide, and drying to obtain dried strawberry tissue slices; the thickness of the strawberry tissue slice is 90-100 mu m. In the present invention, the method of freezing preferably comprises freezing using liquid nitrogen. The operation of freezing is particularly preferably: after the strawberry samples are picked, the strawberry samples are wrapped by tinfoil and then are frozen in liquid nitrogen, and after freezing, the strawberry samples are stored in a refrigerator at minus 80 ℃. The slicing and sample preparation method provided by the invention is simple, the sample is not required to be fixed and wrapped by other substances, the sample can be directly sliced, the obtained slice has higher integrity, and the target object is better retained. In the invention, before slicing, the slicing also preferably comprises thawing, wherein the thawing temperature is preferably-18 to-20 ℃, and the time is preferably 3 to 5 min. In the invention, the slicing machine is preferably a Leica CM1950 freezing microtome for slicing, the temperature of the freezing microtome is preferably-19 ℃, the thickness of the strawberry tissue slice is preferably 90-100 mu m, and further preferably 90 mu m, the strawberry tissue slice is obtained, and the strawberry tissue slice is adhered to a conductive glass slide for subsequent operation. In the invention, the drying method preferably comprises vacuum drying, and the drying time is preferably 3-5 min. The operation of unfreezing the strawberries can effectively avoid the fracture of the strawberry tissues, the thickness of the selected slices is beneficial to the ionization degree of the strawberry tissues, and the effect of mass spectrum imaging is improved.
After the dried strawberry tissue slices are obtained, coating a matrix for assisting laser desorption ionization on the dried strawberry tissue slices to obtain matrix-coated strawberry tissue slices; the substrate comprises 9-aminoacridine; the method of coating a substrate comprises: sublimating 9-aminoacridine on the strawberry tissue slices, and spraying 1mL of methanol aqueous solution containing 30% methanol by volume percentage on the strawberry tissue slices after sublimation. In the invention, during sublimation, a sublimation device (iMLayer) is preferably adopted to automatically set a mode to sublimate 9-AA on the strawberry tissue slices, and the time is preferably 30 min; after sublimation, 1mL of methanol aqueous solution with 30% methanol by volume is preferably sprayed uniformly on the strawberry tissue slices by using a handheld spray gun, and the methanol aqueous solution is preferably sprayed on the conductive glass slide (25 x 75mm) stuck with the strawberry tissue slices in ten times. The 9-AA used in the invention and the coating mode of spraying after sublimation can effectively improve the response of vitamin C ionization.
After the strawberry tissue slice coated with the matrix is obtained, the strawberry tissue slice coated with the matrix is ionized in a mass spectrometer, and a mass spectrum image of vitamin C on the strawberry tissue slice is obtained through processing of data processing software. In the invention, the data processing software comprises Imaging MS Solution software; the ionization also preferably comprises scanning and secondary mass spectrometry scanning of vitamin C parent ions in a primary mass spectrometry anion mode in sequence. The scanning in the primary mass spectrometry anion mode preferably comprises: the mass-to-charge ratio range of scanning is preferably 50-300, the laser energy is preferably 75, the diameter of a laser spot is preferably 25um, and the ion mass-to-charge ratio is preferably 170.02; the secondary mass spectrometry scan preferably comprises: the mass-to-charge ratio of the precursor ions is preferably 170.02, scanning is carried out in a negative ion mode, the scanning mass-to-charge ratio range is preferably 50-300, the laser energy is preferably 75, the diameter of a laser spot is preferably 25um, and the mass-to-charge ratio of the sub-ions is preferably 115.00. The mass spectrum scanning condition provided by the invention can effectively improve the response of the target object.
In the invention, after the mass spectrum image is obtained, preferably, the method further comprises the step of performing comparative analysis after the mass spectrum image is superposed with the optical image; the method for preparing the optical image preferably comprises: and (3) placing the dried strawberry tissue slices in an imaging mass spectrometer with laser for detection to obtain optical images of the strawberry tissue slices. The invention can coincide the optical image and the mass spectrum image, and realize the accurate positioning of the distribution of the vitamin C in the strawberry.
To further illustrate the present invention, a method for mass spectrometry imaging of vitamin C in strawberry tissue according to the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
The mass spectrum imaging method of vitamin C in strawberry tissues comprises the following steps:
(1) collecting strawberry samples in a greenhouse of a strawberry base in a south town Honghougou in Changshu district, Beijing City, 5 months in 2019, wrapping strawberry fruits by tinfoil after picking, placing in liquid nitrogen for quick freezing, placing in a refrigerator at the temperature of-80 ℃ for storage and use, slicing the strawberries by using a freezing slicer after freezing, setting the temperature of the freezing slicer to be-19 ℃, setting the slicing thickness to be 90 mu m, adhering strawberry tissue slices at different picking times on the same conductive glass slide after slicing, and performing vacuum pumping and drying to obtain dried strawberry tissues;
(2) coating an auxiliary laser desorption ionization matrix on the dried strawberry tissue slices; selecting 9-aminoacridine (9-aminoacridine,9-AA) for coating, wherein the coating method comprises the following steps: firstly sublimating 9-AA by using a sublimation device (iMLayer), and spraying 1mL of methanol aqueous solution with the volume percentage of 30% on strawberry tissue slices by using a handheld spray gun after sublimation;
(3) ionizing the strawberry tissue slices coated with the matrix in a mass spectrometer; in the negative ion mode, laser irradiation is carried out under the condition of primary mass spectrum, the diameter of the laser is 25 mu m, and the light intensity is 75; extracting a primary imaging graph of vitamin C in strawberry tissues through data processing software; and carrying out laser irradiation under the condition of secondary mass spectrum to obtain a secondary ion mass spectrum scanning image. The results are shown in fig. 1 to fig. 6, wherein fig. 1 and fig. 2 are mass spectrum images of strawberries at the 5 th day of picking, fig. 3 and fig. 4 are mass spectrum images of strawberries at the 6 th hour of picking, and fig. 5 and fig. 6 are secondary ion mass spectrum scanning imaging images of strawberries at the 6 th hour of picking.
As can be seen from fig. 1 to fig. 6, vitamin C has a relatively obvious distribution difference in strawberry tissues at different harvesting times, which indicates that the mass spectrometry image method provided by the present invention is reliable, and changes in vitamin C distribution in strawberry tissues under different harvesting conditions are more visually visualized.
Comparative example 1
1) The same mass spectrometry imaging method as in example 1 was used, except that in the step (3), the substrate coating method was spray coating, 9-AA was prepared into a 10mg/mL solution and 1, 5-diaminonaphthalene (1,5-DAN) using methanol, and 1,5-DAN substrate was a saturated 1,5-DAN methanol solution, and 1mL of each was added to a manual spray gun for spray coating; in the step (4), a vitamin C standard substance (namely, 1 mu L of 1000mg/mL vitamin C aqueous solution is dripped on a glass slide to replace a strawberry tissue slice) is used, and a mass spectrogram obtained by detecting the vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser analysis is utilized. The results are shown in FIGS. 7 and 8.
2) The mass spectrometry imaging method the same as that of example 1 is adopted, and the difference is that the matrix coating method in the step (3) is spraying, 9-AA is prepared into a 10mg/mL solution and 1, 5-diaminonaphthalene (1,5-DAN), 1,5-DAN matrix is a saturated 1,5-DAN methanol solution, 1mL of each solution is added into a manual spray gun for spraying, and a mass spectrogram obtained by detecting vitamin C in a strawberry slice by using an ionization time-of-flight mass spectrometer after laser resolution. The results are shown in FIGS. 9 and 10.
3) The same mass spectrometry imaging method as in example 1 was used, except that in step (3), the substrate was coated by spray coating, the substrate was selected to be dihydroxybenzoic acid (DHB), and the DHB substrate was 50mg/mL DHB solution containing 1mL of 70% methanol aqueous solution as a solvent; in the step (3), a vitamin C standard substance (namely, 1 mu L of 1000mg/mL vitamin C aqueous solution is dripped on a glass slide to replace a strawberry tissue slice) is used, and a mass spectrogram obtained by detecting the vitamin C standard substance by using an ionization time-of-flight mass spectrometer after laser analysis is utilized. And (4) setting to be in a positive ion mode. The results are shown in FIG. 11.
As shown by the comparison of 1) -3), the vitamin C response is better and has no obvious difference by using 9-AA and 1,5-DAN matrixes, but the 9-AA image effect is best.
Comparative example 2
1) The same mass spectrometry imaging method as in example 1 was used, except that in the step (3), the substrate coating method of the strawberry tissue slices of the blank control group was spray coating, 9-AA was prepared into a 10mg/mL solution using methanol, and 1mL of the solution was added to a manual spray gun for spray coating. The results are shown in FIG. 12.
2) The same mass spectrometry imaging method as in example 1 was used, except that in step (3), the method of matrix coating of the strawberry tissue sections of the blank control group was sublimation, and sublimation was performed using a sublimation apparatus (iMLayer). The results are shown in FIG. 13.
As is clear from comparison of 1) to 2), 2) has a feature of small and uniform crystal grains and better reproducibility than 1) without an organic reagent. As can be seen from a comparison of fig. 12 and 13, the sublimation-coated substrate provided in fig. 2) had a high response value.
Comparative example 3
1) The same mass spectrometry imaging method as in example 1 was used, except that in step (3), the substrate was coated by sublimation + spray coating, and after sublimation of the substrate 9-AA, the substrate was sprayed with 1mL of an aqueous methanol solution containing 70% by volume of methanol. The results are shown in FIG. 14.
2) The same mass spectrometry imaging method as in example 1 was used, except that in the step (3), the method of coating the substrate was sublimation, and the sublimation was carried out using a sublimation apparatus (iMLayer). The results are shown in FIG. 15.
In comparison with fig. 15, fig. 14 shows a low matrix effect and a better imaging effect (high response). The comparison determined that the final substrate was coated in a two-step process.
Comparative example 4
The same mass spectrometry imaging method as in example 1 was used except that spraying was performed in which the methanol aqueous solution used in step (3) had methanol concentrations of 10%, 20%, 40%, 50%, 60%, 70%, 80% and 90% by volume, respectively.
The response values of example 1 and comparative example 4 were counted, and the statistical results are shown in fig. 16 to 26. As can be seen from fig. 16 to 26, when the volume percentage concentration of methanol in the methanol aqueous solution is 30%, the response value is the highest, which is the condition for optimum mass spectrometry imaging.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method for mass spectrometry imaging of vitamin C in strawberry tissue, comprising the steps of:
(1) freezing strawberry fruits, slicing to obtain strawberry tissue slices, adhering the strawberry tissue slices to a conductive glass slide, and drying to obtain dried strawberry tissue slices;
(2) coating a matrix for assisting laser desorption ionization on the dried strawberry tissue slices to obtain matrix-coated strawberry tissue slices; the substrate comprises 9-aminoacridine; the method of coating a substrate comprises: sublimating 9-aminoacridine on the strawberry tissue slices, and spraying 1mL of methanol aqueous solution with the volume percentage of 30% methanol on the strawberry tissue slices after sublimation is finished;
(3) and ionizing the strawberry tissue sections coated with the matrix in a mass spectrometer, and processing by data processing software to obtain a mass spectrum image of the vitamin C on the strawberry tissue sections.
2. The method of mass spectrometry imaging according to claim 1, wherein the method of freezing in step (1) comprises freezing using liquid nitrogen.
3. The mass spectrometry imaging method according to claim 1, wherein the step (1) further comprises thawing at a temperature of-18 to-20 ℃ for 3 to 5min before slicing.
4. The method of mass spectrometry imaging according to claim 1, wherein the drying in step (1) comprises vacuum drying for 3-5 min.
5. The mass spectrometry imaging method of claim 1, wherein the thickness of the tissue section of the strawberry in the step (1) is 90-100 μm.
6. The mass spectrometry imaging method of claim 1, wherein the ionization operation in step (3) comprises sequentially performing a primary mass spectrometry negative ion mode scan and a secondary mass spectrometry scan on the vitamin C parent ions.
7. The mass spectrometry imaging method of claim 6, wherein the mass-to-charge ratio of the primary mass spectrometry negative ion mode is 50-300, the laser energy is 75, the laser spot diameter is 25 μm, and the ion mass-to-charge ratio is 170.02; in the secondary mass spectrum scanning, the mass-to-charge ratio of precursor ions is 170.02, scanning is performed in a negative ion mode, the scanning mass-to-charge ratio range is 50-300, the laser energy is 75, the diameter of a laser spot is 25 mu m, and the mass-to-charge ratio of sub-ions is 115.00.
8. The method of mass spectrometry of claim 1, wherein the data processing software of step (3) comprises ImagingMS Solution software.
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| CN113219044A (en) * | 2021-03-09 | 2021-08-06 | 中国科学院南京土壤研究所 | Mass spectrum imaging target object using 2, 5-dihydroxybenzoic acid as matrix and mass spectrum imaging method thereof |
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