A method for glucose mass spectrometry imaging based on in situ derivatization technique

Abstract

The application discloses a kind of glucose mass spectrum imaging methods based on in situ derivatization technology, belong to mass spectrum imaging detection technical field, the method is: preparation sample section and affix to conductive carrier glass;Derivatization reagent and catalyst are dissolved in volatile organic solvent, preparation derivative reagent system solution, derivative reagent system solution is sprayed on the surface of tissue section and is derived to carry out reaction;After the surface of derived section is coated with matrix, it is identified and analyzed using MALDI-MSI method;Derivatization reagent structure is as follows:In the application, the trace carbonyl-containing compound in biological tissue can be mildly and quickly derived in situ by the derivatization reagent, the mass spectrum signal response of target compound molecule is significantly improved, and the analyte delocalization (diffusion) phenomenon is obviously inhibited. The derived tissue section is analyzed using the MALDI-MSI technology, the trace carbonyl-containing compound in the biological tissue section is realized high spatial resolution in situ visual characterization, and the problems existing in the prior art are effectively solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mass spectrometry imaging detection, and particularly relates to a glucose mass spectrometry imaging method based on in-situ derivatization technology. BACKGROUND

[0002] Monosaccharides play an indispensable role in many important life activities, such as energy supply, signal transduction, etc., and are widely involved in various pathways of basic operation of the organism, and are the most important basic molecules containing carbonyl chemical structure. Representatively, in the growth process of tumors, an important pathological change is the reprogramming of glucose metabolism, and the abnormal glucose metabolism behavior of biological tissues can meet the growth needs of cancer cells. Therefore, in-situ spatial distribution analysis of trace endogenous monosaccharides in biological tissues can better provide new analysis technology support for monitoring the metabolic pathways, physiological functions, abnormal physiological behaviors and disease processes of monosaccharides.

[0003] The inherent high hydrophilicity, no characteristic optical signal, low ionization efficiency and low natural abundance of monosaccharide molecules seriously restrict the detection sensitivity of monosaccharides in complex biological tissues. The derivatization technology of the carbonyl group in the structure of monosaccharide compounds is an effective strategy to improve the detection sensitivity, and 1-phenyl-3-methyl-5-pyrazolone and 8-aminopyrene-1, 3, 6-trisulfonic acid trisodium salt have been developed into mature reagents, but they need a tedious solution derivatization process, which is difficult to meet the needs of in-situ spatial distribution analysis of samples. Although the conventional histochemical staining technology such as PAS method can realize visual detection, the original spatial distribution information of monosaccharide molecules is significantly distorted due to the solubility migration effect in the multi-step solvent fixation and washing process.

[0004] Matrix-assisted laser desorption ionization-mass spectrometry imaging technology (MALDI-MSI) has become an important visualization tool in the fields of biological medicine, judicial identification, plant science and food science in recent years, due to its molecular "soft" ionization, high spatial resolution and the advantages of integrating compound molecular mass information and spatial and temporal distribution information. However, when directly performing MALDI-MSI analysis on trace monosaccharides in biological tissue sections, the ionization efficiency and signal response of monosaccharide molecules are generally low. Although some derivatization MALDI-MSI methods for monosaccharides in tissue sections have been published, due to the relatively long incubation time of the derivatization reaction and the application mode of the derivatization reagent, the analyte in the tissue section shows obvious spatial displacement. The diffusion displacement of the analyte in some published derivatization MALDI-MSI methods for monosaccharides in tissue sections can reach 0.3-1 mm. This uncontrolled diffusion effect weakens the spatial fidelity of mass spectrometry imaging, and introduces significant uncertainty for the accurate spatial positioning of trace monosaccharides in biological tissue sections. SUMMARY

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides a glucose mass spectrometry imaging method based on in-situ derivatization technology. The derivatization reagent in this invention uses coumarin as the parent structure and a hydrazine group as the functional group. This reagent is used to in-situ derivatize trace amounts of carbonyl compounds (such as monosaccharides) in biological tissues, significantly improving the mass spectrometry signal response of the target compound molecules and markedly suppressing analyte delocalization (diffusion). Accurate spatial resolution at the micrometer scale can be achieved. MALDI-MSI technology is used to analyze the derivatized tissue sections, realizing in-situ visualization and characterization of trace monosaccharides in biological tissue sections, effectively solving the problems existing in existing technologies.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A glucose mass spectrometry imaging method based on in-situ derivatization technology includes the following steps:

[0008] (1) Prepare micron-sized sample slices and attach them to a conductive glass slide;

[0009] (2) The derivatization reagent and proton donor catalyst are dissolved in a volatile organic solvent to prepare a derivatization reagent system solution. Then, the derivatization reagent system solution is sprayed onto the surface of tissue sections to carry out derivatization reaction on compounds containing carbonyl groups.

[0010] (3) The MALDI matrix was coated on the surface of the derivatized slices, and then the MALDI-MSI method was used for identification and analysis.

[0011] The derivatizing reagent has the following structure:

[0012]

[0013] (I)

[0014] Wherein, R1-R2 is one of hydrogen, C1 alkyl, C2 alkyl and C3 alkyl; R3-R6 is one of hydrogen, C1 alkyl, C2 alkyl, C3 alkyl, hydroxyl, amino and halogen; n≥0;

[0015] The catalyst includes one of formic acid and acetic acid, or a combination of one of formic acid and acetic acid with one of benzoic acid, p-toluenesulfonic acid, α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid proton donors.

[0016] Furthermore, the thickness of the tissue sections in step (1) is 10-60 μm.

[0017] Further, the concentration of the derivatization reagent in the derivatization reagent system solution in step (2) is 1-50 mM, and the concentration of the catalyst is 0.1-5 M.

[0018] Further, the volatile organic solvent in step (2) is acetonitrile or methanol.

[0019] Further, the derivatization reaction process in step (2) includes natural drying after spraying the derivatization reagent system solution on the surface of the tissue section; or heating and incubating at 25-50℃ in an air atmosphere for 0.5-4 h after spraying the derivatization reagent system solution on the surface of the tissue section; or heating and incubating in an organic vapor atmosphere containing 10-50% volatile proton donor at 25-50℃ for 0.5-4 h after spraying the derivatization reagent system solution on the surface of the tissue section.

[0020] Further, the MALDI matrix in step (3) is 2, 5-dihydroxybenzoic acid, 1, 5-naphthalene diamine or alpha-cyano-4-hydroxycinnamic acid.

[0021] Further, in the MALDI-MSI identification and analysis in step (3), the optical image is collected at 40-1.25 magnification or under a wide-angle lens; the m / z range is 100-1000, and the collision gas of the secondary mass spectrum is argon.

[0022] 1. The derivatization reagent used in the present application has a simple synthesis process, which can be completed in a solution system at room temperature without complicated separation and preparation.

[0023] 2. The derivatization reagent involved in the present application has both a benzopyrone structure with high hydrophobicity and a tertiary amine structure with high mass spectrometry ionization efficiency in the molecular structure, and the introduction of the above structures can significantly improve the mass spectrometry detection sensitivity of monosaccharide molecules in the positive ion mode.

[0024] 3. The derivatization reagent involved in the present application can react with the carbonyl group of monosaccharide in the tissue section under the condition of adding an appropriate amount of proton donor molecule as a reaction catalyst, which is mild and fast compared with traditional derivatization detection technology.

[0025] 4. The in-situ derivatization process of trace carbonyl compounds in the biological tissue section in the present application effectively limits the delocalization (diffusion) of the target analyte by controlling the proportion of the non-volatile proton donor catalyst applied to the surface of the sample section.

[0026] 5. The derivatization reagent involved in the present application is not only suitable for MALDI-MSI analysis, but also suitable for direct mass spectrometry analysis, liquid chromatography-mass spectrometry analysis and other analysis methods, and the application range of the method is wide.

[0027] 6、The application can effectively improve the detection sensitivity of carbonyl compounds in tissue section samples, realize the visualization analysis of monosaccharides in human tumor tissues, and provide guidance for the clinical diagnosis and treatment of cancer, so that the method developed by the application has good practical popularization and application value in the fields of clinical diagnosis and plant research. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum characterization diagram of 7-(diethylamino) coumarin-3-formyl hydrazine (DCCH);

[0029] Figure 2 It is the mass spectrum characterization diagram of 7-(diethylamino) coumarin-3-formyl hydrazine (DCCH);

[0030] Figure 3 It is the MALDI-MS / MS mass spectrum diagram and fragment ion structure diagram of the derivative generated after glucose reacts with DCCH;

[0031] Figure 4 It is the optical image of a pig liver tissue section and the mass spectrum imaging diagram of the distribution of endogenous glucose labeled by DCCH;

[0032] Figure 5 It is the superimposed analysis diagram of the optical image of a pig liver tissue section and the mass spectrum imaging diagram of the distribution of endogenous glucose labeled by DCCH;

[0033] Figure 6 It is the optical image of a tumor pathological tissue section and the mass spectrum imaging diagram of the distribution of endogenous glucose labeled by DCCH. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application, that is, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments.

[0035] Therefore, the following detailed description of the embodiments of the present application provided herein is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] It is to be understood that the terms "first" and "second" and similar relating terms are used merely to distinguish one entity or action from another, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Also, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises... " does not, without more limitations, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0037] The features and nature of the present application will become more apparent from the detailed description set forth below, taken in conjunction with the accompanying drawings.

[0038] Example 1

[0039] A carbonyl derivatization reagent 7-(diethylamino)coumarin-3-formhydrazide (DCCH) has the following structure:

[0040]

[0041] The synthesis method is as follows: accurately weigh 7-(diethylamino) coumarin-3-carboxylic acid ethyl ester 5.8 g, and add it to a 150 mL round-bottom flask containing hydrazine hydrate 6.5 mL (80%), anhydrous ethanol 60 mL, stir at room temperature for 1 h, after the reaction is completed, the whole round-bottom flask is placed in ice water for 30 min, the precipitate is filtered to obtain the product, the product is an orange solid, the yield is about 80%.

[0042] The structure of the probe is characterized by nuclear magnetic resonance hydrogen spectrum, 1 H NMR (400 MHz, CDCl3) δ (ppm): 9.72 (s, 1H), 8.67 (s, 1H), 7.43 (d, J = 8.96 Hz, 1H), 6.65 (dd, J = 2.50 Hz, 8.95 Hz, 1H), 6.49 (d, J = 2.46 Hz, 1H), 3.92 (s, 2H), 3.46 (q, J = 7.14 Hz, 4H), 1.24 (t, J = 7.11 Hz, 6H). The spectral peak at chemical shift 3.92 ppm belongs to the proton in the -NH2 group, and the nuclear magnetic resonance hydrogen spectrum is shown in Figure 1 The molecular mass is verified by high-resolution mass spectrometry, as shown in Figure 2HRMS (ESI) m / z: calcd. for [M+H] + 276.1343, observed 276.1336; [M+Na] + 298.1162, observed 298.1162.

[0043] Example 2

[0044] A carbonyl derivatization reagent 7-amino-4-methylcoumarin-3-acetylhydrazine has the following structural formula:

[0045]

[0046] The synthesis method is as follows: accurately weigh 7-amino-4-methylcoumarin-3-acetic acid ethyl ester 5.3 g, and add it into a 150 mL round-bottom flask containing hydrazine hydrate 7.0 mL (80%), anhydrous ethanol 60 mL, stir at room temperature for 1 h, after the reaction is completed, the whole round-bottom flask is placed in an ice water bath for 30 min, the precipitate is filtered to obtain the product, which is an orange solid, and the yield is about 65%.

[0047] Test Example

[0048] I. Take the carbonyl derivatization reagent in Example 1 as an example, carry out the target plate derivatization reaction of glucose and 7-(diethylamino)coumarin-3-formylhydrazine (DCCH) and MALDI-MS / MS analysis of the derivative product, and the specific operation is as follows:

[0049] Accurately weigh 1.8 mg of glucose standard in a 10 mL cuvette, add a mixed solution of acetonitrile / water (1:1, v / v) 10 mL, dissolve thoroughly, and prepare a glucose standard stock solution with a concentration of 1 mM;

[0050] Accurately weigh 5.5 mg of DCCH in a 10 mL cuvette, add a mixed solution of acetonitrile / acetic acid (7:3, v / v) 10 mL, vortex thoroughly after dissolving thoroughly, and prepare a DCCH derivatization reagent system solution with a DCCH concentration of 2 mM, wherein acetic acid is used as a catalyst for monosaccharide-DCCH derivatization reaction;

[0051] Accurately weigh 100.0 mg of 2, 5-dihydroxybenzoic acid (DHB) in a 10 mL cuvette, add a mixed solution of methanol / water (7:3, v / v) 10 mL, dissolve thoroughly, and prepare a MALDI matrix solution with a concentration of 10 mg / mL;

[0052] The specific chemical derivatization process of glucose is as follows: 2 μL of glucose stock solution is spotted on a metal target plate, and after it is naturally volatilized, 2 μL of DCCH derivatization reagent solution is spotted, and then the metal target plate coated with the reactant is incubated in an incubator, the incubation time is 5 h, and the incubation temperature is 50°C, and after the incubation is completed, 2 μL of DHB matrix solution is spotted; after the above operation is completed, the sample point is completely dried, and then MALDI-MSI analysis is performed.

[0053] The MALDI-MSI data are collected by a Shimadzu iMScope QT imaging mass spectrometer, which is equipped with a Nd:YAG laser with a wavelength of 355 nm. For the above-mentioned samples, the use parameters of the instrument are set as follows: the laser irradiation diameter is 0; the laser intensity is 45; the spatial resolution is 45×45 μm 2 ; the detector voltage is 1.98 kV; the sample voltage is 4.5 kV; the data acquisition range is m / z 150-500; the collision gas in the collision-induced dissociation process is argon; the optimal collision energy is adjusted in the interval of 15-30%; DHB is selected for internal mass calibration, the mass calibration range is m / z 50-1000, and MS / MS analysis is performed on the derivatization product of glucose, and the specific spectrum is shown in Figure 3 . The spectrum shows that the [M+Na] + addition ion peak of the glucose-DCCH derivatization product and its fragment ion peak (m / z 283.1048, 340.1261, 298.1177, 143.0322), wherein the characteristic fragment ion is m / z 340.1261 which contains the molecular structure of glucose and DCCH at the same time, clearly proving the molecular structure of the glucose-DCCH derivatization product.

[0054] II. Derivatization and MALDI mass spectrometric analysis of endogenous monosaccharides in pig liver tissue sections

[0055] The 7-(diethylamino)coumarin-3-formylhydrazine (DCCH) derivatization and MALDI mass spectrometric analysis process of endogenous monosaccharides in pig liver tissue sections is as follows:

[0056] (1) The pig liver tissue stored at -80°C is taken, and a continuous frozen section with a thickness of 20 μm is prepared by using a sectioning machine;

[0057] (2) The prepared pig liver tissue section is quickly transferred to an indium tin oxide (ITO) glass slide, and is dried in a vacuum at room temperature for 15 minutes;

[0058] (3) precisely weigh 27.5 mg of DCCH and 0.61 g of benzoic acid into a 10 mL cuvette, add 10 mL of acetonitrile solution, vortex well, and ultrasonic for 15 minutes to obtain a DCCH concentration of 10 mM and benzoic acid concentration of 0.5 M derivative reagent system solution;

[0059] (4) take two consecutive frozen sections of pig liver for standby, one of which is sprayed with 2 mL of DCCH derivative reagent system solution on the surface of the pig liver tissue section by using a spray gun in two times, and the other is not treated;

[0060] (5) place the pig liver tissue section sprayed with DCCH derivative reagent system solution in a constant temperature incubator, set the incubator temperature to 30℃, and set the incubation time to 2 h;

[0061] (6) after incubation, place the two tissue sections in Shimadzu iMlayer matrix sublimation instrument for DHB matrix coating by sublimation, set the heating temperature to 180℃, and set the matrix coating thickness to 1.5 μm;

[0062] (7) use Shimadzu iMScope QT imaging mass spectrometer to collect MALDI-MSI images, and the relevant parameters are the same as above;

[0063] (8) with the help of data processing software, target extraction of glucose and target ions of glucose after DCCH derivation are performed to in situ image the distribution characteristics of glucose in pig liver tissue sections, see Figure 4 .

[0064] It can be found that compared with the pig liver tissue section without DCCH derivation treatment, the mass spectrum signal of endogenous glucose in the pig liver tissue section treated by DCCH derivation is greatly enhanced (glucose: [M+Na] + 203.05, [M+K] + 219.03; glucose-DCCH: [M+Na] + 460.17, [M+K] + 476.14). After superimposing and analyzing the obtained MALDI-MSI images with the optical micrograph of the tissue, no obvious analyte delocalization phenomenon is observed, and accurate spatial resolution of the analyte at the micron scale can be achieved, see Figure 5 .

[0065] III. Derivatization and MALDI mass spectrometry analysis of endogenous monosaccharides in tumor pathological tissue sections

[0066] The 7-(diethylamino)coumarin-3-formylhydrazine (DCCH) derivatization and MALDI mass spectrometry analysis process of endogenous monosaccharides in tumor pathological tissue sections is as follows:

[0067] (1) Take the tumor pathological tissue stored at -80℃, and use a microtome to prepare continuous frozen sections with a thickness of 30 μm;

[0068] (2) The prepared tumor pathological tissue sections are quickly transferred to an indium tin oxide (ITO) glass slide and dried in a vacuum for 15 minutes at room temperature;

[0069] (3) 110.0 mg of DCCH and 1.54 g of 2, 5-dihydroxybenzoic acid are precisely weighed into a 10 mL cuvette, 10 mL of acetonitrile solution is added, vortexed, ultrasonicated for 15 minutes, to obtain a derivative reagent system solution with a concentration of 40 mM for DCCH and 1 M for 2, 5-dihydroxybenzoic acid;

[0070] (4) Two continuous frozen sections of tumor pathological tissue are taken for use, one of which is sprayed with 6 mL of DCCH derivative reagent system solution on the surface of the tumor pathological tissue section in 6 times using a spray gun or other spraying device, and the other is not treated;

[0071] (5) The tumor pathological tissue section sprayed with the DCCH derivative reagent system solution is placed in a constant temperature incubator, the incubator temperature is set to 30℃, and the incubation time is set to 2 h;

[0072] (6) After incubation, the two tumor pathological tissue sections are placed in a Shimadzu iMlayer matrix sublimation instrument for DHB matrix coating by sublimation, with a heating temperature of 180℃ and a matrix coating thickness of 1.5 μm;

[0073] (7) The iMScope QT imaging mass spectrometry microscope of Shimadzu is used to collect the MALDI-MSI image, and the relevant parameters remain the same as above;

[0074] (8) With the help of data processing software, the distribution characteristics of glucose in the tumor pathological tissue section are imaged in situ by targeting extraction of glucose and target ions after DCCH derivation of glucose, as shown in Figure 6 .

[0075] It can be found that the MALDI-MSI imaging image of endogenous glucose in the tumor pathological tissue section after DCCH derivation shows no obvious analyte delocalization phenomenon, and clear glucose metabolism differences are shown between cancer tissue, peritumoral tissue and paracancerous tissue, with good analysis effect.

Claims

1. A method of glucose mass spectrometry imaging based on in situ derivatization technology, characterized by, The method comprises the following steps: (1) preparing a sample slice with a thickness of 10-60 μm and attaching the sample slice to a conductive slide; (2) dissolving a derivatization reagent and a catalyst for a proton donor in a volatile organic solvent to prepare a derivatization reagent system solution, wherein the concentration of the derivatization reagent in the derivatization reagent system solution is 1-50 mM, and the concentration of the catalyst is 0.1-5 M; Then, the derivatization reagent system solution is sprayed on the surface of the tissue slice to perform a derivatization reaction on the compound containing a carbonyl group; (3) applying a MALDI matrix on the surface of the slice after the derivatization treatment, and then performing identification analysis by using a MALDI-MSI method; The derivatization reagent has the following structure: （Ⅰ） wherein R1-R2 are one of hydrogen, C1 alkyl, C2 alkyl and C3 alkyl; R3-R6 are one of hydrogen, C1 alkyl, C2 alkyl, C3 alkyl, hydroxyl, amino and halogen; and n=0. The catalyst comprises one of formic acid and acetic acid, or a combination of one of formic acid and acetic acid and one of benzoic acid, p-toluenesulfonic acid, alpha-cyano-4-hydroxycinnamic acid and 2, 5-dihydroxybenzoic acid as a proton donor.

2. The in situ derivatization-based method of glucose mass spectrometric imaging according to claim 1, wherein, The volatile organic solvent in step (2) is acetonitrile or methanol.

3. The in situ derivatization-based method for glucose mass spectrometric imaging according to claim 1, wherein, The derivatization reaction process in step (2) comprises naturally drying after the derivatization reagent system solution is sprayed on the surface of the tissue slice; or heating and incubating at 25-50°C in an air atmosphere for 0.5-4 h after the derivatization reagent system solution is sprayed on the surface of the tissue slice; or heating and incubating in an organic vapor atmosphere containing 10-50% volatile proton donor at 25-50°C for 0.5-4 h after the derivatization reagent system solution is sprayed on the surface of the tissue slice.

4. The in situ derivatization-based method of glucose mass spectrometric imaging according to claim 1, wherein, The MALDI matrix in step (3) is 2, 5-dihydroxybenzoic acid, 1, 5-naphthalene diamine or alpha-cyano-4-hydroxycinnamic acid.

5. The in situ derivatization-based method of glucose mass spectrometric imaging according to claim 1, wherein, In the identification analysis by using the MALDI-MSI method in step (3), an optical image is collected at a magnification of 40-1.25 or under a wide-angle lens; the m / z range is 100-1000, and the collision gas of the secondary mass spectrum is argon.

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