Application of 1-chloro-4-hydrazinophthalazine reactive matrix in in situ analysis of monosaccharides using MALDI-MSI

Abstract

The application discloses application of 1-chloro-4-hydrazinyl phthalazine as a reactive matrix in monosaccharide MALDI MSI in-situ analysis. The monosaccharide includes glucose, fructose, fucose and arabinose. The application develops 1-chloro-4-hydrazinyl phthalazine (CHP) as a reactive matrix, which is used for derivatization of monosaccharide in biological tissues and in-situ differentiation of monosaccharide isomers. Taking carrot as a model, monosaccharide on the carrot tissue is derivatized, and a MALDI-MSI experiment is carried out. It is worth noting that characteristic fragments generated by secondary mass spectrometry of the derivatization product can be used for further distinguishing aldoses and ketoses, thereby differentiating monosaccharide isomers and revealing different spatial distributions of monosaccharide isomers in organisms.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry detection technology, specifically involving the use of 1-chloro-4-hydrazinophthalazine as a reactive matrix for in-situ analysis of monosaccharides in the field of MALDI MSI detection. Background Technology

[0002] Matrix-assisted laser desorption / ionization (MALDI) mass spectrometry (MSI) is a powerful analytical technique that can be used for the visualization of numerous components and to reveal the spatial distribution of endogenous biomolecules and exogenous drug molecules in tissues, thus providing important information for research on disease mechanisms. In this technique, the matrix determines the sensitivity of analyte detection and is therefore crucial. Consequently, researchers have been continuously dedicated to developing novel matrices to improve the analytical performance of MALDI MSI.

[0003] Monosaccharides are a class of simple carbohydrate molecules found in living organisms, possessing a variety of biological functions and roles, including: 1. serving as an energy source for cellular metabolism; 2. acting as important structural components within organisms; 3. participating in intercellular signal transduction; and 4. playing a crucial role in disease progression. The distribution and function of monosaccharides vary across different regions of biological tissues. Therefore, visualizing the spatial distribution of monosaccharides in biological tissues is meaningful and can be used to reveal their roles and functional mechanisms in biological processes. For example, glucose and fructose are both hexoses with the same molecular formula (C6H2O). 12 While both glucose and fructose are monosaccharides (O6), their molecular structures differ; glucose is an aldose, while fructose is a ketose. Therefore, their chemical properties and biological functions are also quite different. In-situ detection of monosaccharides in organisms can help us understand the metabolism and function of sugars within the body, thus providing a deeper understanding of related physiological functions.

[0004] Currently, the most commonly used method for analyzing monosaccharides using mass spectrometry is liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). For example, monosaccharides can be derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP), converting them into derivatized products with high mass spectrometric signal responses, thereby improving the detection sensitivity and selectivity of LC-ESI MS. However, this technique cannot provide in-situ information. Based on MALDI MSI analysis technology, researchers have developed sugar derivatization matrices such as Girard's reagent T and P, which use hydrazine groups as reactive groups to react with aldehyde or carbonyl groups in sugars for derivatization, introducing charge centers to improve ionization efficiency and enabling in-situ analysis of monosaccharides. However, in these analyses, Girard's reagents themselves do not have a matrix effect, requiring the introduction of an additional matrix, which may complicate the mass spectrum. Hirofumi et al. applied MALDI MSI technology to strawberry analysis, revealing the distribution of different substances in various parts of the fruit. The experimental results showed that hexoses (such as glucose and fructose) were distributed throughout the strawberry slice. In this work, DHB was used as the matrix. Since glucose and fructose have the same molecular weight and do not have characteristic fragment ions, they cannot be identified separately by MALDI MSI, and in situ detection of monosaccharide isomers remains difficult. Summary of the Invention

[0005] The object of this invention is to provide new uses for 1-chloro-4-hydrazinyl-Phthalazine (CHP).

[0006] The novel application of 1-chloro-4-hydrazinyl-Phthalazine (CHP) provided by this invention is its use as a reactive matrix in matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) in-situ analysis of monosaccharides.

[0007] In the application, the monosaccharides include glucose, fructose, fucose, and arabinose.

[0008] This invention also provides a monosaccharide matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) in-situ analysis method.

[0009] The monosaccharide matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) in-situ analysis method provided by this invention includes the following steps:

[0010] 1) Prepare a reactive matrix solution of 1-chloro-4-hydrazinophthalazine;

[0011] 2) The obtained reactive matrix 1-chloro-4-hydrazidophthalazine solution was sprayed onto the biological sample slices, vacuum dried, and then MALDI-MSI detection was performed to obtain the MALDI imaging results of the biological sample slices. Based on the mass spectrum imaging results of the MALDI-MS / MS of the CHP-derived monosaccharide standard, the monosaccharide distribution composition of the biological sample slices was determined in situ and each monosaccharide isomer was distinguished in situ, revealing the different spatial distributions of monosaccharides and monosaccharide isomers in the organism.

[0012] In step 1) of the above method, the 1-chloro-4-hydrazidophthalazine is dissolved in an acetonitrile / acetic acid (7:3, v / v) solvent at a concentration of 4 mg / ml to obtain a reactive matrix solution;

[0013] In step 2), the biological sample slices may specifically be at least one of carrot slices, codonopsis slices, licorice slices, hawthorn slices, jujube slices, strawberry slices, and apple slices.

[0014] The spraying was performed using a matrix sprayer on biological sample slices. The parameters of the matrix sprayer were as follows: spraying mode CC (cross-flow), nozzle flow rate 50 ml / min, glass plate temperature 45℃, nozzle temperature 25℃, nozzle pressure 0.15 MPa, nozzle speed 1500 mm / min, and spraying voltage 5000 V.

[0015] MALDI mass spectrometry imaging was performed in positive ion mode, with a mass detection range of m / z 50-1000 and a resolution of 200 μm. The imaging data of each pixel was obtained by laser scanning 200 times with a frequency of 2000 Hz.

[0016] 1-Chloro-4-hydrazinophthalazine and monosaccharide standards were prepared into solutions, mixed, reacted, and the resulting mixture was transferred onto a MALDI target, dried, and detected by MALDI-MS and MALDI-MS / MS to obtain the MALDI-MS and MALDI-MS / MS mass spectra of the CHP-derived monosaccharide standards.

[0017] In MALDI MSI experiments, the reactive matrix 1-chloro-4-hydrazinophthalazine serves not only as the matrix for MALDI but also as a derivatization reagent for the analyte. It reacts with the functional groups of the analyte, resulting in derivatized products with higher ionization efficiency. Using a reactive matrix simplifies the mass spectrometry imaging workflow, eliminating the need for additional matrix spraying and solving the problem of cumbersome sample pretreatment. The advantages are as follows: (1) It reduces the dispersion and loss of molecules in the tissue caused by multiple sprayings. (2) Multiple sprayings may result in thick crystals on the slices, making it impossible to obtain the mass spectrometry signal from the slices. The reactive matrix can reduce this situation. (3) The interaction between the derivatization reagent and the matrix can produce complex mass spectra that are difficult to interpret. The mass spectra of derivatizations using a reactive matrix are often cleaner and easier to analyze.

[0018] In summary, 1-chloro-4-hydrazinophthalazine facilitates the derivatization of sugars, improves detection sensitivity, omits the need for further matrix coating, simplifies sample pretreatment, produces clean mass spectra, and can distinguish between glucose and fructose. Undoubtedly, this work will greatly enhance the performance of MALDI MSI in analyzing monosaccharides.

[0019] This invention develops 1-chloro-4-hydrazinyl-Phthalazine (CHP) as a reactive matrix for the derivatization of monosaccharides in biological tissues and the in-situ differentiation of monosaccharide isomers. Using carrots as a model, monosaccharides on carrot tissues were derivatized, and MALDI-MSI experiments were performed. Notably, the characteristic fragments generated by secondary mass spectrometry of the derivatization products can be further used to distinguish aldoses from ketoses, thereby differentiating monosaccharide isomers and revealing the different spatial distributions of monosaccharide isomers in organisms. Attached Figure Description

[0020] Figure 1 The image shows the MALDI-MS mass spectrum of the CHP-derived monosaccharide standard in Example 1 of this invention (CHP itself as the matrix), in positive ion mode without the addition of other matrices. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0021] Figure 2 The image shows the MALDI-MS mass spectrum of a Girard reagent T-derived monosaccharide standard in positive ion mode with DHB (2,5-dihydroxybenzoic acid) as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0022] Figure 3The image shows the MALDI-MS mass spectrum of a monosaccharide standard derivatized with Girard's reagent T, in positive ion mode without the addition of any other matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose

[0023] Figure 4 MALDI-MS mass spectrum of underivative monosaccharide standards, in positive ion mode, with DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0024] Figure 5 The reaction mechanism and MS / MS fragmentation mechanism of CHP chemically derivatized monosaccharide standards.

[0025] Figure 6 CHP-derived monosaccharide standard [M+Na] + The MALDI-MS / MS mass spectrum shows that the substance itself acts as the matrix in positive ion mode. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose

[0026] Figure 7 Girard's reagent T-derived monosaccharides [M] + The MALDI-MS / MS mass spectrum, in positive ion mode, shows DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0027] Figure 8 It is a monosaccharide [M+Na] + The MALDI-MS / MS mass spectrum, in positive ion mode, uses DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0028] Figure 9 The mass spectrum of CHP-derived carrot slices obtained by MALDI MS in situ detection.

[0029] Figure 10 MALDI-MS / MS mass spectra of carrot slices derived in situ from CHP.

[0030] Figure 11 MALDI imaging results of CHP-derived carrot slices. (A) Histological structure of the carrot slice. (B) Photograph of the carrot slice. (C) Ion image of glucose diagnostic ion m / z 143. (D) Ion image of fructose diagnostic ion m / z 110. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] The mass spectrometer used in the following examples was a MALDI-TOF-MS (Bruker Ultraflex, Bremen, Germany), equipped with a solid-state Nd:YAG / 355nm SmartBeam laser as the ion source. MALDI-MS experiments were performed in positive ion mode, and data were recorded and processed using FlexAnalysis and FlexImaging software (Bruker Daltonics).

[0034] Girard reagent T and DHB were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0035] Example 1

[0036] The reactive matrix 1-chloro-4-hydrazidophthalazine and the monosaccharide standard were dissolved in acetonitrile / acetic acid (7:3, v / v) at concentrations of 10 mM and 5 mM, respectively. 5 μL of the reactive matrix solution was mixed with 5 μL of the monosaccharide solution, vortexed, and reacted for 3 min. 1 μL of the mixture was then transferred to a MALDI target (MTP 384 target plate ground steel, Bruke, Bremen, Germany), allowed to air dry, and used for MALDI-MS and MALDI-MS / MS experiments.

[0037] Figure 1 The MALDI-MS mass spectrum of a CHP-derived monosaccharide standard (CHP itself as the matrix) is shown in positive ion mode without the addition of any other matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose

[0038] As shown in the figure, CHP can derivatize monosaccharides, improving the sensitivity of monosaccharide detection, and no additional matrix is ​​required, resulting in a clean spectrum that is easy to interpret.

[0039] The derivatization reagent Girard's reagent T and the monosaccharide standard were dissolved in acetonitrile / acetic acid (7:3, v / v) at concentrations of 10 mM and 5 mM, respectively. The MALDI matrix DHB (2,5-dihydroxybenzoic acid) was dissolved in acetonitrile / water (7:3, v / v) at a concentration of 25 mg / mL. 5 μL of Girard's reagent T solution was mixed with 5 μL of the monosaccharide solution, vortexed, and reacted for 3 min. 1 μL of the mixture was transferred to a MALDI target, allowed to air dry, and then 1 μL of the DHB matrix solution was added to the crystals of the analyte. After drying, the crystals were used for MALDI-MS and MALDI-MS / MS experiments.

[0040] Figure 2 The image shows the MALDI-MS mass spectrum of a monosaccharide standard derivatized with Girard's reagent T, in positive ion mode with DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0041] The derivatization reagent Girard's reagent T and the monosaccharide standard were dissolved in acetonitrile / acetic acid (7:3, v / v) at concentrations of 10 mM and 5 mM, respectively. The MALDI matrix DHB was dissolved in acetonitrile / water (7:3, v / v) at a concentration of 25 mg / mL. 5 μL of Girard's reagent T solution was mixed with 5 μL of monosaccharide solution, vortexed, and reacted for 3 min. 1 μL of the mixture was transferred to a MALDI target, allowed to air dry, and then used for MALDI-MS experiments.

[0042] Figure 3 The image shows the MALDI-MS mass spectrum of a monosaccharide standard derivatized with Girard's reagent T, in positive ion mode without the addition of any other matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose

[0043] Monosaccharide standards were dissolved at a concentration of 5 mM in acetonitrile / acetic acid (7:3, v / v), and MALDI matrix DHB was dissolved at a concentration of 25 mg / mL in acetonitrile / water (7:3, v / v). 5 μL of DHB solution was mixed with 5 μL of monosaccharide solution, vortexed, and 1 μL of the mixture was transferred to a MALDI target and allowed to air dry. After drying, the mixture was used for MALDI-MS and MALDI-MS / MS experiments.

[0044] Figure 4 MALDI-MS mass spectrum of underivative monosaccharide standards, in positive ion mode, with DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0045] Example 2

[0046] MS / MS experiments were performed on the obtained derivatized products using LIFT technology on MALDI-TOF-MS. MALDI MS / MS experiments were conducted in positive ion mode, with a precursor ion detection range of m / z 50–1000 and an isolation window set to ±2 Da for the precursor. MS / MS mass spectra were obtained by laser ablation at 2000 Hz for 5000 cycles, and the imaging data were recorded and processed using FlexAnalysis software (BrukerDaltonics).

[0047] Figure 6 CHP-derived monosaccharide standard [M+Na] + The MALDI-MS / MS mass spectrum shows that the substance itself acts as the matrix in positive ion mode. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose

[0048] Figure 7 Girard's reagent T-derived monosaccharides [M] + The MALDI-MS / MS mass spectrum, in positive ion mode, shows DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0049] Figure 8 It is a monosaccharide [M+Na] + The MALDI-MS / MS mass spectrum, in positive ion mode, uses DHB as the matrix. (A) Glucose (B) Fructose (C) Fucose (D) Arabinose.

[0050] Example 3

[0051] MALDI mass spectrometry imaging experiments were performed using carrot slices as a model. The reactive matrix 1-chloro-4-hydrazidophthalazine was dissolved at a concentration of 4 mg / ml in acetonitrile / acetic acid (7:3, v / v). The prepared matrix solution was sonicated for 15 min to remove dissolved air from the matrix solution.

[0052] Reactive matrix was sprayed onto carrot slices using a VIKTOR (Beijing, CN) matrix sprayer. After spraying, the slices were dried in a vacuum desiccator for 30 min before being used for MALDI-MSI experiments. The matrix sprayer parameters were: spraying mode CC (cross-flow), nozzle flow rate 50 ml / min, slide temperature 45℃, nozzle temperature 25℃, nozzle pressure 0.15 MPa, nozzle speed 1500 mm / min, and spraying voltage 5000 V. MALDI mass spectrometry imaging was performed in positive ion mode, with a precursor ion mass detection range of m / z 50-1000 and a resolution of 200 μm. Imaging data for each pixel was obtained by laser scanning 200 times at a frequency of 2000 Hz. FlexImaging (Bruker Daltonics) was used to record and process the imaging data. A mass window of ±1.0 Da was selected to generate the imaging results, and the TIC method was used for normalization.

[0053] Figure 9 This image shows the in-situ MALDI MS detection results of CHP-derived carrot slices. Derivatization can be completed simply by spraying the reactive matrix directly onto the biological tissue sample, eliminating the need for additional matrix spraying, which simplifies the tedious sample processing procedure. Furthermore, the resulting mass spectra are clean, facilitating analysis by researchers.

[0054] Figure 10 MALDI-MS and MALDI-MS / MS mass spectra of CHP-derived carrot slices in positive ion mode.

[0055] Figure 11 MALDI imaging results of CHP-derived carrot slices. (A) Histological structure of the carrot slice. (B) Photograph of the carrot slice. (C) Ion image of glucose diagnostic ion m / z 143. (D) Ion image of fructose diagnostic ion m / z 110.

[0056] Depend on Figure 11 It is known that using 1-chloro-4-hydrazidophthalazine of the present invention as a reactive matrix for MALDI-MS detection can distinguish monosaccharide isomers in carrot slices, thereby revealing the spatial distribution of monosaccharide isomers in carrots.

[0057] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MSI) in-situ analysis method for monosaccharides, comprising the following steps: 1) Prepare a reactive matrix solution of 1-chloro-4-hydrazinophthalazine; 2) The obtained reactive matrix 1-chloro-4-hydrazidophthalazine solution was sprayed onto the biological sample slices, vacuum dried, and then MALDI-MSI detection was performed to obtain the MALDI imaging results of the biological sample slices. Based on the mass spectrometry imaging results of MALDI-MS / MS, the monosaccharide distribution of the biological sample slices was determined in situ and each monosaccharide isomer was distinguished in situ, revealing the different spatial distributions of monosaccharides and monosaccharide isomers in the organism. The monosaccharides include glucose, fructose, fucose, and arabinose.

2. The method of claim 1, wherein: In step 1), the 1-chloro-4-hydrazidophthalazine is dissolved in an acetonitrile / acetic acid solvent at a concentration of 4 mg / ml to obtain a reactive matrix solution.

3. The method of claim 1, wherein: In step 2), the biological sample slices are at least one of carrot slices, codonopsis slices, licorice slices, hawthorn slices, jujube slices, strawberry slices, and apple slices.

4. The method according to claim 1, characterized in that: The coating was applied to the biological sample slides using a matrix sprayer. The parameters of the matrix sprayer were: spraying mode was cross-flow CC, nozzle flow rate was 50 ml / min, and glass tray temperature was 45°C. C, Nozzle temperature is 25 C, nozzle pressure is 0.15 MPa, nozzle speed is 1500 mm / min, and spraying voltage is 5000 V.

5. The method according to claim 1, characterized in that: MALDI mass spectrometry imaging was performed in positive ion mode, with a mass detection range of m / z 50-1000 and a resolution of 200 μm. The imaging data for each pixel was obtained by laser scanning 200 times with a frequency of 2000 Hz.

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