A MALDI reaction-type matrix for analyzing reducing sugars, 5-methoxy-2-hydroxybenzhydrazide

Abstract

The application relates to a MALDI reaction type matrix 5-methoxy-2-hydroxybenzhydrazide for analyzing reducing sugar, and belongs to the technical field of sugar biochemical analysis. The application provides the application of 5-methoxy-2-hydroxybenzhydrazide as a MALDI reaction type matrix in combination with 2,5-dihydroxybenzoic acid in the analysis of reducing sugar. The 5-methoxy-2-hydroxybenzhydrazide is a reaction type matrix and has a hydrazine group, so that the hydrazine group can react with the hemiacetal end of the reducing sugar. The 2,5-dihydroxybenzoic acid is a catalyst, which can significantly promote the efficient reaction of 5-methoxy-2-hydroxybenzhydrazide and reducing sugar. The 5-methoxy-2-hydroxybenzhydrazide / 2,5-dihydroxybenzoic acid composite matrix provided by the application can form a uniform co-crystal with the reducing sugar, has good signal reproducibility (RSD < 10%), and has good linearity in the range of 0.2-20 pmol / ul (R 2 >0.999).

Description

Technical Field

[0001] This application relates to the field of glycochemical analysis technology, and in particular to a MALDI reactive matrix 5-methoxy-2-hydroxybenzoyl hydrazide for analyzing reducing sugars. Background Technology

[0002] Reducing sugars are a class of carbohydrates with hemiacetal reducing ends, widely distributed in living organisms, including monosaccharides, oligosaccharides, polysaccharides, and those derived from glycoproteins. N - Polysaccharides, etc. Among them, N Glycans are widely involved in vital life activities, closely related to protein folding, cell adhesion, intercellular signal transduction, and recognition. Their highly complex structure and function endow them with a vast information capacity, which cannot currently be simply deduced from the genetic code. N Efficient analysis of polysaccharides is of great significance for glycobiology research.

[0003] Carbohydrates, including reducing sugars, are highly polar, lack chromophores, have low ionization efficiency, and exhibit strong structural heterogeneity, making direct mass spectrometry detection difficult and limiting their sensitivity. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), with its advantages of soft ionization, strong salt tolerance, small sample volume, and convenient detection, has become an important method for analyzing carbohydrates, including reducing sugars. In MALDI detection, a MALDI matrix is ​​typically used to assist in the ionization of the analyte. However, traditional organic matrices such as 2,5-dihydroxybenzoic acid (DHB) suffer from problems such as uneven co-crystallization, poor signal reproducibility, limited detection sensitivity, and unsuitability for small molecular weight ranges (MW < 700 Da), thus limiting the effective characterization and qualitative and quantitative analysis of reducing sugars using MALDI technology.

[0004] To improve detection efficiency, MALDI reactive matrices have been developed. These matrices combine the functions of a MALDI matrix with the ability to derivatize reducing sugars. They can react directly with reducing sugars on the MALDI target plate through a solvent drying process after spotting, introducing easily ionized tag compounds to assist in the ionization of reducing sugars, greatly simplifying the MALDI analysis procedure. Existing reactive matrices such as 3-aminoquinoline (3-AQ), 2,5-dihydroxybenzoylhydrazine (DHBH), and 2-hydrazinophthalic acid (2-HTA) have all improved the detection of reducing sugars by MALDI to some extent. However, the development of these reactive matrices currently relies heavily on trial and error, lacking systematic theoretical guidance, resulting in low screening efficiency and difficulty in applying the experience of successful cases to the design of other novel matrices.

[0005] In fact, the performance of MALDI reactive matrices is closely related to their intrinsic physicochemical properties. To achieve efficient on-target derivatization and assisted ionization, an ideal matrix must meet multiple conditions: first, it needs strong ultraviolet absorption at commonly used laser wavelengths (e.g., 355 nm) to effectively transfer energy; second, it needs good solubility to ensure uniform mixing with the sample; third, its acidity (pKa) needs to be suitable for catalyzing derivatization reactions; and finally, the charge strength at the terminal of reactive groups (e.g., hydrazide groups) directly affects its reactivity with reducing sugar hemiacetals. Therefore, if a set of rational design principles based on key physicochemical parameters can be established, and these parameters can be directionally controlled to guide the molecular design and screening of matrices, it is hoped that the limitations of current empirical trial-and-error methods can be overcome, significantly improving the development efficiency of high-performance reactive matrices. Summary of the Invention

[0006] In view of this, this application provides a MALDI-reactive matrix, 5-methoxy-2-hydroxybenzoyl hydrazine, for the analysis of reducing sugars. Based on a highly efficient target derivatization system constructed from a 5-methoxy-2-hydroxybenzoyl hydrazine / 2,5-dihydroxybenzoic acid composite matrix, it effectively solves the problems of harsh derivatization conditions, uneven crystallization, and insufficient quantitative accuracy in the analysis of reducing sugars using existing MALDI-reactive matrices. The structure of 5-methoxy-2-hydroxybenzoyl hydrazine simultaneously possesses a hydrazine reactive group and a phenolic hydroxyl group. The hydrazine group can undergo a nucleophilic addition reaction with the hemiacetal terminus of reducing sugars to generate a stable hydrazone, while the phenolic hydroxyl group enhances its UV absorption (355 nm) and proton transfer ability under MALDI conditions. Simultaneously, the introduction of the methoxy group further optimizes the electron distribution, improving the reactivity of the N atom at the hydrazine terminus and the matrix performance. 2,5-Dihydroxybenzoic acid, as an organic acid, when combined with 5-methoxy-2-hydroxybenzoyl hydrazine in a certain proportion, can significantly promote the reaction between the hydrazine group and the reducing sugar. At the same time, the two form an acid-base pair, which improves the uniformity of co-crystallization of the matrix and the reducing sugar, and enhances the reproducibility and quantitative accuracy of detection. This can effectively overcome the defects of the existing technologies.

[0007] The first aspect of this application provides the application of 5-methoxy-2-hydroxybenzoyl hydrazine as a MALDI reactive matrix in the analysis of reducing sugars.

[0008] A second aspect of this application also provides the application of 5-methoxy-2-hydroxybenzoyl hydrazine as a MALDI reactive matrix in combination with 2,5-dihydroxybenzoic acid in the analysis of reducing sugars. Specifically, 5-methoxy-2-hydroxybenzoyl hydrazine is the reactive matrix, possessing a hydrazine reactive group that can react with the hemiacetal terminus of reducing sugars. 2,5-Dihydroxybenzoic acid acts as a catalyst, significantly promoting the efficient reaction between 5-methoxy-2-hydroxybenzoyl hydrazine and reducing sugars.

[0009] Preferably, the 5-methoxy-2-hydroxybenzoylhydrazine is as shown in formula (1):

[0010] Equation (1).

[0011] Preferably, the molar ratio of 5-methoxy-2-hydroxybenzoylhydrazine to 2,5-dihydroxybenzoic acid is 1:0.25 to 1:4.

[0012] Preferably, the reducing sugar is a glycoside with a hemiacetal reducing end, and the glycoside with a hemiacetal reducing end is selected from monosaccharides, oligosaccharides, polysaccharides, or those released from glycoproteins. N - Polysaccharides. Specifically, the reducing sugars include, but are not limited to, glucose, maltose oligosaccharides, dextran, and those released from glycoproteins. N - Polysaccharides.

[0013] Preferably, the application in reducing sugar analysis is to perform derivatization qualitative and quantitative detection of reducing sugars, including the following steps:

[0014] (1) Prepare a 5-methoxy-2-hydroxybenzoyl hydrazine solution and a 2,5-dihydroxybenzoic acid solution, and mix them in equal volumes to obtain a composite matrix solution;

[0015] (2) Spot the reducing sugar solution to be tested and the composite matrix solution at the same position on the MALDI target plate, mix them evenly, and obtain the target plate after spotting;

[0016] (3) Place the spotted target plate at 45~65℃ for 20~40 minutes to carry out on-target derivatization reaction and dry it to obtain the dried sample;

[0017] (4) Perform MALDI-MS analysis on the dried sample.

[0018] Addressing the shortcomings of existing technologies, this application first systematically analyzes the core physicochemical parameters affecting the performance of MALDI reactive matrices, including UV absorption intensity at 355 nm, molar intrinsic solubility, pKa, and the negative charge strength of the N atom at the terminal of the hydrazide group. Based on this design framework, using DHB, a commonly used MALDI matrix for carbohydrates, as the parent matrix, different substituents were introduced to regulate the above parameters, and a compound with excellent comprehensive performance (5-methoxy-2-hydroxybenzoylhydrazide 5MeO2HBH) was screened and obtained. When combined with DHB, this compound can achieve efficient derivatization and highly sensitive detection of reducing sugars under mild conditions, providing a powerful tool for high-throughput, high-precision qualitative and quantitative analysis in glycobiology research, especially in plant glycomics. Furthermore, the design and screening strategy established in this application provides a clear theoretical basis and referable synthetic principles for the subsequent development of MALDI reactive matrices.

[0019] Preferably, in step (1), the concentration of the 5-methoxy-2-hydroxybenzoyl hydrazine solution is 0.05~0.20 mol / L, and the solvent of the 5-methoxy-2-hydroxybenzoyl hydrazine solution is methanol; the concentration of the 2,5-dihydroxybenzoic acid solution is 0.025~0.125 mol / L, and the solvent of the 2,5-dihydroxybenzoic acid solution is methanol.

[0020] Preferably, in step (3), the temperature of the derivatization reaction is 50°C and the time is 30 minutes.

[0021] Preferably, in step (4), the MALDI-MS analysis uses positive ion reflectance mode.

[0022] Specifically, the application in reducing sugar analysis involves the derivatization and qualitative and quantitative detection of reducing sugars, including the following steps:

[0023] 1. Reagent preparation: Prepare a 5-methoxy-2-hydroxybenzoyl hydrazine solution with a concentration of 0.05~0.20 mol / L using analytical grade or chromatographic grade methanol as the solvent; prepare a 2,5-dihydroxybenzoic acid solution with a concentration of 0.025~0.125 mol / L using analytical grade or chromatographic grade methanol as the solvent; mix the two solutions at a volume ratio of 1:1 to prepare a composite matrix solution, wherein the molar ratio of 5-methoxy-2-hydroxybenzoyl hydrazine to 2,5-dihydroxybenzoic acid in the composite matrix solution is 1:0.25~4, more preferably 1:4; prepare an aqueous solution of the reducing sugar to be tested using ultrapure water or double-distilled water as the solvent;

[0024] 2. Spotting: Take equal volumes of reducing sugar aqueous solution and composite matrix solution, and spot them sequentially onto the same spotting well of the MALDI polishing target plate. The spotting volume of the reducing sugar aqueous solution to be tested is 0.5~1.0 μL, and the spotting volume of the composite matrix solution is 0.5~1.0 μL. Repeat the pipetting 10~20 times to mix well, and obtain the target plate after spotting.

[0025] 3. Target plate derivatization reaction: The target plate after spotting is reacted at 45~65℃ for 20~40 min until the sample spot is completely dry. Preferably, the target plate after spotting is placed at 50℃ for 30 min.

[0026] 4. Mass spectrometry analysis: MALDI-TOF MS positive ion reflectance mode was used for detection.

[0027] A third aspect of this application also provides a kit for the MALDI-MS detection of reducing sugars, comprising the above-mentioned 5-methoxy-2-hydroxybenzoylhydrazine and the above-mentioned 2,5-dihydroxybenzoic acid.

[0028] Compared with the prior art, this application has the following advantages:

[0029] 1. Rational structural design: The rational design of 5-methoxy-2-hydroxybenzoyl hydrazide uses the 2,5-dihydroxybenzoic acid skeleton structure as a template, introduces methoxy groups to optimize photoelectric properties and reactivity, and introduces hydrazine groups to provide efficient derivatization ability;

[0030] 2. High reactivity: Under the catalysis of 2,5-dihydroxybenzoic acid, 5-methoxy-2-hydroxybenzoylhydrazine can achieve complete derivatization of reducing sugars within 30 min at 50℃ (derivatization efficiency > 99%), and the reaction conditions are mild;

[0031] 3. Wide applicability: The 5-methoxy-2-hydroxybenzoylhydrazine / 2,5-dihydroxybenzoic acid composite matrix has low background interference, making it suitable not only for the detection of macromolecules. N - Polysaccharides are also suitable for detecting small molecule oligosaccharides and even monosaccharides;

[0032] 4. Excellent quantitative performance: The 5-methoxy-2-hydroxybenzoylhydrazine / 2,5-dihydroxybenzoic acid composite matrix can form a uniform co-crystallization with reducing sugars, exhibiting good signal reproducibility (RSD < 10%) and good linearity in the range of 0.2–20 pmol / μL (R0). 2 > 0.999);

[0033] 5. Suitable for complex samples: The 5-methoxy-2-hydroxybenzoyl hydrazine / 2,5-dihydroxybenzoic acid composite matrix has been successfully applied to plant fruits. N - Highly sensitive and accurate qualitative and quantitative analysis of polysaccharides.

[0034] 6. This application not only provides an efficient tool for reducing sugar analysis, but more importantly, it establishes a rational design principle for MALDI reactive matrices based on key physicochemical parameters, providing theoretical guidance and screening basis for the development of new matrices. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the description of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 Physicochemical parameters of four hydroxybenzoyl hydrazine derivatives, including UV absorption intensity at 355 nm, molar intrinsic solubility, pKa, and negative charge intensity of the N atom at the hydrazine terminal group;

[0037] Figure 2Schematic diagram of the chemical synthesis of four hydroxybenzoylhydrazine derivatives;

[0038] Figure 3 5-Methoxy-2-hydroxybenzoylhydrazine 1 H NMR spectrum;

[0039] Figure 4 5-Methoxy-2-hydroxybenzoylhydrazine 13 C NMR spectrum;

[0040] Figure 5 MALDI mass spectra of reducing sugars detected using DHB and four hydroxybenzoyl hydrazine derivatives as matrices; Figure 5 (a) in the text is maltohexaose (G6); Figure 5 (b) in the text represents dextran 2000 (D2K);

[0041] Figure 6 Effects of different target plate derivatization reaction conditions on the derivatization yield and co-crystallization morphology of 5MeO2HBH / DHB and DHBH / DHB; Figure 6 In the figure, (a) represents the molar ratio of DHB to the reactive matrix; Figure 6 (b) in the figure represents the reaction temperature; Figure 6 (c) in the equation represents the reaction time. Figure 6 (d) in the figure represents the co-crystallization morphology of DHB and reactive matrix in different molar ratios; Figure 6 (e) in the figure is the MALDI-MSI diagram of the major ionic form of the G6 derivatization product;

[0042] Figure 7 MALDI mass spectra of dextran and oligosaccharides of different molecular weights were detected using a 5MeO2HBH / DHB composite matrix. Figure 7 (a) shows the detection of dextran D3K and D4K in DHB, 5MeO2HBH and 5MeO2HBH / DHB matrices; Figure 7 (b) shows the detection of oligosaccharides G1~G6 using a 5MeO2HBH / DHB matrix;

[0043] Figure 8 Comparison of the limits of detection (LOD) of the 5MeO2HBH / DHB composite matrix with DHB for detecting G7, NGA3, NA2 and Man-8 standards; MALDI mass spectra of detection using the DHB matrix ( Figure 8 (a) and (c) in the image); MALDI mass spectra detected using the 5MeO2HBH / DHB composite matrix ( Figure 8 (b) and (d) in the middle.

[0044] Figure 9Comparison of the limits of detection (LOD) of the 5MeO2HBH / DHB composite matrix with DHB for detecting G7, NGA3, NA2 and Man-8 standards; MALDI mass spectra of detection using the DHB matrix ( Figure 9 (e) and (g) in the image); MALDI mass spectra detected using the 5MeO2HBH / DHB composite matrix ( Figure 9 (f) and (h) in the middle.

[0045] Figure 10 Detection of 5MeO2HBH / DHB composite matrix N -Performance evaluation of polysaccharide standards; Figure 10 In the figure, (a) shows the normalized ionic strength and RSD value of G7 detected by DHB and 5MeO2HBH / DHB. Figure 10 (b) to Figure 10 (d) represents the detection of 5MeO2HBH / DHB. N - Quantitative standard curves of polysaccharide standards NGA3(b), NA2(c), and Man-8(d);

[0046] Figure 11 : Secondary fragmentation mode of G11 derived from 5MeO2HBH; Figure 11 (a) shows the MALDI secondary mass spectrum of G11 detected by DHB and 5MeO2HBH / DHB matrix; Figure 11 (b) in the figure represents the fragmentation pattern of 5MeO2HBH-labeled maltodextrin;

[0047] Figure 12 : N-glycan mass spectrum of wild-type tomato fruit during the color-breaking stage;

[0048] Figure 13 : N-glycan mass spectrum of mutant tomato fruit;

[0049] Figure 14 : Quantitative comparison of the content of 18 sugar types between mutants and wild types. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0052] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0053] The "MALDI-MS" mentioned in this application refers to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), abbreviated as MALDI-MS. The working principle of MALDI is to irradiate the co-crystallization formed by the sample and matrix with a laser. The matrix absorbs energy from the laser and transfers it to the sample molecules, causing them to ionize. MALDI is a soft ionization technique suitable for the determination of biomolecules.

[0054] The "reactive matrix" mentioned in this application refers to a conventional matrix that assists in the ionization of sugar chains while simultaneously reacting rapidly with their reducing end hemiacetals to covalently bind ionized tags and improve ionization efficiency.

[0055] The “target plate derivatization” described in this application refers to the simultaneous occurrence of sample spotting and derivatization reaction on the target plate, which greatly simplifies the operation steps and is compatible with the high-throughput characteristics of MALDI.

[0056] The derivatization efficiency described in this application is equal to the peak intensity of the derivatized product / (peak intensity of the derivatized product + peak intensity of the underrivatized product).

[0057] The matrix-assisted laser desorption / ionization time-of-flight mass spectrometer used in the following examples is a UlrafleXtreme™ MALDI-TOF / TOF MS (Bruker Daltonic, Germany), employing a 355 nm Nd:YAG laser. Detection was performed in positive ion reflectance mode. Positive ion reflectance mode parameters were: accelerating voltage, 25.00 kV; delayed extraction voltage, 22.30 kV; delayed extraction time, 130 ns; reflector voltage 1, 26.50 kV; reflector voltage 2, 13.50 kV; lens voltage, 7.50 kV; frequency, 1000 Hz. A 384 polished steel target was used, and mass spectrometry data analysis was performed using Bruker Flexanalysis 3.4 software.

[0058] The following abbreviations or foreign terms are used throughout this application:

[0059] 5MeO2HBH, 5-methoxy-2-hydroxybenzoylhydrazine;

[0060] ddH2O, deionized water;

[0061] DHB, 2,5-dihydroxybenzoic acid;

[0062] DHBH, 2,5-dihydroxybenzoyl hydrazide;

[0063] D 2K, dextran 2000;

[0064] D 3K, dextran 3000;

[0065] ESI, electrospray mass spectrometry;

[0066] G1, glucose;

[0067] G2, maltobiose;

[0068] G3, maltotriose;

[0069] G4, maltotetraose;

[0070] G5, maltopentose;

[0071] G6, maltohexaose;

[0072] G7, maltoheptaose;

[0073] HRMS, High Resolution Mass Spectrometry;

[0074] Hz, Hertz;

[0075] IS, internal standard;

[0076] LOD, limit of detection;

[0077] MALDI, matrix-assisted laser desorption / ionization;

[0078] MeOH, methanol;

[0079] Min, minute;

[0080] MP, melting point;

[0081] MR, molar ratio;

[0082] MS, mass spectrometry;

[0083] MW, molecular weight;

[0084] m / z, mass-to-charge ratio;

[0085] NMR, nuclear magnetic resonance;

[0086] PA, proton affinity;

[0087] PNGase A, N-glycosidase A;

[0088] RSD, Relative Standard Deviation;

[0089] Rt, room temperature;

[0090] S / N, signal-to-noise ratio;

[0091] SD, standard deviation;

[0092] Tis-HCl, tris(hydroxymethyl)aminomethane hydrochloride;

[0093] μL, microliter;

[0094] V, Volt;

[0095] ACE, acetone;

[0096] ACN, acetonitrile.

[0097] Example 1: Four hydroxybenzoyl hydrazine derivatives as N - Calculation and investigation of physicochemical parameters of polysaccharide MALDI reactive matrix

[0098] 1. Theoretical Calculation

[0099] Density functional theory (DFT) was used to optimize the ground-state geometry and calculate the negative charge distribution of the N-terminal N-atom of the hydrazide group at the B3LYP / 6-31G(d) level using the Gaussian 09 software suite. No restrictions were placed on bonds, bond angles, or dihedral angles in the calculations, allowing all atoms to move freely. A polarized continuum model (PCM) was employed to account for solvent effects. Natural layout analysis (NPA) and Malikian population analysis (MPA) were performed based on the optimized geometry.

[0100] 2. Ultraviolet absorption intensity detection

[0101] The ultraviolet absorption spectra were acquired using a Shimadzu UV-1800 dual-beam UV-Vis spectrophotometer. The instrument was first baseline-calibrated using MeOH as a blank, and then a matrix solution of the same concentration was prepared using MeOH as a solvent. The absorption spectra in the wavelength range of 250–450 nm were scanned and recorded.

[0102] 3. Molar intrinsic solubility and pKa

[0103] Using the SciFinder database, the molar intrinsic solubility and pKa of four hydroxybenzoyl hydrazine derivatives were calculated and statistically analyzed.

[0104] In Example 1, Figure 1 Physicochemical parameters of four hydroxybenzoyl hydrazine derivatives were analyzed. First, the UV absorption intensity at the MALDI laser wavelength of 355 nm was examined. DHBH showed the highest value, followed by 5MeO2HBH, which is beneficial for their better absorption and transfer of laser energy, promoting the desorption and ionization of sugar analytes. Conversely, 5Me2HBH showed the weakest UV absorption intensity at 355 nm, which is unfavorable for its use as a MALDI matrix for carbohydrate detection. Figure 1Secondly, regarding intrinsic solubility, consistent with our conjecture, both DHBH and 5MeO2HBH inherit the good solubility of DHB, which facilitates their better solubility in water. N - The polysaccharide analyte was well mixed and reacted without precipitation; while 5Br2HBH, possibly due to the induced electron-withdrawing effect of the bromine atom and steric hindrance, had the lowest solubility among the four matrices. Figure 1 pKa mainly reflects the ability of a compound to donate a proton. The reaction of 5MeO2HBH with reducing sugars is essentially a reaction of a weakly acid-catalyzed acylhydrazine group and a hemiacetal, in which DHBH and 5MeO2HBH exhibit relatively weak acidity. Figure 1 Furthermore, the negative charge intensity of the terminal N atom of the hydrazide group primarily affects the matrix and... N - The reactivity of polysaccharides, among which 5Me2HBH and 5MeO2HBH have the highest calculated negative charge strength, and therefore the highest theoretical reactivity ( Figure 1 In summary, among the four derivatives of 5MeO2HBH, 5MeO2HBH exhibits the best overall theoretical performance.

[0105] Example 2: Chemical Synthesis and Characterization of Four Hydroxybenzoylhydrazine Derivatives

[0106] 1. Synthesis

[0107] Weigh out 0.66 g of methyl 5-methylsalicylate (4 mmol), 0.92 g of methyl 5-bromosalicylate (4 mmol), 0.68 g of methyl 5-hydroxysalicylate (4 mmol), and 0.73 g of methyl 5-methoxysalicylate (4 mmol) and place them in four 50 mL round-bottom flasks. Add 20 mL of EtOH to each flask and stir to dissolve. Then add 668 μL of 85% hydrazine hydrate (12 mmol) to each flask. Stir at room temperature (25 °C) for more than 6 hours until most of the raw materials are converted into hydrazide products.

[0108] 2. Separation and purification

[0109] After each reaction was complete, the solution volume was concentrated to nearly half its original volume by rotary evaporation in a 45°C water bath, resulting in the precipitation of a significant amount of solid product. The solid product was filtered and washed thoroughly with large amounts of water and appropriate amounts of ethanol to remove excess hydrazine hydrate and residual raw materials as much as possible. It was then recrystallized with 50% MeOH to obtain the target product, which was continuously dried in a vacuum drying oven at 60°C for more than 6 hours to obtain four hydroxybenzoyl hydrazine derivatives, including 5-methyl-2-hydroxybenzoyl hydrazine (5Me2HBH), 5-bromo-2-hydroxybenzoyl hydrazine (5Br2HBH), 2,5-dihydroxybenzoyl hydrazine (DHBH), and 5MeO2HBH.

[0110] 3. Nuclear magnetic resonance characterization

[0111] Four synthesized hydroxybenzoyl hydrazine derivatives were characterized by NMR.

[0112] In Example 2, Figure 2 Schematic diagram of the chemical synthesis of four hydroxybenzoylhydrazine derivatives; Figure 3 It is 5-methoxy-2-hydroxybenzoylhydrazine 1 H NMR spectrum; Figure 4 It is 5-methoxy-2-hydroxybenzoylhydrazine 13 C NMR spectrum. Taking 5MeO2HBH as an example, its 1 H NMR and 13 The C NMR spectrum results are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.98(s, 1H), 10.04 (s, 1H), 7.36 (d, J = 3.0 Hz, 1H), 6.99 (dd, J = 9.0, 3.0 Hz,1H), 6.83 (d, J = 8.9 Hz, 1H), 4.63 (s, 2H), 3.71 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 167.64, 153.57, 151.47, 120.67, 118.13, 114.06, 110.44, 55.61. The spectral signals are clean and correspond one-to-one with their structural information, indicating that the separation and purification method in this embodiment can obtain the target product with high purity.

[0113] Example 3: Four hydroxybenzoyl hydrazine derivatives as N - Performance evaluation of polysaccharide MALDI reactive matrix

[0114] (1) Prepare 100 pmol / μL G6 sugar solution and 1 mg / mL D2K sugar solution respectively, and store them in a refrigerator at -20℃;

[0115] (2) Using methanol as solvent and the four hydroxybenzoyl hydrazine derivatives synthesized in Example 2 as matrix, prepare 0.1 mol / L matrix solutions and store them in a -20℃ refrigerator.

[0116] (3) Prepare a 0.1 mol / L DHB methanol solution and store it in a refrigerator at -20℃;

[0117] (4) Take 0.5 μL of G6 sugar solution or D2K sugar solution from step 1 and 0.5 μL of the four hydroxybenzoyl hydrazine derivative matrix solutions from step 2, and spot them sequentially onto the same well of a polished steel target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix thoroughly. Vacuum dry at 15°C;

[0118] (5) As a control, take 0.5 μL of G6 sugar solution or D2K sugar solution from step 1 and 0.5 μL of 0.1 mol / L DHB methanol solution from step 2, and spot them sequentially onto the same well of a polished steel target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix thoroughly. Vacuum dry at 15°C;

[0119] (6) The target plate is sent into the MALDI mass spectrometer, and data acquisition is performed using the positive ion reflectance mode;

[0120] In Example 3, Figure 5 MALDI mass spectra of reducing sugars detected using DHB and four hydroxybenzoyl hydrazine derivatives as matrices.

[0121] Figure 5 (a) shows the MALDI mass spectrum of maltohexasose (G6) detected under non-derivative conditions using DHB and four hydroxybenzoylhydrazine derivatives as matrices. 1013.317 Da corresponds to [G6 + Na]. + As can be seen from the peak, under the controlled low-temperature vacuum conditions of this embodiment, the four hydroxybenzoyl hydrazine derivatives do not react with the reducing ends of the sugar chains, which facilitates the examination of the characteristics of 5MeO2HBH itself as a matrix. Among the four hydroxybenzoyl hydrazine derivatives, 5Me2HBH has the lowest S / N ratio for detecting G6, while 5MeO2HBH has the best detection effect, followed by DHBH, and both are comparable to DHB in detection effect. Figure 5 (b) shows the detection of dextran 2000 (D2K) under non-derivative conditions using DHB and four hydroxybenzoyl hydrazine derivatives as matrices. 5Me2HBH and 5Br2HBH exhibit low detection sensitivity and are unsuitable as MALDI matrices for carbohydrate detection. In contrast, 5MeO2HBH and DHBH show better detection sensitivity than DHB, which is consistent with... Figure 1 Both 5MeO2HBH and DHBH exhibit high UV absorption intensities at 355 nm, consistent with each other, making them suitable as high-performance MALDI matrices for analyzing carbohydrates and subsequent derivatization reactions. It is evident that 5MeO2HBH and DHBH are significantly superior to DHB, a commonly used MALDI matrix for carbohydrates, and both possess good intrinsic molar solubility. Figure 1These two matrices are ideal for detecting carbohydrates. Subsequent examples will use these two matrices for further comparative analysis.

[0122] Example 4: 5MeO2HBH as N - Optimization of reaction conditions for polysaccharide MALDI reactive matrix

[0123] 1. Optimization of the molar ratio of DHB to 5MeO2HBH in the composite matrix

[0124] (1) Prepare a series of DHB solutions with concentrations of 0.01 mol / L, 0.025 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L in MeOH;

[0125] (2) Take the 0.1 mol / L 5MeO2HBH matrix solution from Example 3 and mix it with DHB solutions of different concentrations from step (1) in equal volumes to obtain a series of composite matrix solutions with molar ratios of DHB to 5MeO2HBH of 1:10, 1:4, 1:2, 1:1, 2:1, 3:1, 4:1 and 5:1.

[0126] (3) As a control, a DHBH solution with a concentration of 0.1 mol / L was prepared in MeOH and mixed with a series of DHB solutions of different concentrations in step (1) to obtain a series of composite matrix solutions with a molar ratio of DHB to DHBH of 1:10, 1:4, 1:2, 1:1, 2:1, 3:1, 4:1 and 5:1.

[0127] (4) Take 0.5 μL of the G6 sugar solution from Example 3 and 1 μL of the composite matrix solution from step (2) or step (3), and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix. React at 30°C for 30 min;

[0128] (5) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflectance mode;

[0129] In Example 4, Figure 6 (a) and Figure 6 (d) shows the effect of different molar ratios of DHB to reactive matrix on the derivatization efficiency and co-crystallization morphology of reducing sugar G6. It can be seen that when the molar ratios of DHB to DHBH and 5MeO2HBH are 3:1 and 1:4, respectively, G6 exhibits the highest derivatization efficiency and the best co-crystallization uniformity between the composite matrix and G6. Furthermore, under the same reaction conditions, the derivatization efficiency of 5MeO2HBH / DHB is significantly higher than that of DHBH / DHB.

[0130] 2. Optimization of reaction temperature for target derivatization

[0131] (1) Take the 0.1 mol / L 5MeO2HBH matrix solution from Example 3 and the 0.025 mol / L DHB solution from Step 1 and mix them evenly to obtain the 5MeO2HBH / DHB composite matrix solution;

[0132] (2) As a control, take an equal volume of 0.1 mol / L DHBH matrix solution from Example 3 and 0.3 mol / L DHB solution from Step 1 and mix them evenly to obtain DHBH / DHB composite matrix solution;

[0133] (3) Take 0.5 μL of G6 sugar solution from Example 3 and 1 μL of 5MeO2HBH / DHB and DHBH / DHB composite matrix solutions from step (1) or step (2), and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray 10 times on the MALDI target plate to mix. React at 35 ℃, 50 ℃, 65 ℃ and 80 ℃ for 25 min respectively;

[0134] (4) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0135] In Example 4, Figure 6 (b) shows the effect of reaction temperature on the derivatization efficiency of G6. With increasing reaction temperature, the derivatization yields of G6 for both composite matrices generally increased. However, it is clear that the derivatization efficiency of the 5MeO2HBH / DHB composite matrix is ​​higher than that of the DHBH / DHB composite matrix. The former can achieve a reaction yield of over 95% at a reaction temperature of 50℃, while the latter requires a reaction temperature of 80℃ to achieve the same level of reaction. This indicates that the reactivity of 5MeO2HBH is much higher than that of DHBH, which can be explained by their different negative charge intensity distributions in their structures. Figure 1 The calculated negative charge intensity of the terminal N atom of the acylhydrazine group in 5MeO2HBH is higher than that in DHBH. This may be because the methoxy group of the former has a stronger electron-donating ability through conjugation than the hydroxyl group of the latter. This conjugation effect can increase the electron cloud density of the acylhydrazine group, thereby improving the reactivity of the acylhydrazine group.

[0136] 3. Optimization of reaction time for target derivatization

[0137] (1) Take 0.5 μL of the G6 sugar solution from Example 3 and 1 μL of the 5MeO2HBH / DHB and DHBH / DHB composite matrix solutions from Step 2, and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix. React at 50 °C for 15, 30, 45 and 60 min, respectively;

[0138] (2) The target plate was sent into the MALDI mass spectrometer and data acquisition was performed using the positive ion reflectance mode;

[0139] In Example 4, Figure 6 (c) shows the effect of reaction time on G6 derivatization efficiency. It is evident that the 5MeO2HBH / DHB composite matrix achieves the highest derivatization efficiency and product signal-to-noise ratio at a reaction time of 30 min. In contrast, the DHBH / DHB composite matrix still fails to achieve complete G6 derivatization at 60 min. This demonstrates that 5MeO2HBH possesses excellent reactivity and is therefore suitable for biological samples. N The analysis of polysaccharides has advantages.

[0140] In Example 4, Figure 6 (e) shows the MALDI-MSI chromatograms of the main ionic forms of G6 derivatized products determined using different composite matrices. The sodium ion addition peak of DHBH-labeled G6 ([G6+DHBH+Na]+, 1163 Da) was collected in the DHBH / DHB composite matrix, while the sodium ion addition peak of 5MeO2HBH-labeled G6 ([G6+5MeO2HBH+Na]+, 1177 Da) was collected in the 5MeO2HBH / DHB composite matrix, both achieving complete derivatization through a thorough derivatization reaction. The ion signal of DHBH-labeled G6 is uniformly dispersed throughout the co-crystallization of the composite matrix, while the ion signal of 5MeO2HBH-labeled G6 is relatively concentrated in the center of the co-crystallization, exhibiting higher intensity, while the ion signal collected at the edges is very weak. This indicates that the 5MeO2HBH / DHB composite matrix has a certain concentration and enrichment effect on the analyte, which is beneficial for better analysis and detection of carbohydrates with relatively low concentrations.

[0141] Example 5: Qualitative performance evaluation of 5MeO2HBH / DHB composite matrix

[0142] Qualitative determination of dextran and oligosaccharides of different molecular weights using a 1.5MeO2HBH / DHB composite matrix.

[0143] (1) Prepare 40 pmol / μL solutions of G1, G2, G3, G4, G5, and G6 sugars, 1 mg / mL solutions of D3K sugars, and 1 mg / mL solutions of D4K sugars, and store them at -20°C.

[0144] (2) Take 0.5 μL of the sugar solutions G1, G2, G3, G4, G5, G6, D3K, and D4K from step (1) and 1 μL of the DHB, 5MeO2HBH, and 5MeO2HBH / DHB composite matrix solutions from Example 3 and Example 4, respectively, and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix thoroughly. React at 50°C for 30 min to ensure complete derivatization of the reducing sugars;

[0145] (3) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0146] In Example 5, Figure 7 MALDI mass spectra of dextran and oligosaccharides of different molecular weights were detected in a 5MeO2HBH / DHB composite matrix. Figure 7 (a) shows the MALDI-MS results for two dextran mixtures (D3K and D4K) with different degrees of polymerization. Compared with using DHB or 5MeO2HBH matrices alone, the composite matrix 5MeO2HBH / DHB significantly enhances the ionization efficiency of dextran due to target-plate reaction and derivatization, resulting in a significant increase in ion signal intensity and S / N ratio. Figure 7 (b) shows the MALDI-MS detection results for oligosaccharides G1, G2, G3, G4, G5, and G6. Matrix background is the main reason for interference and inhibition of MALDI-MS detection of small molecule oligosaccharides. The 5MeO2HBH / DHB composite matrix can efficiently derivatize and reduce oligosaccharides on the MALDI target plate, thereby producing an overall molecular weight shift, avoiding matrix background peaks, and facilitating the analysis of various small molecule oligosaccharides, including glucose. Therefore, the 5MeO2HBH / DHB composite matrix is ​​suitable for both dextran and small molecule oligosaccharides.

[0147] Qualitative detection of reducing properties of 2.5MeO2HBH / DHB composite matrix N - Polysaccharides

[0148] (1) Prepare standard sugar solutions of G7, NGA3, NA2 and Man-8 at 40 pmol / μL respectively, and then dilute them to a series of concentrations of 10 pmol / μL, 4 pmol / μL, 2 pmol / μL, 1 pmol / μL, 0.1 pmol / μL and 0.05 pmol / μL respectively to obtain sugar solutions of G7, NGA3, NA2 and Man-8 at gradient concentrations, and store them in a refrigerator at -20℃;

[0149] (2) Take 0.5 μL of the G7, NGA3, NA2 or Man-8 sugar solution from step (1) and 1 μL of the 5MeO2HBH / DHB composite matrix solution from Example 4, and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray the solution onto the MALDI target plate 10 times to mix thoroughly. React at 50℃ for 30 min to ensure complete reaction of the reducing sugars;

[0150] (3) As a control, take 0.5 μL of the G7, NGA3, NA2 or Man-8 sugar solution from step (1) and 0.5 μL of the 0.1 mol / L DHB methanol solution from Example 3, and spot them sequentially onto the same well of the target plate. Use a pipette to directly pipette the MALDI target plate 10 times to mix. Allow to air dry at room temperature for 30 min;

[0151] (4) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0152] In Example 5 Figure 8 and 9 The limits of detection (LODs) for G7, NGA3, NA2, and Man-8 standards were determined using 5MeO2HBH / DH and DHB as MALDI matrices, respectively. Figure 8 (a), (c) and Figure 9 In the diagram, (e) and (g) represent detection using the DHB matrix, while Figure 8 (b), (d) and Figure 9 In the figures (f) and (h), the detection was performed using the 5MeO2HBH / DHB composite matrix. Direct detection of [M+Na] using the DHB matrix was also performed. + Ion signals, detection and acquisition of derivatization of the 5MeO2HBH / DHB composite matrix [M+5MeO2HBH+Na] + Ion signals. Specific LOD results are summarized in Table 1. When using DHB as the matrix to detect G7, NGA3, NA2, and Man-8, the detection limits were all approximately 500 fmol, while the detection limit using the 5MeO2HBH / DHB composite matrix was as low as approximately 25 fmol. This demonstrates that the analytical method developed based on the 5MeO2HBH / DHB composite matrix is ​​effective for... N - Polysaccharides have higher detection sensitivity.

[0153] Table 1. Limits of Detection (LOD) for G7, NGA3, NA2, and Man-8 using DHB and 5MeO2HBH / DHB composite matrices.

[0154]

[0155] Example 6: Quantitative Performance Study of 5MeO2HBH / DHB Composite Matrix

[0156] (1) Prepare a G7 sugar solution with a concentration of 4 pmol / μL as an internal standard solution and store it in a refrigerator at -20℃;

[0157] (2) Take the standard sugar solutions of NGA3, NA2 and Man-8 with a concentration of 40 pmol / μL from Example 5, and dilute them to 10 pmol / μL, 4 pmol / μL, 1 pmol / μL and 0.4 pmol / μL respectively to obtain NGA3, NA2 and Man-8 sugar solutions of various gradient concentrations, and store them in a refrigerator at -20℃.

[0158] (3) Take 0.5 μL of standard sugar solutions of different concentrations of NGA3, NA2 or Man-8 from step (2), 0.5 μL of G7 internal standard solution from step (1), and 1 μL of DHB from Example 3 or MeO2HBH / DHB composite matrix solution from Example 4, and spot them sequentially onto the same well of the target plate. Use a pipette to directly aspirate and spray the MALDI target plate 10 times to mix, and react at 50°C for 30 min. Insert the target plate into the MALDI mass spectrometer, collect data in positive ion reflectance mode, and randomly sample 45 times from each well and take the average value.

[0159] In Example 6, Figure 10 Detection of 5MeO2HBH / DHB composite matrix N -Performance evaluation of glycan standards. Using G7 as the model analyte, [G7+Na] samples were collected. + Ions and [G7+5MeO2HBH +Na] + The signal intensity of the ions, with RSD values ​​of 84.08% and 8.11%, indicates that the optimized 5MeO2HBH / DHB composite matrix has good signal reproducibility. Figure 10 (a) in the middle. Figure 10 (b) to Figure 10 (d) represents the detection of 5MeO2HBH / DHB. N - Quantitative standard curves for polysaccharide standards NGA3(b), NA2(c), and Man-8(d). R values ​​of the three quantitative curves. 2 All values ​​are greater than 0.999, indicating that within this linear response range (200~20000 fmol), the derivatives based on the 5MeO2HBH / DHB composite matrix... N - The ratio of the ionic signal intensity of the glycan to the internal standard and N - The amount of polysaccharide showed a good linear positive correlation, which is conducive to its subsequent quantitative analysis in real samples. N - Polysaccharides laid the foundation for quantitative methodologies.

[0160] Example 7: Detection of secondary mass spectrometry fragmentation patterns of N-glycosides using a 5MeO2HBH / DHB composite matrix

[0161] (1) Take the 1 mg / mL D2K sugar solution in Example 3 as the test sample, and the 0.1 mol / L DHB methanol solution in Example 3 and the 5MeO2HBH / DHB solution in Example 4 as the MALDI matrix;

[0162] (2) Spot 0.5 μL of the D2K solution from step (1) and 0.5 μL of the 5MeO2HBH / DHB solution onto the same well on the target plate. Use a pipette to repeatedly aspirate and pipette the solution onto the MALDI target plate 10 times to mix it thoroughly. React at 50°C for 30 min.

[0163] (3) As a control, 0.5 μL of the D2K solution and 0.5 μL of the DHB solution from step (1) were sequentially spotted onto the same well of the target plate. The mixture was then directly pipetted onto the MALDI target plate 10 times to mix it, and reacted at 50°C for 30 min.

[0164] (4) The target plate is sent into the MALDI mass spectrometer and data acquisition is performed using the positive ion reflection mode.

[0165] In Example 7, Figure 11 To detect the secondary fragmentation mass spectrum of D2K using DHB and 5MeO2HBH / DHB respectively, G11 in D2K was selected as the precursor [G11+5MeO2HBH+Na]. + Ions were subjected to MALDI-MS 2 Fragmentation. The 5MeO2HBH-labeled G11 ion can break at both ends of the sugar chain to produce a series of specifically identifiable and classifiable B-type and Y-type ions with distinct mass differences, and can also generate a series of [unclear - possibly related to tag loss]. 0,2 Type A transcyclic fragmentation ions provide structural information on the abundance of glycosidic bond linkage types. Figure 11 (a)). However, the underivatively derivatized G11 ions, when directly fragmented in DHB, tend to break at the glycosidic bond, producing only single, indistinguishable B / Z and C / Y type ion fragments due to their identical molecular weight. Figure 11 (b)). In summary, derivatized and fragmented glycans based on the 5MeO2HBH / DHB composite matrix can generate more diverse and easily identifiable types of ionic fragments, which can be used to deduce clearer structural features of carbohydrates.

[0166] Example 8: 5MeO2HBH / DHB composite matrix for use in fruit of tomato type II α-mannosidase gene mutant N - Polysaccharide analysis

[0167] 1. Preparation of 5MeO2HBH / DHB composite matrix and internal standard

[0168] (1) The 5MeO2HBH / DHB solution from Example 4 was used as the MALDI matrix;

[0169] (2) Using 40 pmol / μL G7 from Example 5 as the stock solution, 20 pmol / μL G7 was prepared as the internal standard for the detection of N-glycan group in actual samples;

[0170] 2. Extraction of total protein from tomato fruit

[0171] Wild-type and type II α-mannosidase gene mutant fruits were used as materials. 1 g of tomato fruit samples from different genetic backgrounds were weighed and total protein was extracted by heating in a 65°C water bath for 10 minutes with three times the volume of buffer Y. After centrifugation at 12,000 rpm (20°C, 20 minutes), the supernatant was collected and cold phenol buffer (pH 7.5–7.9) was added. The mixture was shaken for 1 minute and centrifuged again at 12,000 rpm (4°C, 15 minutes), retaining the lower phenol phase and the middle protein layer. The mixture was washed twice with cold buffer Z. Finally, the precipitate was precipitated overnight at -20°C with five times the volume of 0.1 M ammonium acetate methanol solution. The precipitate was washed twice with MeOH and ACE, and then vacuum-dried to obtain protein particles, which were stored at -80°C. Buffer Y is composed of 1 M Tris-HCl (pH 8.2), 1% (v / v) β-mercaptoethanol, 10% (w / v) SDS, 0.1 M PMSF, 0.5 M EDTA, 0.5 M EGTA and 1× protease inhibitor, while buffer Z is 50 mM Tris HCl (pH 8.2).

[0172] 3. Tomato fruit N - Release and purification of glycans

[0173] Take 1 mg of protein from each of the total protein groups from step 2 and dissolve it in 0.01 M Tris-HCl buffer (pH 8.2). Denature the protein by heating at 100°C for 10 minutes. Then add 30 μg trypsin, 30 μg chymotrypsin, and 2 μL of 0.2 M CaCl2 solution and digest overnight at 37°C. Neutralize and acidify the solution to pH 4.0 with 0.1 M citrate-phosphate buffer. Then add 0.5 μL of PNGase A and incubate at 37°C for 24 hours. The released protein... NThe polysaccharide was collected and purified using a C18 solid-phase extraction column (500 mg packing material, 3 mL ddH2O as eluent) and a graphitized carbon solid-phase extraction column (250 mg packing material, 2 mL 50% ACN as eluent). The purified polysaccharide was then purified. N - The polysaccharide was dried by vacuum centrifugation and reconstituted in 25 μL of ddH2O, then stored at -20°C for later use.

[0174] 4. Target derivatization

[0175] Add 0.5 μL of G7 internal standard solution from step 1 and 0.5 μL of G7 internal standard solution from step 3. N - The polysaccharide sample solution and 1 μL of the 5MeO2HBH / DHB composite matrix solution from step 1 were sequentially spotted into the same well on a polished steel target plate. The mixture was then repeatedly pipetted 10 times onto the MALDI target plate to ensure thorough mixing. The reaction was carried out at 50°C for 30 minutes.

[0176] 5. MALDI-MS mass spectrometry analysis

[0177] The target plate was fed into the MALDI mass spectrometer, and data acquisition was performed using the positive ion reflectance mode.

[0178] In Example 8, Figure 12-14 Analysis of N-glycosides in the fruit of tomato type II α-mannosidase gene mutant using a 5MeO2HBH / DHB composite matrix. Figure 12 and Figure 13 These are the N-glycan mass spectra of wild-type and mutant tomato fruits during the color-breaking stage, respectively, with a total of 18 glycoform structures detected. Figure 14 This is a quantitative comparison chart of the content of 18 sugar types in mutants and wild type. Compared with wild type, mutant #24 showed a significant increase in the content of one oligomannose N-glycan, XylFucMan3GlcNAc2(H3N2F1X1), and a significant decrease in the content of three other N-glycans, namely oligomannose XylFucMan2GlcNAc2(H2N2F1X1), complex GlcNAcXylFucMan3GlcNAc2(H3N3F1X1), and complex GlcNAc2XylFucMan3GlcNAc2(H3N4F1X1). Since the conversion between the H3N2F1X1 and H2N2F1X1 glycoforms involves the hydrolysis of α-mannose residues, which is directly related to the enzyme function of α-Man, it is speculated that the loss of the type II α-mannosidase gene function in the mutant may cause the increase of substrate H3N2F1X1 and the decrease of product H2N2F1X1.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

Application of 1,5-methoxy-2-hydroxybenzoylhydrazine as a MALDI reactive matrix in combination with 2,5-dihydroxybenzoic acid in the analysis of reducing sugars.

2. The application according to claim 1, characterized in that, The 5-methoxy-2-hydroxybenzoylhydrazine is as shown in formula (1): Equation (1).

3. The application according to claim 1, characterized in that, The molar ratio of 5-methoxy-2-hydroxybenzoylhydrazine to 2,5-dihydroxybenzoic acid is 1:0.25 to 1:

4.

4. The application according to claim 1, characterized in that, The reducing sugar is a glycoside with a hemiacetal reducing terminus, and the glycoside with a hemiacetal reducing terminus is selected from monosaccharides, oligosaccharides, polysaccharides, or released from glycoproteins. N - Polysaccharides.

5. The application according to claim 1, characterized in that, The application in reducing sugar analysis involves the derivatization, qualitative and quantitative detection of reducing sugars, including the following steps: (1) Prepare a 5-methoxy-2-hydroxybenzoyl hydrazine solution and a 2,5-dihydroxybenzoic acid solution, and mix them in equal volumes to obtain a composite matrix solution; (2) Spot the reducing sugar solution to be tested and the composite matrix solution at the same position on the MALDI target plate, mix them evenly, and obtain the target plate after spotting; (3) Place the spotted target plate at 45~65℃ for 20~40 minutes to carry out on-target derivatization reaction and dry it to obtain the dried sample; (4) Perform MALDI-MS analysis on the dried sample.

6. The application according to claim 5, characterized in that, In step (1), the concentration of the 5-methoxy-2-hydroxybenzoyl hydrazine solution is 0.05~0.20 mol / L, and the solvent of the 5-methoxy-2-hydroxybenzoyl hydrazine solution is methanol; the concentration of the 2,5-dihydroxybenzoic acid solution is 0.025~0.125 mol / L, and the solvent of the 2,5-dihydroxybenzoic acid solution is methanol.

7. The application according to claim 5, characterized in that, In step (3), the derivatization reaction is carried out at a temperature of 50°C for 30 minutes.

8. The application according to claim 5, characterized in that, In step (4), the MALDI-MS analysis uses positive ion reflectance mode.

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