Application of 2,4-dicarboxyphenylhydrazine as a reactive matrix for mass calibration and N-glycan analysis in negative ion mode of MALDI mass spectrometry

Abstract

The present invention discloses an application of a reactive matrix 2,4-dicarboxyphenylhydrazine in mass calibration and N-glycan analysis in the negative ion mode of a MALDI mass spectrometer. 2,4-dicarboxyphenylhydrazine reacts with the hemiacetal at the end of a reducing sugar to form a hydrazone. The reaction has high derivatization efficiency and utilizes a target plate for online derivatization, which is simple to operate. Utilizing this derivatization strategy, derivatized products can be detected in the negative ion mode. The carboxylate groups in 2,4-dicarboxyphenylhydrazine significantly improve the ionization efficiency of reducing sugars in the negative ion mode. The present invention uses dextran derivatized with 2,4-dicarboxyphenylhydrazine as a mass calibrant for a MALDI mass spectrometer. The mass difference between adjacent signal peaks is 162Da, corresponding to one glucose residue, thereby achieving precise calibration in the negative ion mode simultaneously. In addition, the DCPH / DHB composite matrix can significantly improve the cocrystallization uniformity with the analyte and improve the accuracy of quantitative detection of N-glycans.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry detection, and in particular to the application of 2,4-dicarboxyphenylhydrazine (DCPH) as a reactive matrix in mass calibration and N-glycan analysis in a MALDI (matrix-assisted laser desorption ionization) mass spectrometry negative ion mode. Background Art

[0002] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is widely used to determine the mass of macromolecules such as proteins, polysaccharides, and polymers. Accurate mass-to-charge ratio calibration is a prerequisite for mass spectrometry analysis, so choosing the right mass calibrant is crucial.

[0003] Compounds used as mass calibrants should have the following characteristics: (1) precise spectral characteristics and accurate molecular weight; (2) abundant spectral peaks within the calibration range and appropriate difference between adjacent mass spectral peaks; (3) easy to prepare and inexpensive; (4) easy to store and have a long shelf life; (5) high purity. Currently, substances commonly used as calibrants include proteins and peptide mixtures, but the separation and purification costs of such calibrants are high, they are sensitive to the environment, and have high requirements for storage conditions; metal clusters, such as [Cs n I n-1 ] + and [Cs n-1 I n ] - Cluster compounds can be used for MALDI-MS calibration in both positive and negative ion modes, but the peak intensity decreases significantly with increasing molecular weight. Polymers such as polyethylene glycol and poly(ethylene glycol) have been used by T. Gruending et al. to calibrate MALDI mass spectrometry mass numbers using polyalanine, which allows calibration within the m / z range of 1000 to 3500. However, calibration for positive and negative ions requires the use of DHB and CHCA matrices, respectively, which is cumbersome. Therefore, the design and development of novel MALDI mass spectrometry calibrants is of great significance. Summary of the Invention

[0004] The present invention provides 2,4-dicarboxyphenylhydrazine (DCPH) as a reactive matrix and Dextran 3000 (D3K) as a reducing dextran, and the polymer is derivatized through a hydrazone reaction. The reaction has high derivatization efficiency and can obtain a strong deprotonation signal of the derivatized product in negative ion mode, which can be used for mass calibration of MALDI mass spectrometry. At the same time, secondary fragmentation of the hydrazone product can obtain rich fragment information, which is then used for mass calibration of secondary mass spectrometry. The mixed use of the acidic matrix DHB and DCPH can significantly improve the derivatization efficiency and detection sensitivity of carbohydrate compounds. Among them, DHB is a catalyst and DCPH is a reactive matrix. DHB and 3-HBA are both excellent matrices for carbohydrates. Therefore, excess derivatization reagents do not need to be separated, simplifying the operation steps. In addition, DCPH itself has strong crystal uniformity. When used in combination with DHB, it can still maintain good co-crystallization uniformity with the analyte, thereby improving the reproducibility and accuracy of quantitative analysis. The structural formula of DCPH described in the present invention is shown in formula (1):

[0005]

[0006] Application of 2,4-dicarboxyphenylhydrazine (DCPH) as a reactive matrix for mass calibration and N-glycan analysis in negative ion mode of MALDI (matrix-assisted laser desorption ionization) mass spectrometry.

[0007] In the present invention, 2,4-dicarboxyphenylhydrazine reacts with the hemiacetal at the end of a reducing sugar to form a hydrazone. This reaction has high derivatization efficiency and utilizes a target plate for online derivatization, making it easy to operate. Using this derivatization strategy, the derivatized product can be detected in negative ion mode. Furthermore, the carboxylate groups in 2,4-dicarboxyphenylhydrazine significantly increase the ionization efficiency of reducing sugars in negative ion mode. The present invention uses dextran derivatized with 2,4-dicarboxyphenylhydrazine as a mass calibrant for MALDI mass spectrometry. The mass difference between adjacent signal peaks is 162 Da, corresponding to a single glucose residue, enabling precise calibration in negative ion mode. Furthermore, the DCPH / DHB composite matrix can significantly improve the uniformity of cocrystallization with the analyte, enhancing the accuracy of quantitative detection of N-glycans.

[0008] The application of DCPH of the present invention as a reactive matrix in mass calibration and N-glycan analysis in the negative ion mode of MALDI mass spectrometry comprises the following specific steps:

[0009] (1) Reagent preparation

[0010] A DHB (2,5-dihydroxybenzoic acid) methanol solution was prepared, and DCPH (2,4-dicarboxyphenylhydrazine) was dissolved in an equal volume of a mixed solvent of methanol and dimethyl sulfoxide to obtain a DCPH solution. Then, equal volumes of the DHB methanol solution and the DCPH solution were mixed to obtain a DCPH / DHB composite matrix solution. Dextran (D3K) was dissolved in water to obtain a dextran aqueous solution.

[0011] (2) Sample

[0012] Take equal volumes of dextran aqueous solution and DCPH / DHB composite matrix solution, spot them on the same point of the target plate of the MALDI-MS instrument, and mix them by pipetting directly on the target plate to obtain the spotted target plate;

[0013] (3) Target plate derivatization reaction

[0014] The target plate after spotting is reacted at 45-65°C for 20-40 minutes, and the sample spot is completely dried to obtain a target plate containing a mass calibrant;

[0015] (4) Mass spectrometry calibration

[0016] The target plate containing the mass calibrant is sent into the MALDI-MS mass spectrometer, data is collected in negative ion mode and mass calibration is performed. The calibration parameters obtained after the instrument calibration will be automatically used to calibrate the mass spectrometry detection results of the actual sample.

[0017] In step (1), the methanol used to dissolve the matrix DHB (2,5-dihydroxybenzoic acid) is an analytical grade or chromatographic grade solvent;

[0018] The concentration of the DCPH solution is 0.02-0.10 mol / L, more preferably 0.04 mol / L;

[0019] The concentration of the DHB solution is 0.025-0.125 mol / L, more preferably 0.1 mol / L;

[0020] The water used to dissolve dextran is ultrapure water or double-distilled water;

[0021] The dextran is Dextran 3000 (D3K), which is a reducing dextran. The concentration of the dextran in the dextran aqueous solution is 0.5 to 2 mg / mL, more preferably 1 mg / mL.

[0022] In step (2), the sample volume of the dextran aqueous solution is 0.2 to 0.9 μL, more preferably 0.5 μL;

[0023] The spotting volume of the DCPH / DHB composite matrix solution is 0.5 to 1.5 μL, more preferably 1.0 μL;

[0024] Mix by pipetting directly on the target plate. Repeat pipetting 10 to 20 times using a pipette.

[0025] In step (3), the spotted target plate is reacted at 54-56° C. for 28-32 minutes. More preferably, the spotted target plate is reacted at 55° C. for 30 minutes.

[0026] In step (4), the detection mode adopted by the MALDI-MS mass spectrometer is the reflectron mode under the negative ion mode;

[0027] On the other hand, the present invention also provides a method for measuring and analyzing actual samples after instrument calibration using the derivatized D3K.

[0028] The term "MALDI mass spectrometry" as used herein refers to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), also known as MALDI mass spectrometry. MALDI operates by irradiating a co-crystallized thin film formed by a sample and a matrix with laser light. The matrix absorbs energy from the laser and transfers it to the sample molecules, ionizing them. It is a soft ionization technique suitable for the analysis of biomacromolecules.

[0029] The "reactive matrix" described in the present invention refers to a conventional matrix that assists in the ionization of reducing sugars and can react rapidly with the reducing end hemiacetal thereof to covalently bind to the ionization tag, thereby improving the ionization efficiency.

[0030] The derivatization efficiency described in the present invention = derivatization product peak intensity / (derivatization product peak intensity + underivatized peak intensity).

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) DCPH contains a reactive hydrazine group in its structure, which reacts with polysaccharides to form hydrazones. This reaction is carried out under weak acid conditions, resulting in high derivatization efficiency. In addition to being a derivatization reagent, the reactive matrix DCPH can also serve as a matrix-assisted sample ionization, so excess derivatization reagent does not need to be purified.

[0033] (2) DCPH and DHB form a composite matrix, which works together to improve the sample ionization efficiency. DHB is an organic acid matrix that can significantly catalyze the hydrazone reaction. The target plate derivatization method provided by the present invention is simple to operate and greatly improves efficiency;

[0034] (3) The DCPH structure contains carboxyl groups, which are prone to losing protons and emitting peaks in the negative ion mode. The method provided by the present invention can significantly improve the detection sensitivity of reducing sugars in the negative ion mode, overcoming the problem of low detection sensitivity of polysaccharides in the negative ion mode;

[0035] (4) In the present invention, a strong derivatization product peak can be detected in the negative ion mode, achieving accurate mass calibration of MALDI-MS in the negative ion mode, and a characteristic fragment peak with a mass difference of 162 Da can be obtained in the secondary mass spectrometry, so secondary mass spectrometry calibration can also be performed;

[0036] (5) The samples used in the present invention are simple to prepare, widely available, inexpensive, and easy to store. The calibration method is simple to operate, and the use of dextran as a calibrant has a uniform mass distribution and high accuracy.

[0037] (6) The strong crystal uniformity of DCPH itself can improve the accuracy and reproducibility of quantitative detection (RSD < 10%). The DCPH / DHB composite matrix is ​​suitable for the qualitative and quantitative detection of N-glycans, and has a good linear relationship in the range of 0.2 to 20 pmol / μL. 2 >0.997. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the synthesis process of DCPH of the present invention;

[0039] Figure 2 The NMR spectrum and high-resolution ESI mass spectrum of DCPH of the present invention are shown below: Figure 2 A is DCPH 1 H NMR spectrum; Figure 2 B is DCPH 13 C NMR spectrum; Figure 2 C is the high-resolution ESI mass spectrum of DCPH in negative ion mode;

[0040] Figure 3 Schematic diagram of the principle of DCPH derivatization of reducing sugars of the present invention;

[0041] Figure 4 is the derivatization efficiency of maltoheptaose (G7) under different reaction conditions; Figure 4 A Different DCPH concentrations; Figure 4 B Different DHB concentrations; Figure 4 C different reaction temperatures; Figure 4 D Different reaction times;

[0042] Figure 5 is the MALDI mass spectrum of D3K derivatized under standard conditions; Figure 5 A. Primary mass spectrum in negative ion mode; Figure 5 B. Secondary mass spectrum of the parent ion m / z 3111.992 in negative ion mode;

[0043] Figure 6The detection limits of reducing sugars with different molecular weights using DHB and DCPH / DHB as MALDI matrices are shown; Figure 6 A, E are the detection limits of maltoheptaose (G7); Figure 6 B, F are the detection limits for NGA3; Figure 6 C and G are the detection limits of NA2; Figure 6 D,H are the detection limits of Man-8; Figure 6 AD is the spectrum detected with DCPH / DHB as the matrix; Figure 6 EH is the spectrum detected with DHB as the matrix;

[0044] Figure 7 The signal reproducibility when detecting G7 using DHB and DCPH / DHB as matrices and the quantitative curves for the detection of NGA3, NA2 and Man-8; Figure 7 A is the normalized graph of the mean peak intensity and RSD value of 55 detections in the same well; Figure 7 B is the linear range of DCPH / DHB matrix for detecting NGA3; Figure 7 C is the linear range of NA2 detection using DCPH / DHB matrix; Figure 7 D is the linear range of DCPH / DHB matrix detection of Man-8;

[0045] Figure 8 This is the negative ion MALDI mass spectrum of the target plate derivatization of reducing N-glycans in peach fruit before and after ripening using DCPH / DHB as the matrix after mass calibration; Figure 8 A is the mass spectrum of the reduced N-glycans detected in peach and the attribution of the mass spectrum peaks after amplification and the corresponding sugar chain structure diagram; Figure 8 B is the heat map of the content analysis of different reducing N-glycans in peach fruits before and after ripening after content normalization; Figure 8 C is the changing trend of the relative content of N-glycans during peach fruit ripening. DETAILED DESCRIPTION

[0046] The present invention will be further described in the following examples, but the present invention is not limited to the following examples.

[0047] The samples, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The present invention is described below by means of specific examples, but the present invention is not limited thereto. The specific model of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer used in the following examples is Ulraflex Xtreme TMMALDI-TOF / TOF MS (Bruker Daltonic, Germany) was performed using a 355 nm Nd:YAG laser. Parameters for positive ion reflectron mode were as follows: accelerating voltage, 25.00 kV; delayed extraction voltage, 22.30 kV; delayed extraction time, 150 ns; reflectron voltage 1, 26.50 kV; reflectron voltage 2, 13.50 kV; lens voltage, 8.50 kV; frequency, 1000 Hz. Parameters for negative ion reflectron mode were as follows: accelerating voltage, 20.00 kV; delayed extraction voltage, 17.75 kV; delayed extraction time, 120 ns; reflectron voltage 1, 21.10 kV; reflectron voltage 2, 10.70 kV; lens voltage, 8.50 kV; frequency, 1000 Hz. The target plate used was a 384 polished steel plate (MTP 384 polished steel), and the mass spectrometry data were analyzed using Bruker Flexanalysis 3.4 software.

[0048] The following abbreviations or foreign terms are used throughout this invention:

[0049] DCPH 2,4-dicarboxyphenylhydrazine;

[0050] DHB 2,5-dihydroxybenzoic acid;

[0051] D3K dextran 3000;

[0052] ESI, electrospray mass spectrometry;

[0053] G7 maltoheptaose;

[0054] HRMS, high-resolution mass spectrometry;

[0055] IS, internal standard;

[0056] LOD, limit of detection;

[0057] LOQ, limit of quantification;

[0058] MALDI, matrix-assisted laser desorption ionization;

[0059] MeOH, methanol;

[0060] min,minute;

[0061] MS, mass spectrometry;

[0062] MW, molecular weight;

[0063] m / z, mass-to-charge ratio;

[0064] NMR, nuclear magnetic resonance;

[0065] PNGase A, peptide N-glycosidase A;

[0066] RSD, relative standard deviation;

[0067] S / N, signal-to-noise ratio;

[0068] Tis-HCl, tris(hydroxymethyl)aminomethane hydrochloride;

[0069] μL, microliter;

[0070] V, volt;

[0071] Example 1: Synthesis and characterization of DCPH

[0072] 1. Synthesis of DCPH

[0073] 2,4-Dicarboxybromobenzene (4 mmol) and 85% hydrazine hydrate (16 mmol) were stirred in ethanol (25 mL) at 80°C for about 24 hours. Then, an appropriate amount of 1 mol / L dilute hydrochloric acid was added to the reaction solution until the solution became weakly acidic and a large amount of white solid precipitated. The solid product was then filtered.

[0074] 2. Separation and purification of DCPH

[0075] The filtered white solid was washed with a large amount of water, an appropriate amount of dichloromethane and ethanol in sequence to remove excess hydrazine hydrate and residual reaction raw materials, and then recrystallized with 80% ethanol to obtain the purified target product and dried continuously in a vacuum drying oven at 60°C for 6 hours to obtain the target product 2,4-dicarboxyphenylhydrazine.

[0076] 3. Characterization of DCPH

[0077] DCPH was characterized by NMR and high-resolution ESI.

[0078] In Example 1, Figure 1 Schematic diagram of the synthesis process of DCPH. Figure 2 For DCPH 1 H NMR spectrum (2A), 13 C NMR spectrum (2B) and high-resolution ESI positive ion mode mass spectrum (2C) are as follows 1 H NMR (500MHz, DMSO) δ11.90(s,1H),11.47(s,1H),8.35(s,1H),7.87(d,J=8.8Hz,1H),7.35(d,J=8.8Hz,1H); 13C NMR (126MHz, DMSO) δ167.53(s), 156.34(s), 143.28(s), 127.52(s), 123.32(s), 121.16(s), 111.89(s), 109.83(s); ESI-HRMS for DCPH(m / z):195.0415[MH] - The spectrum signal is clean and corresponds one-to-one with its structural information, which shows that the target product with high purity can be obtained by the separation and purification method in this example.

[0079] Example 2: Optimization of Derivatization Conditions for Reducing Sugar Target Plates Based on DCPH Reactive Matrix .

[0080] 50 pmol / 0.5 μL of maltoheptaose (G7) was prepared as a model compound, and the concentration of DCPH, the concentration of DHB, the reaction temperature, and the reaction time were investigated. The specific steps are as follows:

[0081] (1) Optimization of DCPH concentration: Prepare DHB solution with a concentration of 0.05 mol / L and DCPH solutions with concentrations of 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L and 0.10 mol / L respectively, and mix the DHB solution and the series of DCPH solutions in equal volumes to form DCPH / DHB composite matrix solutions with different DCPH concentrations. Take 0.5 μL G7 and 1 μL composite matrix solution and spot them on the same spot hole of the target plate in turn, mix them, react at 55 ° C for 30 minutes, and then perform MALDI positive and negative ion reflection mode detection. In the positive ion mode, the derivatization efficiency is calculated by the peak intensity, and the S / N of the derivatization product is collected in the negative ion mode. Figure 4 As shown in A, when the concentration of DCPH is 0.04 mol / L, the comprehensive evaluation of derivatization efficiency and product signal-to-noise ratio is the highest;

[0082] (2) DHB concentration: Prepare DCPH solution with a concentration of 0.04 mol / L and DHB solutions with concentrations of 0.025 mol / L, 0.05 mol / L, 0.075 mol / L, 0.1 mol / L and 0.125 mol / L respectively, and mix the DCPH solution and the series of DHB solutions in equal volumes to form DCPH / DHB composite matrix solutions with different DHB concentrations. Take 0.5 μL G7 and 1 μL composite matrix solution and spot them on the same spot hole of the target plate in turn, mix them, react at 55 ° C for 30 minutes, and then perform MALDI positive and negative ion reflection mode detection. In the positive ion mode, the derivatization efficiency is calculated by the peak intensity, and the S / N of the derivatization product is collected in the negative ion mode. Figure 4 As shown in B, when the concentration of DHB is 0.1 mol / L, the comprehensive evaluation of derivatization efficiency and product signal-to-noise ratio is the highest;

[0083] (3) Reaction temperature optimization: Prepare a DCPH solution with a concentration of 0.04 mol / L and a DHB solution with a concentration of 0.1 mol / L, and mix the DCPH solution and DHB solution in equal volumes to form a DCPH / DHB composite matrix solution. Take 0.5 μL of G7 and 1 μL of the composite matrix solution and spot them on the same spot hole of the target plate in turn, and mix them. React at 45, 50, 55, 60 and 65 ° C for 30 minutes, and then perform MALDI positive and negative ion reflection mode detection. In the positive ion mode, the derivatization efficiency is calculated by the peak intensity, and the S / N of the derivatization product is collected in the negative ion mode. Figure 4 As shown in C, when the reaction temperature is 55°C, the comprehensive evaluation of derivatization efficiency and product signal-to-noise ratio is the highest;

[0084] (4) Reaction time optimization: Prepare a DCPH solution with a concentration of 0.04 mol / L and a DHB solution with a concentration of 0.1 mol / L, and mix the DCPH solution and DHB solution in equal volumes to form a DCPH / DHB composite matrix solution. Take 0.5 μL of G7 and 1 μL of the composite matrix solution and spot them on the same spot hole of the target plate in turn, mix them, and react at 55°C for 20, 25, 30, 35 and 40 minutes respectively, and then perform MALDI positive and negative ion reflection mode detection. In the positive ion mode, the derivatization efficiency is calculated based on the peak intensity, and the S / N of the derivatization product is collected in the negative ion mode. Figure 4 As shown in D, when the reaction time is 30 min, the derivatization efficiency and product signal-to-noise ratio can reach the best;

[0085] Example 1 demonstrates that by optimizing the derivatization conditions for reducing sugar targets, derivatization efficiencies exceeding 99% can be achieved. Based on these optimization results, subsequent experiments used a DCPH / DHB composite matrix solution consisting of 0.04 mol / L DCPH and 0.1 mol / L DHB, with the reaction carried out at 55°C for 30 minutes.

[0086] Example 3: DCPHu-derivatized D3K for mass calibration in negative ion mode of MALDI-MS

[0087] (1) Prepare the DCPH / DHB composite matrix solution and 1 mg / mL D3K aqueous solution as described in Example 2. Place 0.5 μL of the D3K solution and 1 μL of the DCPH / DHB composite matrix solution sequentially onto the same well of a target plate, mix thoroughly, and react at 55°C for 30 min. Place the target plate in a mass spectrometer, acquire a primary mass spectrum in negative ion mode, and calibrate the mass spectrometer. Figure 5A is the MALDI mass spectrum of D3K in negative ion mode. The signal peaks are normally distributed, with uniform mass distribution and high accuracy. The mass-to-charge ratio difference between adjacent signal peaks is 162, corresponding to a single glucose residue. The primary mass spectrometry calibration data are listed in Table 1.

[0088] Table 1: Primary calibration data for DCPH-derivatized D3K in negative ion mode

[0089]

[0090] (2) Select the parent ion m / z 3111.992 in the primary mass spectrum and perform secondary mass spectrometry in negative ion mode. Figure 5 B shows its corresponding secondary mass spectrum. Through fragment attribution, it was found that the secondary fragmentation mode of polysaccharide after DCPH derivatization mainly produced a series of 2,4 The single fragmentation pattern of type A trans-ring fragmentation facilitates secondary mass calibration. The mass-to-charge ratio difference between adjacent signal peaks is m / z 162, indicating that the fragment ions differ by a single glucose residue. Because all fragments have precise masses, the method of the present invention can also be used for MALDI-MS secondary mass spectrometry calibration. The secondary mass spectrometry calibration data are listed in Table 2.

[0091] Table 2: Negative ion mode secondary calibration data for DCPH-derivatized D3K

[0092]

[0093] Example 4: Qualitative Investigation of DCPH / DHB Composite Matrix Detection of N-Glycan Standards

[0094] 1. DCPH / DHB as a matrix for detecting N-glycan standards

[0095] (1) Prepare 50 pmol / 0.5 μL of G7 sugar solution, and then dilute to 20 pmol / 0.5 μL, 10 pmol / 0.5 μL, 5 pmol / 0.5 μL, 2 pmol / 0.5 μL, 1 pmol / 0.5 μL, 500 fmol / 0.5 μL and 50 fmol / 0.5 μL to obtain G7 sugar solutions of various concentrations, which can be stored in a refrigerator at 4°C.

[0096] (2) Prepare 20 pmol / 0.5 μL standard sugar solutions of NGA3, NA2, and Man-8, respectively, and then dilute them to 10 pmol / 0.5 μL, 5 pmol / 0.5 μL, 2 pmol / 0.5 μL, 1 pmol / 0.5 μL, 500 fmol / 0.5 μL, and 50 fmol / 0.5 μL to obtain NGA3, NA2, and Man-8 sugar solutions of various gradient concentrations, which can be stored in a -20°C refrigerator;

[0097] (3) Take 0.5 μL of the G7 sugar solution in step (1) or the NGA3, NA2, or Man-8 standard sugar solution in step (2) and 1 μL of the DCPH / DHB composite matrix solution in Example 3, and spot them on the same well of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix. React at 55°C for 30 minutes to ensure that the sugar standard can react completely;

[0098] (4) As a control, take 0.5 μL of the G7 sugar solution in step (1) or the standard sugar solution of NGA3, NA2, or Man-8 in step (2) and 0.5 μL of the 0.05 mol / L LDHB methanol solution in Example 2, and spot them on the same well of the target plate. Use a pipette to repeatedly pipette 10 times directly onto the MALDI target plate to mix. Dry naturally at room temperature for 30 min.

[0099] (5) The target plate is sent to the MALDI mass spectrometer, and the negative ion reflectron mode is selected for data acquisition.

[0100] In Example 4 Figure 6 The detection limits of different N-glycan standards were determined using DCPH / DHB and DHB as MALDI matrices. Figure 6 AD is the spectrum detected with DCPH / DHB as the matrix, Figure 6 EH is the spectrum obtained using DHB as the matrix. Specific detection limits are summarized in Table 1. When using DHB as the matrix for G7, NGA3, NA2, and Man-8, the detection limits were all 500 fmol, while when using DCPH / DHB as the matrix, the detection limits were as low as 50 fmol. This indicates that using DCPH / DHB as the matrix provides the best detection results and is suitable for the detection of oligosaccharides and polysaccharides.

[0101] Example 5: Quantitative investigation of the stability and linear range of the method using DCPH / DHB as the matrix

[0102] (1) Prepare a 2 pmol / 0.5 μL G7 sugar solution as the internal standard solution and store it in a refrigerator at 4°C.

[0103] (2) Take the 20 pmol / 0.5 μL standard sugar solutions of NGA3, NA2 and Man-8 in Example 4 and dilute them to 10 pmol / 0.5 μL, 5 pmol / 0.5 μL, 2 pmol / 0.5 μL, 1 pmol / 0.5 μL, 500 fmol / 0.5 μL and 200 fmol / 0.5 μL, respectively, to obtain NGA3, NA2 and Man-8 sugar solutions of various gradient concentrations, which can be stored in a refrigerator at -20°C;

[0104] (3) Take 0.5 μL of the G7 sugar solution in Example 2, 1 μL of the DHB matrix solution in Example 2, or the DCPH / DHB composite matrix solution in Example 3, and spot them on the same well of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix, and react at 55°C for 30 minutes. The target plate is sent to the MALDI mass spectrometer, and the negative ion reflectron mode is used to sample 55 times in the same well.

[0105] (4) Take 0.5 μL of NGA3 sugar solution of different concentrations in step (2), 0.5 μL of G7 internal standard solution in step (1), and 1 μL of DCPH / DHB composite matrix solution in Example 3, and spot them on the same well of the target plate in sequence. Use a pipette to repeatedly aspirate 10 times directly on the MALDI target plate to mix, and react at 55°C for 30 minutes. The target plate is sent to the MALDI mass spectrometer, and data is collected in negative ion reflection mode. Each well is randomly sampled 10 times and the average value is taken;

[0106] (5) Take 0.5 μL of NA2 sugar solution of different concentrations in step (2), 0.5 μL of G7 internal standard solution in step (1), and 1 μL of DCPH / DHB composite matrix solution in Example 3, and spot them on the same well of the target plate in sequence. Use a pipette to repeatedly aspirate 10 times directly on the MALDI target plate to mix, and react at 55°C for 30 minutes. The target plate is sent to the MALDI mass spectrometer, and data is collected in negative ion reflection mode. Each well is randomly sampled 10 times and the average value is taken;

[0107] (6) Take 0.5 μL of different concentrations of Man-8 sugar solution in step (2), 0.5 μL of G7 internal standard solution in step (1), and 1 μL of DCPH / DHB composite matrix solution in Example 3, and spot them on the same sample hole of the target plate in sequence. Use a pipette to repeatedly aspirate 10 times directly on the MALDI target plate to mix, and react at 55°C for 30 minutes. The target plate is sent to the MALDI mass spectrometer, and data is collected in negative ion reflection mode. Each sample hole is randomly sampled 10 times and the average value is taken;

[0108] In Example 5, Figure 7 A represents the signal reproducibility for G7 detection using DHB and DCPH / DHB as matrices. By normalizing the signal intensities at 55 points to the mean, it is clear that the G7 ion intensity remains essentially stable throughout the test using DCPH / DHB as the matrix, with a relative standard deviation of 6.96%, significantly lower than the control matrix (68.19%). This demonstrates the robustness of the method using DCPH / DHB as the matrix, contributing to improved quantitative analysis accuracy. Figure 7BD was used as the internal standard. In the negative ion reflectance mode, the linear ranges of NGA3, NA2 and Man-8 were investigated respectively. It was found that they showed good linear relationships in the range of 0.2 to 20 pmol / 0.5 μL. 2 >0.996. It can be seen that using DCPH / DHB as a matrix can reliably quantitatively analyze protein glycans of different molecular weights, and the DCPH / DHB-based target plate derivatization strategy can achieve qualitative and quantitative detection of protein glycans.

[0109] Example 6: Quantitative Analysis of Reducing N-Glycans in Peach Fruit Before and After Ripening Using DCPH / DHB

[0110] The homemade N-glycans in the present invention adopt the standard glycoprotein N-glycan preparation method, and the PNGase A enzymatic method is used to prepare the N-glycans. The specific steps are as follows:

[0111] (1) Total protein extraction. Weigh 1 g of peach fruit sample and extract it with buffer solution Y [1 mol / L Tris-HCl (pH 8.2), 10% (w / v) sodium dodecyl sulfate, 1% (v / v) β-mercaptoethanol, 0.5 mol / L ethylene glycol ditetraacetic acid, 0.5 mol / L ethylenediaminetetraacetic acid, 0.1 mol / L phenylmethylsulfonyl chloride and 1× protease inhibitor] at 65°C. Centrifuge at 12000 rpm for 20 min at room temperature. Transfer the supernatant to a new centrifuge tube. Add an appropriate amount of cold phenol, vortex for 1 min, centrifuge at 12000 rpm at 4°C for 20 min, and discard the supernatant. Add cold buffer solution Z (50 mmol / L Tris-HCl, pH 8.2) and wash twice. Then add 0.1 mol / L cold ammonium acetate methanol solution and precipitate at -20°C overnight.

[0112] (2) Enzymatic hydrolysis. The extracted protein was dissolved in 0.01 mol / L Tris-HCl (pH 8.2), heated at 100°C for 10 min to denature the protein, and then cooled to room temperature. 30 μg of trypsin, 30 μg of chymosin, and 2 μL of 0.2 mol / L calcium chloride were added, and the mixture was incubated at 37°C for 24 h for enzymatic hydrolysis. 0.5 μL of PNGase A was then added, mixed, and incubated at 37°C for 24 h for enzymatic hydrolysis.

[0113] (3) Purification. The sample obtained by enzymatic hydrolysis was centrifuged, and the dissociated reducing N-glycans were purified using a C18 solid phase extraction column and a graphitized carbon solid phase extraction column in sequence. The C18 solid phase extraction column was used to remove impurity peptides, proteins and other contaminants. The purification process was as follows: 4 mL of methanol was used to activate the column, and 4 mL of ultrapure water was used to balance the column. The supernatant was added to the column, and the column was eluted with ultrapure water three times, 1 mL each time, and the eluate was collected. Then, a graphitized carbon solid phase extraction column was used to remove salts and small molecular polar compounds. The specific process was as follows: 3 mL of acetonitrile and 1 mL of 50% acetonitrile aqueous solution were used to activate the column in sequence, and 3 mL of ultrapure water was used to balance the column. The sugar solution collected during the C18 purification process was loaded and eluted with 5 mL of ultrapure water to remove impurities. Elute with 2 mL of 50% acetonitrile aqueous solution and collect the eluted sugar solution. Remove the solvent by vacuum centrifugation at room temperature and redissolve in 25 μL of ultrapure water. Store in a refrigerator at -20°C.

[0114] (4) Take 0.5 μL of the sugar solution in step (2), 0.5 μL of the G7 internal standard solution in Example 5, and 1 μL of the DCPH / DHB composite matrix solution in Example 3, and sequentially spot them on the same well of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix, and react at 55°C for 30 minutes. Send the target plate to the MALDI mass spectrometer, and collect data in negative ion reflectron mode;

[0115] (5) Mass calibration was performed using the internal standard method. The data of various N-glycans detected were normalized, and then the relative content of each N-glycan in peaches before and after ripening was calculated.

[0116] In Example 6, Figure 8 This is the negative ion MALDI mass spectrum of the target plate derivatization of reducing N-glycans in peach fruit before and after ripening using DCPH / DHB as the matrix; Figure 8 A is the mass spectrum of the reduced N-glycans detected in peach and the attribution of the mass spectrum peaks after amplification and the corresponding sugar chain structure diagram; Figure 8 B is a heatmap showing the content analysis of different reducing N-glycans in peach fruit before and after ripening after normalization. The results demonstrated that the DCPH / DHB matrix successfully enabled highly sensitive qualitative and quantitative analysis of 24 N-glycans in peach fruit samples, with mannose and complex N-glycans being the predominant types. Figure 8 C shows the trend of changes in the relative content of N-glycans during peach fruit ripening. Eight N-glycans showed a significant increase in relative content, and one N-glycan showed a significant decrease in relative content. These may be important factors regulating peach fruit ripening, providing a reference and analytical basis for the potential correlation between fruit ripening and N-glycome expression.

[0117] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. Application of 2,4-dicarboxyphenylhydrazine as a reactive matrix for mass calibration and N-glycan analysis in negative ion mode of MALDI mass spectrometry.

2. Application of 2,4-dicarboxyphenylhydrazine as a reactive matrix and 2,5-dihydroxybenzoic acid as a catalyst in mass calibration and N-glycan analysis in the negative ion mode of MALDI mass spectrometry.

3. The use according to claim 1, characterized in that The 2,4-dicarboxyphenylhydrazine is shown in formula (1):

4. The use according to claim 1, characterized in that Specifically include: (1) Reagent preparation DHB is 2,5-dihydroxybenzoic acid, a DHB methanol solution is prepared, DCPH is 2,4-dicarboxyphenylhydrazine, DCPH is dissolved in a mixed solvent of methanol and dimethyl sulfoxide to obtain a DCPH solution, the DHB methanol solution and the DCPH solution are then mixed to obtain a DCPH / DHB composite matrix solution, and dextran is dissolved in water to obtain a dextran aqueous solution; (2) Sample Take equal volumes of dextran aqueous solution and DCPH / DHB composite matrix solution, spot them on the same spotting well of the target plate of the MALDI-TOF-MS instrument, and mix them directly by pipetting on the target plate to obtain the spotted target plate; (3) Target plate derivatization reaction The target plate after spotting is reacted at 45-65°C for 20-40 minutes, and the sample spot is completely dried to obtain a target plate containing a mass calibrant; (4) Mass spectrometry calibration The target plate containing the mass calibrant is sent into the MALDI-TOF-MS mass spectrometer, and data is collected and mass calibrated in positive and negative ion modes respectively. The calibration parameters obtained after the instrument calibration will be automatically used to calibrate the mass spectrometry detection results of the actual sample.

5. The use according to claim 4, characterized in that In step (1), the concentration of 2,4-dicarboxyphenylhydrazine in the DCPH solution is 0.02 to 0.10 mol / L.

6. The use according to claim 4, characterized in that In step (1), the concentration of 2,5-dihydroxybenzoic acid in the DHB methanol solution is 0.025-0.125 mol / L.

7. The use according to claim 4, characterized in that In step (1), the glucan is a reducing glucan, and the concentration of glucan in the glucan aqueous solution is 0.5-2 mg / mL.

8. The use according to claim 4, characterized in that In step (2), the sample volume of the dextran aqueous solution is 0.2 to 0.9 μL; In step (2), the spotting volume of the DCPH / DHB composite matrix solution is 0.5 to 1.5 μL.

9. The use according to claim 4, characterized in that In step (2), pipetting is performed directly on the target plate to mix the mixture. Repeated pipetting is performed 10 to 20 times directly on the target plate using a pipette.

10. The use according to claim 4, characterized in that In step (3), the spotted target plate is reacted at 54-56° C. for 28-32 minutes.

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