A MALDI reactive matrix for analysis of reducing sugars: 2-nitro-4-carboxyphenylhydrazine

Abstract

The present invention relates to the field of sugar biochemical analysis, and discloses the application of 2-nitro-4-carboxyphenylhydrazine (NCPH) alone as a reactive matrix and in combination with 2,5-dihydroxybenzoic acid (DHB) (NCPH / DHB) for derivatization qualitative and quantitative detection of reducing sugars. NCPH has a hydrazine reactive group, and as a reactive matrix, it is easy to react with the hemiacetal end of reducing sugars, which can significantly improve its ionization efficiency; it can also assist in the ionization of reducing sugars, and the excess NCPH does not need to be separated. DHB is an organic acid that can greatly catalyze the derivatization reaction and further improve the detection effect of reducing sugars. After NCPH is combined with DHB, a truly complete derivatization detection of reducing sugars with a derivatization yield of more than 99% can be achieved at a reaction temperature of 55°C; it can also significantly improve the co-crystallization uniformity with the analyte, and improve the accuracy of quantitative detection of reducing sugars.

Description

Technical Field

[0001] The invention relates to the field of sugar biochemical analysis, and in particular to a MALDI reaction type matrix 2-nitro-4-carboxyphenylhydrazine for analyzing reducing sugars. Background Art

[0002] Reducing sugars are a type of reducing sugar with a hemiacetal reducing end. They are widely found in nature and are rich in variety, including small oligosaccharides such as glucose and maltose, large glucans composed of a series of glucose with different polymerization degrees, and oligosaccharides released by enzymatic hydrolysis of glycoproteins. N -Glycans ( N -glycan) and so on. Among them, N- Glycans are N- Glycosylation and protein forming glycosidic bonds with reducing sugars play an important role in the life activities of organisms (Xu, CC, Davis, TWNg., Glycosylation-Directed Quality Control of Protein Folding. Nature Reviews Molecular Cell Biology, 2015, Volume 16, 742-752). N- Characterization of glycans can reveal N- The dynamic changes of glycosylation in organisms are of great significance to the study of glycobiology.

[0003] However, reducing sugars have almost no fluorescent groups and chromophores, lack of easily ionized sites, and therefore have extremely low ionization efficiency. In addition, they have highly complex heterogeneity and other disadvantages, so it is very difficult to directly analyze and detect them (Kailemia, MJ, Ruhaak, LR, Lebrilla, CB, Oligosaccharide Analysis By Mass Spectrometry: A Review Of Recent Developments. Analytical Chemistry, 2014, Volume 86, 196). It is often necessary to introduce easily ionized polar groups into their structures through chemical derivatization to fundamentally improve their ionization efficiency. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) has been widely used in the detection of various highly heterogeneous biomacromolecules such as proteins, nucleic acids, and reducing sugars due to its advantages of soft ionization, strong salt resistance, low sample consumption, and convenient sample preparation (Cai Zongwei, Liu Shuying, Applications of MALDI TOF spectroscopy, Topics in Current Chemistry, 2013, 331). 2,5-Dihydroxybenzoic acid (DHB) is the most widely used MALDI matrix for detecting reducing sugars, but it easily forms irregular needle-shaped crystals with the analyte on the target plate, resulting in uneven sample distribution, poor signal reproducibility, and limited detection sensitivity, which seriously restricts its qualitative and quantitative analysis of reducing sugars (Jang, KS, Kim, YH, Rapid and robust MALDI-TOF MStechniques for microbial identification: a brief overview of their diverseapplications, Journal of Microbiology, 2018, Volume 56, 209-216).

[0004] Reactive matrices are reagents that integrate the functions of derivatization and MALDI matrices. They can be chemically derivatized with reducing sugars on a MALDI target plate and then directly used for mass spectrometry analysis, which further simplifies the experimental process and improves work efficiency. It is well known to those skilled in the art that the ideal derivatization yield of completely derivatized reducing sugars is 100%, and the derivatization yield is positively correlated with the reaction temperature and reaction time. The higher the reaction temperature or the longer the reaction time, the higher the derivatization yield (Zhao, XY, Guo, C., Huang, Y., Huang, LL, Ma, G., Liu, YQ, He, Q., Wang, HW, Chen, KS, Pan, YJ, Combination strategy of reactive and catalytic matrices for qualitative and quantitative profiling of N-glycans in MALDI-MS. Analytical Chemistry, 2019, Volume 91,9251-9258; Ouyang D, Huang H, He Y, et al. Utilization of hydralazine as areactive matrix for enhanced detection and on-MALDI-target derivatization of saccharides. Chinese Chemical Letters, 2024, Volume 35, 386-391.). The higher the derivatization yield, the more thorough the derivatization of reducing sugars by the reactive matrix, and the more conducive to improving the accuracy of quantitative analysis. If the derivatization reaction is not thorough enough, then in the detection and analysis process of reducing sugars in complex biological samples, the underivatized reducing sugar raw materials will seriously increase the complexity of the spectral analysis, greatly reduce the accuracy of quantitative analysis, and cause the experimental results to fail to reflect the real sample data. It can be seen that the closer the derivatization yield is to 100%, the better the accuracy of quantitative analysis. Therefore, the prior art can only increase the derivatization yield as much as possible by increasing the reaction temperature or extending the reaction time so that it is infinitely close to the ideal value of 100%. However, on the one hand, extending the reaction time will greatly reduce the reaction efficiency and increase the cost; on the other hand, for biological samples, the lower the reaction temperature, the milder it is, and the more conducive it is to the detection of biological samples. The higher the reaction temperature, the more it will cause degradation, destruction or other complex side reactions to the sugar analytes in the biological samples, which is not conducive to the quantitative analysis and detection of biological samples. Therefore, in the quantitative analysis and detection of biological samples, improving the derivatization yield and reducing the reaction temperature and reaction time are contradictory requirements.

[0005] The existing reducing sugar detection to improve the derivatization yield is based on the premise of increasing the reaction temperature. Too high a reaction temperature will inevitably damage the biological sample to be detected. Even so, when the reaction temperature reaches above 65°C in the existing reducing sugar detection, the derivatization yield can only reach more than 95%, which is still far from the ideal value of 100%, and cannot be called true complete derivatization. For example, 2, 5-dihydroxybenzoylhydrazide (DHBH) developed by the applicant in the previous research is a classic reactive matrix for detecting reducing sugars. The combined matrix strategy composed of it and the acidic matrix DHB as a catalyst can achieve complete derivatization of reducing sugars under the reaction conditions of 65°C and 30 minutes. It should be clarified that the complete derivatization considered in this article refers to a derivatization yield of more than 95%; if the derivatization yield is to be increased to more than 99%, the reaction temperature is required to be increased to more than 80°C (see Figure 1 , from Figure S6 in the Supporting Information of the following references: Zhao, XY, Guo, C., Huang, Y., Huang, LL, Ma, G., Liu, YQ, He, Q., Wang, HW, Chen, KS, Pan, YJ, Combination strategy of reactive and catalytic matrices for qualitative and quantitative profiling of N -glycans in MALDI-MS. Analytical Chemistry, 2019, Volume 91, 9251-9258). Although a derivatization yield of more than 95% can basically meet the prerequisite for quantitative analysis, with the development of glycobiology research, improving the accuracy of quantitative analysis has put forward higher requirements for the derivatization yield of reactive matrices. A derivatization yield of 95% can no longer meet the requirements, and a derivatization yield of more than 99% or even 100% is required. The necessary means to improve the derivatization yield in the existing technology is to increase the reaction temperature; when the reaction temperature reaches above 80°C, it is inevitable that the sugar analytes in the biological sample will be degraded, destroyed or bring other complex side reactions, which is not conducive to the quantitative analysis and detection of biological samples.

[0006] Similarly, other reactive matrices reported for the derivatization of reducing sugars can only achieve a derivatization yield of more than 95%, and the reaction temperature is often higher than 60 °C, such as 3-aminoquinoline (3-AQ, Kaoru K., Yuko F., Shinichi I., Sadanori S., Koichi T., Highly sensitive MALDI analyses of glycans by a new aminoquinoline-labeling method using 3-aminoquinoline / α-cyano-4-hydroxycinnamic acid liquid matrix. Analytical Chemistry, 2011, Volume 83, 3663-3667), hydrazinonicotinic acid (HYNIC, Jiao J., Yang L., Zhang Y., Hydrazinonicotinic acid derivatization for selective ionization and improved glycan structure characterization by MALDI-MS. Analyst, 2015, Volume 140, 5475-5480), 2-phenyl-3-(p-aminophenyl) Acrylonitrile (PAPAN, Ling, L., Xiao, CS, Ma, Y., Jiang, LY, Wang, S., Guo, LM, Jiang, SM, Guo, X. H,. 2-Phenyl-3-(p-Aminophenyl) acrylonitrile: a reactive matrix for sensitive and selective analysis of glycans by MALDI-MS. Analytical Chemistry, 2019, Volume 91, 8801-8807; Chinese patent CN108398482A) and quinoline-3-carboxylic acid hydrazide (Q3CH, Zhao, XY, Huang, Y., Ma, G., Liu, YQ, Guo, C., He, Q., Wang, HW, Liao, JC, Pan, YJ,Parallel on-target derivatization for mass calibration and rapid profiling of. N -glycans by MALDI-TOF MS. Analytical Chemistry, 2020, Volume 92, 991-998), etc., and these reactive matrices are used in mammals N -Analysis of glycans. Compared with mammals, plants have N - The glycosylation pattern is very different from that of mammals (see Chinese patent CN117969642B), such as the core glycosylation and terminal glycosylation epitopes are obviously different, which makes the relatively mature N - Glycosylation research methods are difficult to expand and apply in plants, resulting in N -The research on glycosylation is relatively lagging behind. Secondly, the abundance of glycoproteins in plants is lower, and the sugar chain enrichment, purification and even characterization process is often easily interfered by a large number of plant-derived pollutants such as pigments and pectins (Nagashima Yukihiro, Antje von Schaewen, Hisashi Koiwa. Function of N -glycosylation in plants, Plant Science, 2018, Volume 274, 70-79). N - Glycan characterization is more difficult.

[0007] In summary, there are two problems with the existing developed reactive matrices for the derivatization analysis and detection of reducing sugars: (1) In order to increase the derivatization yield, the necessary means is to increase the reaction temperature. The usual reaction temperature is above 60°C. In order to achieve a derivatization yield of more than 99%, the reaction temperature even needs to be above 80°C. However, for biological samples, increasing the reaction temperature will inevitably cause degradation, destruction or other complex side reactions to the sugar analytes in the biological samples, which is not conducive to the quantitative analysis and detection of biological samples; (2) Even if the reaction temperature is increased during the detection of existing reducing sugars, it is impossible to achieve true complete derivatization. When the reaction temperature of the existing reactive matrices reaches above 60°C, the derivatization yield can only reach more than 95%, which is still far from the ideal value of 100% and cannot be called true complete derivatization. Therefore, the existing reactive matrices for the derivatization analysis and detection of reducing sugars cannot achieve true complete derivatization under relatively mild conditions (below 60°C), and cannot meet the growing demand for biological reducing sugars, especially those in plants that are more difficult to detect. NThe need for highly sensitive qualitative and highly accurate quantitative analysis of glycans has limited the in-depth research and application of glycobiology. N -MALDI reaction matrix for glycan analysis and detection has important value and significance in improving its matrix performance and comprehensive detection effect. Summary of the invention

[0008] In view of the problems existing in the existing MALDI reaction matrix of reducing sugar, the present invention provides a MALDI reaction matrix 2-nitro-4-carboxyphenylhydrazine for analyzing reducing sugar. 2-nitro-4-carboxyphenylhydrazine (NCPH) is developed and applied for the first time as a MALDI reaction matrix for reducing sugar analysis, which is an ideal matrix for detecting reducing sugar; and the detection effect of reducing sugar can be further improved by compounding NCPH with DHB (NCPH / DHB). The present invention overcomes the technical bias of the prior art that the biological sample must be damaged at high temperature by increasing the reaction temperature in order to achieve complete derivatization (i.e., increasing the derivatization yield as close to 100% as possible). The present invention uses NCPH / DHB compounding to achieve true complete derivatization of reducing sugar under relatively mild conditions. Compared with the prior art, the reactive matrix can only achieve a derivatization yield of more than 95% at above 60°C or even above 65°C. In contrast, the present invention, in MALDI mass spectrometry, the efficient target plate derivatization strategy based on NCPH / DHB can achieve truly complete derivatization detection of reducing sugars under reaction conditions with a lower reaction temperature (55°C) than the prior art. The truly complete derivatization in the present invention refers to a derivatization yield of more than 99%, especially for more complex and difficult plants. N -Qualitative and quantitative analysis of glycans with excellent accuracy.

[0009] The first object of the present invention is to provide an application of 2-nitro-4-carboxyphenylhydrazine (NCPH) as a MALDI reactive matrix in reducing sugar analysis. The reducing sugar is defined as a reducing sugar with a hemiacetal reducing end, which may include oligosaccharides such as glucose and maltose, glucans composed of a series of glucose with different polymerization degrees, and N - Polysaccharides, etc.

[0010] The structural formula of NCPH described in the present invention is shown in formula (1): Formula (1)

[0011] The second object of the present invention is to provide the use of a composite matrix of NCPH (2-nitro-4-carboxyphenylhydrazine) and DHB (2,5-dihydroxybenzoic acid) in the analysis of reducing sugars in MALDI mass spectrometry.

[0012] In one embodiment of the present invention, the 2-nitro-4-carboxyphenylhydrazine as a MALDI reactive matrix is ​​preferably combined with 2,5-dihydroxybenzoic acid in a molar ratio of 1:0.5 to 3.125; more preferably, the molar ratio is 1:2.5. NCPH is a reactive matrix having a hydrazine reactive group that can react with the hemiacetal end of a reducing sugar. DHB is an acidic catalyst that can significantly promote the derivatization reaction of NCPH with a reducing sugar.

[0013] The NCPH described in the present invention has the dual functions of a MALDI matrix and a derivatization reagent. It has matrix properties that assist in the ionization of carbohydrates, and its reaction performance is derived from the hydrazine group in its structure, which can undergo a dehydration reaction with the hemiacetal end of a reducing sugar to generate a stable hydrazone, so the excess NCPH does not need to be purified and removed. DHB has the dual functions of a MALDI matrix and a catalyst. As a traditional MALDI matrix for detecting carbohydrates, it can significantly promote the desorption and ionization of reducing sugars, and its catalytic performance is derived from the carboxyl group in its structure, which can increase the acidity of the reaction system and thus greatly catalyze the derivatization reaction, so DHB also does not need to be removed additionally. Therefore, the combined use of NCPH and DHB can significantly improve the reaction efficiency with reducing sugars, and compared with the prior art, a truly complete derivatization reaction with a lower temperature and a higher yield can be achieved on the target plate, which is specifically manifested in that a derivatization yield of more than 99% can be achieved at a reaction temperature of 55°C; at the same time, after mixing NCPH and DHB, a fairly uniform co-crystal morphology can be formed, which is effective in the derivatization of the reducing sugars. N -The derivatization detection process of glycans has good signal reproducibility and quantitative accuracy.

[0014] The application in reducing sugar analysis is to perform derivatization and qualitative and quantitative detection on reducing sugars, and the application specifically comprises the following steps:

[0015] 1. Reagent preparation

[0016] An NCPH solution is prepared with an equal volume mixed solvent of an organic solvent 1 and an organic solvent 2, wherein the NCPH is 2-nitro-4-carboxyphenylhydrazine; a DHB solution is prepared with the organic solvent 1 as a solvent, wherein the DHB is 2, 5-dihydroxybenzoic acid; the NCPH solution and the DHB solution are mixed in equal volumes to prepare an NCPH / DHB composite matrix solution; an acidic aqueous solution of a reducing sugar to be tested is prepared; the organic solvent 1 is selected from any one or more polar organic solvents miscible with water having a polarity greater than or equal to ethanol; the organic solvent 2 is selected from any one or more dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF); in the present invention, because the reducing sugar to be tested is dissolved in water, a solvent with a large polarity can be better miscible with the aqueous solution, and DMSO and DMF have a wide range of solubility, can dissolve a variety of compounds, and are miscible with water and various organic solvents, so the full reaction of the reactive matrix and the sugar analyte can be further enhanced.

[0017] 2. Sample

[0018] Take equal volumes of the aqueous solution of the reducing sugar to be tested and the NCPH / DHB composite matrix solution, spot them in the same spotting hole of the MALDI target plate in sequence, mix them evenly directly on the target plate, and obtain the spotted target plate.

[0019] 3. Target plate derivatization reaction

[0020] The target plate after spotting is placed at 45-65°C for 20-40 min, the mixed sample spot is completely dried, and then MALDI mass spectrometry analysis is performed.

[0021] In one embodiment of the present invention, in step 1, the organic solvent 1 is selected from any one or more of methanol, acetonitrile, and ethanol;

[0022] In one embodiment of the present invention, in step 1, the organic solvent 1 and the organic solvent 2 are analytical grade or chromatographic grade solvents;

[0023] In one embodiment of the present invention, the solvent for preparing the reducing sugar aqueous solution is ultrapure water or double distilled water;

[0024] In one embodiment of the present invention, the concentration of the NCPH solution is 0.02-0.10 mol / L;

[0025] In one embodiment of the present invention, the concentration of the DHB solution is 0.025-0.125 mol / L;

[0026] In one embodiment of the present invention, in step 2, the spotting volume of the reducing sugar aqueous solution to be tested is 0.5-1.5 μL;

[0027] In one embodiment of the present invention, the spotting volume of the NCPH / DHB composite matrix solution is 0.5 to 1.5 μL;

[0028] In one embodiment of the present invention, the aqueous solution of the reducing sugar to be tested and the NCPH / DHB composite matrix solution are mixed by repeatedly pipetting on a polished target plate 10 to 20 times using a pipette;

[0029] In one embodiment of the present invention, the detection mode adopted by the MALDI mass spectrometry analysis is positive ion linear or reflectance mode.

[0030] The third object of the present invention is to provide a kit for in situ labeling of reducing sugars on MALDI target plates with NCPH via hydrazone reaction. The kit can provide one or more containers containing a certain amount of NCPH suitable for in situ labeling of a selected number of reducing sugar MALDI target plates.

[0031] For example, conveniently, the amount of NCPH in the container can be selected to be sufficient to label the reducing sugar samples in the wells of the 384-well MALDI target plate. The NCPH used for labeling is quite small, and ~3 mg of NCPH is usually sufficient to label the reducing sugar samples in all 384 wells of the MALDI target plate. The kit further includes one or more containers, including: (a) a container containing a uniformly recrystallized powder of the NCPH matrix, (b) a container containing a uniformly recrystallized powder of the DHB matrix, (c) a container containing a solvent suitable for the NCPH matrix, (d) a container containing a solvent suitable for the DHB matrix, (e) a container containing an equal volume of the NCPH matrix solution and the DHB matrix solution prepared according to the requirements using the suitable solvent, and (f) a container containing an acidic aqueous solution suitable for the reducing sugar sample to be tested. The container of the uniformly recrystallized powder of the DHB matrix used for compounding NCPH contains 2.5 times the molar equivalent of DHB. Once compounded with NCPH, the optimal compounding ratio can be obtained, that is, the molar ratio of NCPH to DHB is 1:2.5.

[0032] In one embodiment of the present invention, the NCPH matrix recrystallized uniform powder and the DHB matrix recrystallized uniform powder are high-purity powders that have undergone a recrystallization process, and the high purity refers to 99.9% and above.

[0033] In one embodiment of the present invention, the solvent adapted to the NCPH matrix is ​​a mixed solvent of an analytically pure or chromatographic grade organic solvent 1 and an organic solvent 2; the solvent adapted to the DHB matrix is ​​an analytically pure or chromatographic grade organic solvent 1; the organic solvent 1 is selected from any one or more polar organic solvents miscible with water having a polarity greater than or equal to ethanol; the organic solvent 2 is selected from any one or more of dimethyl sulfoxide or N,N-dimethylformamide.

[0034] In one embodiment of the present invention, the acid in the acidic aqueous solution adapted to the reducing sugar sample to be tested is trifluoroacetic acid or hydrochloric acid, and the water is double distilled water or single distilled water.

[0035] In one embodiment of the present invention, the trifluoroacetic acid is of chromatography grade purity.

[0036] The "MALDI mass spectrometry" described in the present invention refers to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), referred to as MALDI mass spectrometry. The working principle of MALDI is to irradiate the co-crystallized film formed by the analyte and the matrix with a pulsed laser of a specific wavelength, and the matrix absorbs energy from the laser and transfers it to the analyte molecules to desorb and ionize them. MALDI is a soft ionization technology suitable for the determination of biomacromolecules.

[0037] The "reactive matrix" described in the present invention refers to a conventional matrix that assists the desorption and ionization of reducing sugars and can undergo a derivatization reaction with the hemiacetal reducing end thereof to covalently bind an ionization tag and improve the ionization efficiency.

[0038] The "target plate derivatization strategy" described in the present invention refers to the simultaneous spotting and derivatization of the analyte on the target plate, which greatly simplifies the operation steps and is compatible with the high-throughput characteristics of MALDI.

[0039] The derivatization yield described in the present invention = derivatization product peak intensity / (derivatization product peak intensity + underivatized peak intensity).

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. NCPH can assist the desorption and ionization of reducing sugars and has the performance as a MALDI matrix.

[0042] 2. In the NCPH / DHB composite matrix, NCPH is a reactive matrix with a hydrazine reactive group that can react with the hemiacetal end of reducing sugars. DHB is an acidic catalyst that can significantly promote the derivatization reaction between NCPH and reducing sugars. NCPH has matrix properties for detecting reducing sugars, so it does not require purification and separation, and is suitable for target plate derivatization to analyze and detect reducing sugars.

[0043] 3. Due to the multiple effects of derivatization, catalysis and promotion of ionization, the NCPH / DHB composite matrix has excellent matrix properties, which can significantly improve the ionization efficiency and detection sensitivity of reducing sugars and expand the quality detection range of reducing polysaccharides.

[0044] 4. The present invention overcomes the technical bias of the prior art that in order to achieve complete derivatization (i.e., to increase the derivatization yield as close to 100% as possible), the reaction temperature must be increased, resulting in the destruction of biological samples at high temperatures. The present invention uses NCPH / DHB composites to achieve truly complete derivatization of reducing sugars under relatively mild conditions. Compared with the reactive matrix of the prior art, which can only achieve a derivatization yield of more than 95% at temperatures above 60°C or even above 65°C, in contrast, in the present invention, the efficient target plate derivatization strategy based on NCPH / DHB in MALDI mass spectrometry can achieve truly complete derivatization detection of reducing sugars under reaction conditions with a lower reaction temperature (55°C) than the prior art. The truly complete derivatization described in the present invention refers to a derivatization yield of more than 99%, especially for more complex and difficult plants. N - The qualitative and quantitative analysis of polysaccharides has excellent accuracy. The combined use of NCPH and DHB can also significantly improve the uniformity of co-crystallization with reducing sugars, with good quantitative detection accuracy and signal reproducibility (RSD <10%), and has a good linear correlation in the range of 0.2 to 20 pmol / μL (R 2 >0.996). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The derivatization yield of reducing sugars by the reactive matrix DHBH at different reaction temperatures in the prior art (Zhao, XY, Guo, C., Huang, Y., Huang, LL, Ma, G., Liu, YQ, He, Q., Wang, HW, Chen, KS, Pan, YJ, Combination strategy of reactive and catalytic matrices for qualitative and quantitative profiling of N -glycans in MALDI-MS. Analytical Chemistry, 2019, Volume 91, 9251-9258). The data in this figure are from the original experimental data records of the inventor's previous research;

[0046] Figure 2 Schematic diagram of the synthesis process of NCPH;

[0047] Figure 3 is the NMR spectrum and high-resolution ESI mass spectrum of NCPH; among them, Figure 3 A in the equation is NCPH 1 H NMR spectrum; Figure 3 B in the equation is NCPH 13 C NMR spectrum; Figure 3 C in the figure is the high-resolution ESI mass spectrum of NCPH in negative ion mode;

[0048] Figure 4 This is the MALDI mass spectrum for detecting maltoheptaose (G7); Figure 4 A in the figure is the MALDI mass spectrum of G7 detected using DHB as a matrix; Figure 4 B in the figure is the MALDI mass spectrum of G7 detected by using NCPH as a matrix;

[0049] Figure 5 The schematic diagram of the target plate derivatization of reducing sugars based on NCPH / DHB;

[0050] Figure 6 The figure shows the effect of different acidic catalysts and NCPH composite matrix on the derivatization reaction of G7;

[0051] Figure 7 This is the effect of NCPH concentration, DHB concentration, temperature and reaction time on the target plate derivatization reaction; Figure 7 A in the figure represents different NCPH concentrations; Figure 7 B in the figure represents different DHB concentrations; Figure 7 C in it is different reaction temperatures; Figure 7 D in the figure is different reaction time;

[0052] Figure 8 The figure is a diagram showing the suitability of using NCPH / DHB as a MALDI matrix to detect oligosaccharides and dextran D3K of different molecular weights; Figure 8 A in the figure is the MALDI mass spectrum of low molecular weight oligosaccharides detected by NCPH / DHB derivatization; Figure 8 B in the figure is the MALDI mass spectrum of D3K detected by NCPH / DHB derivatization;

[0053] Fig. 9 The following are the detection limit diagrams of reducing sugars with different molecular weights using DHB and NCPH / DHB as MALDI matrices respectively; Fig. 9 A and E in the figure are the detection limits of maltoheptaose (G7); Fig. 9 B and F in it are the detection limits for NGA3; Fig. 9 C and G in it are the detection limits of NA2; Fig. 9 D and H in are the detection limits of Man-8; Fig. 9 AD in the figure is the spectrum detected with NCPH / DHB as the matrix; Fig. 9 EH in the figure is the spectrum detected with DHB as the matrix;

[0054] Fig.10 The figure shows the reproducibility of the signal when G7 is detected using DHB and NCPH / DHB as matrices and the quantitative curves of NGA3, NA2 and Man-8 detection; Fig.10 A in the figure is the normalized graph of the mean peak intensity and RSD value of 55 detections in the same spot well; Fig.10 B in the figure is the linear range of NGA3 detection using NCPH / DHB matrix; Fig.10 C in is the linear range of NCPH / DHB matrix detection of NA2; Fig.10 D in is the linear range of NCPH / DHB matrix detection of Man-8;

[0055] Fig.11 To investigate the effect of NCPH / DHB on peach fruit protein N- MALDI mass spectra of glycans after target plate derivatization; Fig.11 A in the figure is the detection N- Mass spectra of glycans and the attribution of mass spectrometry peaks after magnification and the corresponding sugar chain structure diagram; Fig.11 B in the figure is the normalized content of different N- Heat map of glycan content analysis; Fig.11 C in the peach fruit ripening process N- The changing trend of relative content of polysaccharides. DETAILED DESCRIPTION

[0056] The present invention will be further described in the following examples, but the present invention is not limited to the following examples.

[0057] 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.

[0058] The specific model of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer used in the following examples is UlrafleXtreme TMMALDI-TOF / TOF MS (Bruker Daltonic, Germany) was performed using a 355 nm Nd:YAG laser. All detections were performed in positive ion reflectron mode. Positive ion reflectron mode parameters: acceleration voltage, 25.00 kV; delayed extraction voltage, 22.30 kV; delayed extraction time, 150 ns; reflector voltage 1, 26.50 kV; reflector voltage 2, 13.50 kV; lens voltage, 8.50 kV; frequency, 1000 Hz. The target plate used was 384 polished steel (MTP 384polished steel), and mass spectrometry data were analyzed using Bruker Flexanalysis 3.4 software.

[0059] The following abbreviations or foreign terms are used throughout the present invention:

[0060] CA, p-Coumaric acid

[0061] CCA, α-cyanocinnamic acid

[0062] CHCA, α-Cyano-4-hydroxycinnamic acid

[0063] dH 2 O, deionized water;

[0064] D3K, Dextran 3000

[0065] DHB, 2, 5-dihydroxybenzoic acid;

[0066] DHCA, 3, 4-dihydroxycinnamic acid

[0067] DMSO, dimethyl sulfoxide;

[0068] ESI, electrospray mass spectrometry;

[0069] G1, glucose;

[0070] G2, maltose;

[0071] G3, maltotriose;

[0072] G4, maltotetraose;

[0073] G5, maltopentaose;

[0074] G6, maltohexaose;

[0075] G7, maltoheptaose;

[0076] HAc, acetic acid

[0077] HRMS, high-resolution mass spectrometry;

[0078] Hz, Hertz;

[0079] IS, internal standard;

[0080] LOD, limit of detection;

[0081] LOQ, limit of quantitation;

[0082] MALDI, matrix-assisted laser desorption ionization;

[0083] MeOH, methanol;

[0084] min, minute;

[0085] MP, melting point;

[0086] MS, mass spectrometry;

[0087] MW, molecular weight;

[0088] m / z , mass-to-charge ratio;

[0089] NCPH, 2-nitro-4-carboxyphenylhydrazine;

[0090] NMR, nuclear magnetic resonance;

[0091] PNGase A, peptide N- Glycosidase A;

[0092] RSD, relative standard deviation;

[0093] rt, room temperature;

[0094] SA, sinapinic acid;

[0095] S / N, signal-to-noise ratio;

[0096] Tis-HCl, tris(hydroxymethyl)aminomethane hydrochloride;

[0097] μL, microliter;

[0098] V, volt;

[0099] Example 1: Synthesis and characterization of NCPH

[0100] 1. Synthesis of NCPH

[0101] Stir 2-nitro-4-carboxychlorobenzene (1-4 mmol) and 85% hydrazine hydrate (4-16 mmol) in ethanol (5-25 mL) at 15-40 °C for about 3-6 hours, then add an appropriate amount of dilute hydrochloric acid to the reaction solution until the solution becomes weakly acidic and a large amount of orange solid is precipitated, and then filter the solid product.

[0102] 2. Isolation and purification of NCPH

[0103] The filtered orange solid was washed with a large amount of water, an appropriate amount of dichloromethane and ethanol in turn 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 30 to 60 ° C for 2 to 6 hours to obtain the target product 2-nitro-4-carboxyphenylhydrazine.

[0104] 3. Characterization of NCPH

[0105] NCPH was characterized by NMR, high-resolution ESI and melting point analysis.

[0106] In Example 1, Figure 2 Schematic diagram of the synthesis process of NCPH. Figure 3 For NCPH 1 H NMR spectrum ( Figure 3 A) 13 CNMR spectrum ( Figure 3 B) and high-resolution ESI positive ion mode mass spectrum ( Figure 3 The result is as follows 1 H NMR (500 MHz, DMSO) δ 9.56 (s, 1H), 8.58 (s, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.64 (d, J =9.2 Hz, 1H); 13 C NMR (126 MHz, DMSO) δ 165.97 (s), 149.19 (s), 135.50 (s), 128.52 (s), 128.11 (s), 116.61 (s), 114.91 (s); ESI-HRMS for NCPH ( m / z ):196.0356 [MH] - . The spectrum signal is clean and matches its structural information. It can be seen that the separation and purification method in this embodiment can obtain a high-purity target product. Table 1: Melting points of the matrix and the detection limits for G7, NGA3, NA2 and Man-8. In Table 1, the melting point of DHB is 195-196 °C, and the melting point of NCPH is 236-237 °C. It can be seen that NCPH meets the requirements of the high vacuum working environment in MALDI mass spectrometry.

[0107] Table 1

[0108]

[0109] Example 2: NCPH itself as a MALDI matrix for detecting reducing sugars

[0110] (1) Prepare 50 pmol / 0.5 μL G7 sugar solution, which can be stored in a refrigerator at 4°C;

[0111] (2) Prepare a 0.05 mol / L NCPH matrix solution using a mixed solution of equal volumes of MeOH and DMSO as solvent. This solution can be stored in a refrigerator at 4 °C.

[0112] (3) Prepare a 0.05 mol / L DHB methanol solution, which can be stored in a refrigerator at 4°C;

[0113] (4) Take 0.5 μL of the G7 sugar solution in step 1 and 0.5 μL of the NCPH matrix solution in step 2, and spot them on the same spot hole of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix. Dry naturally at 15 °C;

[0114] (5) As a control, take 0.5 μL of the G7 sugar solution in step 1 and 0.5 μL of the DHB matrix solution in step 2, 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. Dry naturally at 15 °C;

[0115] (6) The target plate is sent to the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0116] In Example 2, Figure 4 The MALDI mass spectra of G7 detected using DHB and NCPH as matrices. 1175.370 Da corresponds to [G7+Na] + It can be seen that under the low temperature conditions controlled in this example, NCPH does not react with the reducing end of the sugar chain, which makes it easier to examine the characteristics of NCPH itself as a matrix. Figure 4 The signal-to-noise ratio of B is significantly higher than Figure 4 As shown in A, NCPH itself has the MALDI matrix properties for detecting reducing sugars and is an ideal matrix for detecting reducing sugars.

[0117] Example 3: Combination of NCPH and DHB to construct a target plate derivatization strategy for reducing sugars

[0118] 1. Selection of acidic catalyst in composite matrix

[0119] (1) In ddH 2A 9% HAc solution was prepared in MeOH with a volume ratio of 0.05 mol / L, a DHB solution, a CA solution, a DHCA solution, a CCA solution, a CHCA solution and a SA solution were prepared in MeOH, and the 0.05 mol / L NCPH solution prepared in Example 2 was mixed with all the above acidic solutions in equal volumes to prepare NCPH / HAc, NCPH / DHB, NCPH / CA, NCPH / DHCA, NCPH / CCA, NCPH / CHCA and NCPH / SA composite matrix solutions;

[0120] (2) Take 0.5 μL of the G7 sugar solution in Example 2 and 1 μL of the seven composite matrix solutions in step (1) and spot them on the same spot hole of the target plate in sequence. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix well and react at 60 °C for 60 min.

[0121] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0122] In Example 3, Figure 5 The schematic diagram of the target plate derivatization of reducing sugars based on NCPH / DHB, wherein NCPH is a reactive matrix with a hydrazine reactive group that can react with the hemiacetal end of the reducing sugar. The acidic matrix is ​​a catalyst that can significantly catalyze the derivatization reaction of NCPH and reducing sugars. Excess NCPH can be used as an efficient matrix for MALDI mass spectrometry detection of reducing sugars without purification and separation, and is suitable for the target plate derivatization strategy, which can realize the derivatization detection of reducing sugars. Figure 6 The effect of different acidic catalysts and NCPH composite matrices on the derivatization reaction of G7. It can be seen that the acidic catalyst can significantly catalyze the reaction process to achieve complete derivatization of G7, and the organic acid matrix can significantly enhance the detection effect. From the signal intensity and signal-to-noise ratio of the derivatized product, as well as the matrix background and co-crystal morphology of the composite matrix, it can be seen that the comprehensive detection effect of the NCPH / DHB composite matrix is ​​significantly better than that of other composite matrices.

[0123] 2. Optimization of NCPH concentration in NCPH / DHB composite matrix

[0124] (1) Preparation of NCPH / DHB composite matrix: DHB solution with a concentration of 0.05 mol / L and NCPH 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 were prepared respectively, and the corresponding molar ratios of NCPH to DHB were 1:2.5, 1.25, 0.833, 0.625 and 0.5 respectively. The DHB solution and the series of NCPH solutions were mixed in equal volumes in sequence to form NCPH / DHB composite matrix solutions with different NCPH concentrations;

[0125] (2) Take 0.5 μL of the G7 sugar solution in Example 2 and 1 μL of the NCPH / DHB composite matrix solution in step (1) and spot them on the same spot hole of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix well and react at 55°C for 30 min.

[0126] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0127] In Example 3, Figure 7 A in the figure is the effect of different concentrations of NCPH on the derivatization yield of G7. It can be seen that when the concentration of NCPH is 0.04 mol / L, the comprehensive evaluation of derivatization yield and product signal-to-noise ratio is the highest.

[0128] 3. Optimization of DHB concentration in NCPH / DHB composite matrix

[0129] (1) Preparation of NCPH / DHB composite matrix: NCPH 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 were prepared respectively, and the corresponding molar ratios of NCPH to DHB were 1:0.625, 1.25, 1.875, 2.5, 3.125, respectively. The NCPH solution and the series of DHB solutions were mixed in equal volumes to form NCPH / DHB composite matrix solutions with different DHB concentrations;

[0130] (2) Take 0.5 μL of the G7 sugar solution in Example 2 and 1 μL of the NCPH / DHB composite matrix solution in step (1) and spot them on the same spot hole of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix well and react at 55°C for 30 min.

[0131] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0132] In Example 3, Figure 7 B in the figure shows the effect of different concentrations of DHB on the derivatization yield of G7. It can be seen that when the concentration of DHB is 0.1 mol / L, the comprehensive evaluation of derivatization yield and product signal-to-noise ratio is the highest.

[0133] 3. Optimization of reaction temperature for target plate derivatization

[0134] (1) preparing a NCPH / DHB composite matrix, preparing a 0.04 mol / L NCPH solution and a 0.1 mol / L DHB solution, wherein the corresponding molar ratio of NCPH to DHB is 1:2.5, and mixing the NCPH solution and the DHB solution in equal volumes to form a NCPH / DHB composite matrix solution;

[0135] (2) Take 0.5 μL of the G7 sugar solution in Example 2 and 1 μL of the NCPH / DHB composite matrix solution in step (1) and spot them on the same spot hole of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix. React at 45, 50, 55, 60 and 65 °C for 30 min respectively;

[0136] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0137] In Example 3, Figure 7 C in it is the effect of different reaction temperatures on the derivatization yield of G7. It can be seen that when the reaction temperature is 55 °C, the comprehensive evaluation of the derivatization yield and the product signal-to-noise ratio is the highest. The NCPH / DHB composite matrix of the present application is compared with the reactive matrix 2, 5-dihydroxybenzoylhydrazide (DHBH) previously studied by the inventors (Zhao, XY, Guo, C., Huang, Y., Huang, LL, Ma, G., Liu, YQ, He, Q., Wang, HW, Chen, KS, Pan, YJ, Combination strategy of reactive and catalytic matrices for qualitative and quantitative profiling of N-glycans in MALDI-MS. AnalyticalChemistry, 2019, Volume 91, 9251-9258), this application only requires a lower reaction temperature (55 °C) to achieve true complete derivatization. The true complete derivatization defined in this application is a derivatization yield of more than 99%. The latter requires a higher temperature of 80 °C to achieve the complete derivatization of reducing sugars defined in this article (the complete derivatization defined in this article refers to a derivatization yield of more than 95%, see Figure 1 , the data in this figure comes from the original experimental data records of the inventor’s previous research).

[0138] 4. Optimization of reaction time for target plate derivatization

[0139] (1) preparing NCPH / DHB composite matrix, preparing 0.04 mol / L NCPH solution and 0.1 mol / L DHB solution, and mixing the NCPH solution and the DHB solution in equal volumes to form a NCPH / DHB composite matrix solution;

[0140] (2) Take 0.5 μL of the G7 sugar solution in Example 2 and 1 μL of the NCPH / DHB composite matrix solution in step (1) and spot them on the same spot hole 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 20, 25, 30, 35 and 40 min respectively;

[0141] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0142] In Example 3, Figure 7 The effect of different reaction times on the derivatization yield of G7 in D. It can be seen that when the reaction time is 30 min, the derivatization yield and product signal-to-noise ratio can reach the best.

[0143] Example 4: Qualitative investigation of NCPH / DHB composite matrix for detecting oligosaccharides of different molecular weights

[0144] 1. NCPH / DHB as a matrix to detect oligosaccharides and dextran mixtures of different molecular weights

[0145] (1) Prepare 50 pmol / 0.5 μL of G1, G2, G3, G4, G5, G6 sugar solutions and 1 mg / mL of D3K sugar solution respectively, which can be stored in a refrigerator at 4°C;

[0146] (2) Take 0.5 μL of the G1, G2, G3, G4, G5, G6 sugar solutions and D3K sugar solution in step (1) and 1 μL of the NCPH / DHB composite matrix solution in Example 3, and spot them on the same spot hole of the target plate in sequence. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix. React at 55 °C for 30 min to ensure that the oligosaccharides can react completely;

[0147] (3) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0148] In Example 4, Figure 8 A in the figure is the MALDI mass spectrum of G1, G2, G3, G4, G5 and G6 detected by NCPH / DHB composite matrix. When NCPH / DHB is used as the matrix, the detection of small molecule oligosaccharides has little background interference and a high signal-to-noise ratio. Figure 8 Figure B is the MALDI mass spectrum of D3K qualitatively detected by NCPH / DHB composite matrix. The detection of glucan mixture also has a high signal-to-noise ratio, and each mass spectrum peak can be distinguished and assigned. The difference between adjacent oligosaccharide peaks is 162 Da, corresponding to one glucose residue, and the peak shape shows a standard normal distribution in the mass range of 1000-3000 Da, covering most of the protein. N- Sugar chain. It can be seen that the NCPH / DHB composite matrix has a broad spectrum of applicability to both small molecule oligosaccharides and dextran.

[0149] 2. NCPH / DHB as a matrix test N- Glycan Standards

[0150] (1) Prepare 50 pmol / 0.5 μL of G7 sugar solution, 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 ℃;

[0151] (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 different gradient concentrations, which can be stored in a -20 ℃ refrigerator;

[0152] (3) 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 1 μL of the NCPH / DHB composite matrix solution in Example 3, and spot them on the same spot hole 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 min to ensure that the sugar standard can react completely;

[0153] (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 DHB methanol solution in Example 2, and spot them on the same spot hole of the target plate. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix. Dry naturally at room temperature for 30 min;

[0154] (5) The target plate is sent into the MALDI mass spectrometer, and the positive ion reflection mode is selected for data acquisition.

[0155] In Example 4 Fig. 9 To detect different N- Detection limit of glycan standards. Fig. 9 AD in the figure is the spectrum detected with NCPH / DHB as the matrix. Fig. 9 EH in the figure is the spectrum detected with DHB as the matrix. The specific detection limit results are summarized in Table 1. When G7, NGA3, NA2 and Man-8 were detected with DHB as the matrix, the detection limit was 500 fmol, while when NCPH / DHB was used as the matrix, the detection limit was as low as 50 fmol. It can be seen that NCPH / DHB has the best detection effect and is suitable for the detection of oligosaccharides and polysaccharides.

[0156] Example 5: Quantitative investigation of the stability and linear range of the method using NCPH / DHB as the matrix

[0157] (1) Prepare a G7 sugar solution with a concentration of 2 pmol / 0.5 μL as the internal standard solution, which can be stored in a refrigerator at 4 °C;

[0158] (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 -20 °C refrigerator;

[0159] (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 NCPH / DHB composite matrix solution in Example 3, and spot them on the same spot hole 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 min. Send the target plate to the MALDI mass spectrometer, use the positive ion reflection mode, and sample 55 times in the same spot hole;

[0160] (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 NCPH / 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 pipette 10 times directly on the MALDI target plate to mix, and react at 55 °C for 30 min. Send the target plate to the MALDI mass spectrometer, collect data in positive ion reflection mode, and randomly sample 10 times in each well to take the average value;

[0161] (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 NCPH / 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 pipette 10 times directly on the MALDI target plate to mix, and react at 55 °C for 30 min. Send the target plate to the MALDI mass spectrometer, collect data in positive ion reflection mode, and randomly sample each well 10 times to take the average value;

[0162] (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 NCPH / 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 pipette 10 times directly on the MALDI target plate to mix, and react at 55 °C for 30 min. Send the target plate to the MALDI mass spectrometer, collect data in positive ion reflection mode, and randomly sample 10 times in each well to take the average value;

[0163] In Example 5, Fig.10 A in the figure is the reproducibility of the signal when G7 is detected using DHB and NCPH / DHB as matrices. By normalizing the mean of the signal intensity of 55 points, it can be seen intuitively that when NCPH / DHB is used as the matrix, the G7 ion intensity remains basically stable throughout the test process, with a relative standard deviation of 8.74%, which is significantly lower than the control group matrix (68.19%). It can be seen that when NCPH / DHB is used as the matrix, the method has good stability, which is conducive to improving the accuracy of quantitative analysis. Fig.10BD in the sample was G7 as the internal standard. The linear ranges of NGA3, NA2 and Man-8 were investigated in the positive ion reflection mode. It was found that they showed a good linear relationship in the range of 0.2-20 pmol / 0.5 μL. 2 >0.996. It can be seen that NCPH / DHB can be used as a matrix for reliable quantitative analysis of protein sugar chains of different molecular weights, and the target plate derivatization strategy based on NCPH / DHB can realize the qualitative and quantitative detection of protein sugar chains.

[0164] Example 6: Effect of NCPH / DHB on protein in peach fruit N- Quantitative analysis of glycans

[0165] The present invention is self-made N - Glycans are based on standard glycoproteins N - Preparation method of polysaccharide, using PNGase A enzymatic method N - polysaccharide, the specific steps are as follows:

[0166] Total protein extraction. Weigh 1 g of peach fruit sample and use buffer solution Y [1 mol / L Tris-HCl (pH 8.2), 10% ( w / v ) Sodium lauryl 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] were extracted at 65 ℃, centrifuged at 12000 rpm for 20 min at room temperature, and the supernatant was transferred to a new centrifuge tube. An appropriate amount of cold phenol was added, vortexed for 1 min, centrifuged at 12000 rpm at 4 ℃ for 20 min, and the supernatant was discarded. Cold buffer solution Z (50 mmol / L Tris-HCl, pH 8.2) was added for washing twice, and then 0.1 mol / L cold ammonium acetate methanol solution was added and precipitated at -20°C overnight.

[0167] (2) Enzymatic hydrolysis. Dissolve the extracted protein in 0.01 mol / L Tris-HCl (pH 8.2), heat at 100°C for 10 min to denature the protein, and then cool to room temperature. Add 30 μg trypsin, 30 μg chymosin, and 2 μL of 0.2 mol / L calcium chloride, and incubate at 37°C for 24 h for enzymatic hydrolysis. Then add 0.5 μL PNGase A, mix well, and incubate at 37°C for 24 h for enzymatic hydrolysis.

[0168] (3) Purification. The sample obtained by enzymatic hydrolysis was centrifuged and the reductive nature of the dissociation was analyzed by C18 solid phase extraction column and graphitized carbon solid phase extraction column. N-polysaccharide purification. C18 solid phase extraction column is used to remove impurity peptides, proteins and other contaminants. The purification process is as follows: 4 mL of methanol is used to activate the column, and 4 mL of ultrapure water is used to balance the column. The supernatant is added to the column, and the column is eluted with ultrapure water 3 times, 1 mL each time, and the eluate is collected. Then, a graphitized carbon solid phase extraction column is used to remove impurities from salts and small molecular polar compounds. The specific process is as follows: 3 mL of acetonitrile and 1 mL of 50% acetonitrile aqueous solution are used to activate the column in turn, and 3 mL of ultrapure water is used to balance the column. The sugar solution collected during the C18 purification process is loaded and 5 mL of ultrapure water is used to elute and remove impurities. 2 mL of 50% acetonitrile aqueous solution is used to elute and collect the eluted sugar solution, vacuum centrifuge at room temperature to remove the solvent, and redissolve in 25 μL of ultrapure water. It can be stored in a -20 ℃ refrigerator;

[0169] (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 NCPH / DHB composite matrix solution in Example 3, and spot them on the same spot hole of the target plate in sequence. Use a pipette to repeatedly pipette 10 times directly on the MALDI target plate to mix, and react at 55 °C for 30 min. Send the target plate to the MALDI mass spectrometer, and collect data in positive ion reflection mode;

[0170] (5) Mass calibration by internal standard method. N -glycan data were normalized and then the N -The relative content of polysaccharides in peaches before and after ripening.

[0171] In Example 6, Fig.11 To study the protein of peach fruit before and after ripening using NCPH / DHB as a matrix N -MALDI mass spectra of glycans after target plate derivatization; Fig.11 A in the figure is the detection N - Mass spectra of glycans and the attribution of mass spectrometry peaks after magnification and the corresponding sugar chain structure diagram; Fig.11 B in the figure is the normalized content of different N -Glycan content analysis heat map. The results showed that the NCPH / DHB matrix was successfully used to analyze 24 polysaccharides in peach fruit samples. N -Qualitative and quantitative analysis of polysaccharides, peach fruit N -Glycans are mannose and complex N- Mainly polysaccharides. Fig.11 C in the peach fruit ripening process N- The relative content of polysaccharides in the samples was 8 N- The relative content of polysaccharides showed a significant upward trend and 1 N-The relative content of polysaccharides was significantly decreased, which may be the important influencing factors regulating peach fruit ripening and play an important role in fruit ripening and ripening. N- The potential relevance of glycomic expression is provided.

[0172] Embodiment 7: Reducing Sugar MALDI Target Plate In Situ Labeling Kit

[0173] Preparation of NCPH matrix solution: Add the solvent suitable for NCPH into the container containing the uniformly recrystallized NCPH matrix powder, shake it ultrasonically, fully dissolve it, centrifuge it, and set it aside.

[0174] Preparation of DHB matrix solution: Add the solvent suitable for DHB into the container containing the uniformly recrystallized DHB matrix powder, ultrasonically shake, fully dissolve, centrifuge, and set aside.

[0175] Preparation of NCPH and DHB. The NCPH solution and DHB solution obtained in (1) and (2) are fully shaken and mixed in a matrix compounding container, centrifuged, and set aside.

[0176] Preparation of the solution of reducing sugar to be tested. Redissolve the sample to be tested with an acidic solution suitable for reducing sugar, shake and mix thoroughly, centrifuge, and set aside.

[0177] In Example 7, it is expected that the MALDI matrix configuration and the compounding of NCPH and DHB are performed only at the location where the labeling analysis of the reducing sugar sample is to be performed, and usually the compounding is used immediately to ensure the efficient use of the test kit. The three containers in the test kit have such adaptation convenience and shape and size that the containers are adapted to centrifugal operation. Stably, a container for accommodating uniform powders of NCPH and DHB recrystallization, a container for accommodating a solvent adapted to the NCPH matrix, a solvent adapted to the DHB matrix, and an acidic aqueous solution adapted to the sample to be tested of the reducing sugar, a container for accommodating equal volumes of compounded NCPH solution and DHB solution and fully mixing, or these three containers have the characteristic of avoiding light, so that it can be stably stored for a long time at room temperature. The test kit preferably further includes instructions for using the reagents included, and the instructions include the storage and use conditions of the provided matrix powder and solvent, and precautions.

[0178] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Application of 2-nitro-4-carboxyphenylhydrazine as a MALDI reactive matrix complexed with 2, 5-dihydroxybenzoic acid in reducing sugar analysis.

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

3. The use according to claim 1, characterized in that: The application in reducing sugar analysis is to perform derivatization qualitative and quantitative detection on reducing sugar, which specifically includes the following steps: (1) Reagent preparation: An NCPH solution is prepared with an equal volume mixed solvent of organic solvent 1 and organic solvent 2, wherein NCPH is 2-nitro-4-carboxyphenylhydrazine; a DHB solution is prepared with organic solvent 1 as a solvent, wherein DHB is 2, 5-dihydroxybenzoic acid; the NCPH solution and the DHB solution are mixed in equal volumes to prepare an NCPH / DHB composite matrix solution; an acidic aqueous solution of the reducing sugar to be tested is prepared; the organic solvent 1 is selected from any one or more polar organic solvents miscible with water having a polarity greater than or equal to ethanol; the organic solvent 2 is selected from any one or more dimethyl sulfoxide or N,N-dimethylformamide; (2) Sample: Take equal volumes of the aqueous solution of the reducing sugar to be tested and the NCPH / DHB composite matrix solution, spot them on the same spotting hole of the MALDI target plate, mix them evenly directly on the target plate, and obtain the spotted target plate; (3) Target plate derivatization reaction: The target plate after spotting is placed at 45-65°C for 20-40 min, the mixed sample spot is completely dried, and then MALDI mass spectrometry analysis is performed.

4. The use according to claim 3, characterized in that: In step (1), the concentration of the NCPH solution is 0.02-0.10 mol / L.

5. The use according to claim 3, characterized in that: In step (1), the concentration of the DHB solution is 0.025-0.125 mol / L.

6. The use according to claim 3, characterized in that: In step (3), the detection mode adopted by the MALDI mass spectrometry analysis is positive ion linear or reflectance mode.

7. A kit for in situ labeling of reducing sugars on a MALDI target plate using NCPH via a hydrazone reaction, the kit comprising: (a) a container containing uniform recrystallized powder of NCPH matrix, (b) a container containing uniform recrystallized powder of DHB matrix, (c) a container containing a solvent suitable for NCPH matrix, (d) a container containing a solvent suitable for DHB matrix, (e) a container containing a volume sufficient to mix the NCPH matrix solution and the DHB matrix solution prepared according to the requirements using the suitable solvent, and (f) a container containing an acidic aqueous solution suitable for the reducing sugar sample to be tested.

8. The kit according to claim 7, characterized in that The NCPH matrix recrystallized uniform powder and the DHB matrix recrystallized uniform powder are high-purity powders that have undergone a recrystallization process, and the high purity refers to 99.9% and above.

9. The kit according to claim 7, characterized in that The solvent adapted to the NCPH matrix is ​​a mixed solvent of an analytically pure or chromatographic grade organic solvent 1 and an organic solvent 2; the solvent adapted to the DHB matrix is ​​an analytically pure or chromatographic grade organic solvent 1; the organic solvent 1 is selected from any one or more polar organic solvents miscible with water having a polarity greater than or equal to ethanol; the organic solvent 2 is selected from any one or more of dimethyl sulfoxide or N,N-dimethylformamide.

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