CN108398482B

CN108398482B - Use of 2-phenyl-3- (p-aminophenyl) acrylonitrile as matrix in MALDI-MS analysis of saccharides

Info

Publication number: CN108398482B
Application number: CN201810212509.0A
Authority: CN
Inventors: 国新华; 玲玲; 姜世梅; 马瑶
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2018-03-15
Filing date: 2018-03-15
Publication date: 2021-03-30
Anticipated expiration: 2038-03-15
Also published as: CN108398482A

Abstract

The invention relates to application of 2-phenyl-3- (p-aminophenyl) acrylonitrile as a matrix in MALDI-MS (matrix-assisted laser desorption ionization-mass spectrometry) analysis of saccharides, and the 2-phenyl-3- (p-aminophenyl) acrylonitrile as a reactive matrix has high sensitivity and high selectivity when being used for MALDI-MS analysis of saccharides, thereby solving the problems that the saccharides are low in ionization efficiency in MALDI and are often inhibited by other samples. The 2-phenyl-3- (p-aminophenyl) acrylonitrile can perform non-reductive amination reaction with the aldehyde group of the saccharide, and thus can be used as a saccharide derivatization reagent, so that the ionization efficiency of the saccharide in MALDI-MS is improved, and on the other hand, the 2-phenyl-3- (p-aminophenyl) acrylonitrile can also be used as a MALDI matrix to assist the desorption and ionization of saccharide compounds.

Description

Use of 2-phenyl-3- (p-aminophenyl) acrylonitrile as matrix in MALDI-MS analysis of saccharides

Technical Field

The invention relates to the field of biochemical detection, in particular to application of 2-phenyl-3- (p-aminophenyl) acrylonitrile (2-phenyl-3- (p-aminophenyl) acrylonitrite, PAPAN) as a Matrix in Matrix-Assisted Laser Desorption Ionization time of flight Mass Spectrometry (MALDI-MS) analysis of saccharides.

Background

MALDI-MS technology has been developed rapidly as a soft ionization mass spectrometry since reported by Tanaka et al in 1988, and has the characteristics of high sensitivity, high throughput, high salt tolerance, simple sample preparation and the like, thus playing an important role in biological sample analysis, environmental analysis, clinic and the like. In the MALDI-MS technique, a matrix forms a co-crystal with the sample to be measured, and the matrix absorbs laser energy and transfers it to the analyte to assist the analyte in resolving the ions. Commonly used MALDI matrices are small organic molecules with conjugated structures, such as alpha-cyano-4-hydroxycinnamic acid (CHCA), Sinapic Acid (SA), 3-hydroxypicolinic acid (HPA), 2, 5-dihydroxybenzoic acid (DHB), and the like. In sample analysis, the selection of MALDI matrix plays a key role, e.g. CHCA is commonly used for the analysis of polypeptides and proteins smaller than 10000Da, SA is used for proteins larger than 10000Da, HPA is mostly used for nucleic acid analysis, while DHB is suitable for the analysis of polypeptides as well as polysaccharides. Although there are many studies on the matrix, the choice of the matrix is still empirical, and there are still great challenges especially for some samples that are difficult to ionize and have low abundance, such as saccharides.

Protein glycosylation plays an important role in biological processes such as protein stability, immunogenicity, intracellular biological conduction and the like, and identification of oligosaccharide is very important in glycosylation research. The rapid analysis by MALDI-MS technique, a relatively simple spectrum analysis (MALDI gives rise to a single charge peak) is particularly suitable for carbohydrate analysis. However, MALDI-MS analysis of carbohydrates still presents significant challenges due to their low ionization efficiency. Together with the inhibitory effect caused by other substances having high proton affinity such as polypeptides, sugar analysis is difficult, and therefore, it is urgent to selectively increase the ionization efficiency of sugars.

Methylation and reductive amination are currently common derivatization methods. However, methylation shifts different sugars with different mass-to-charge ratios (m/z), which makes the spectrum analysis difficult, and methylated sugars need to be separated from the reaction system before mass spectrometric detection. In recent years, reductive amination reagents such as 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP) have been reported in succession to improve the efficiency of sugar ionization, but the reductive amination reaction requires the use of the reducing agent NaBCN3 and therefore has been removed before detection.

Disclosure of Invention

The invention provides an application of PAPAN as a matrix in MALDI-MS (matrix assisted laser desorption ionization-mass spectrometry) analysis of saccharides, wherein the PAPAN derivatizes saccharides through a non-reductive amination reaction, so that the ionization efficiency of the saccharides is improved, particularly, the PAPAN has strong absorption at 355nm and can be used as a matrix to assist the ionization of the saccharides, so that excessive reaction derivatization reagents do not need to be separated, and the application of PAPAN as the matrix in MALDI-MS analysis of the saccharides not only improves the sensitivity of saccharide detection, but also improves the selectivity of saccharide detection, and solves the problems that the saccharides are low in ionization efficiency in MALDI and are often inhibited by other samples. The structural formula of the PAPAN is shown as the formula (1):

the application of the PAPAN as the matrix in MALDI-MS analysis of saccharides comprises the following specific steps:

a. mixing and dissolving PAPAN and sugar to be detected in a methanol solution containing 5% acetic acid, and reacting at 65 ℃ for 1h to obtain a mixed solution;

b. and (b) spotting the mixed solution obtained in the step (a) on an anchorchip target plate matched with a MALDI mass spectrum, spotting pure water on the reaction solution, naturally drying to form a uniform sample layer, and performing MALD-MS mass spectrum analysis on the dried sample.

As a more excellent technical scheme of the invention: the concentration of the PAPAN in the step a is 2-5 mg/mL.

As a more excellent technical scheme of the invention: the volume of the mixed solution in the step b is 0.5-1 mu L.

As a more excellent technical scheme of the invention: the pure water in the step b is 0.5-1 mu L.

The synthesis method of PAPAN comprises the following steps: a. first, 20mL of methanol and 0.115g of 0.005mol of metallic sodium were added to a round-bottom flask, and the mixture was stirred until the metallic sodium was completely dissolved and turned into sodium methoxide. Then adding 1.17g and 0.01mol of benzyl cyanide, 1.51g and 0.01mol of p-nitrobenzaldehyde, stirring the mixed solution for 2 hours at room temperature until the raw materials react to generate yellow precipitate, and washing the filtered solid with methanol and water for three times to obtain 2.25g of bright yellow product; b. a round bottom flask was charged with 2.5g of the bright yellow product from step a, 0.01mol, 40mL of ethanol and 11.3g of SnCl, stannous chloride dihydrate, 0.05mol₂·2H₂O, the mixture was refluxed for 0.5 h. With saturated NaHCO₃Neutralized to weak alkalinity, diluted with 50mL of water, and then extracted with chloroform. The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated using a rotary evaporator to give 2.09g of a yellow solid, i.e., PAPAN.

Compared with the prior art, the invention has the following beneficial effects:

the PAPAN has a conjugated structure, is simple to synthesize and low in cost, has strong absorption at the 355nm wavelength of an instrument, can perform non-reductive amination reaction with aldehyde groups of saccharides, is used as a saccharide derivative reagent, improves the saccharide ionization degree in MALDI-MS (matrix-assisted laser desorption ionization-mass spectrometry), has simple reaction steps, does not need other reagents, can serve as a good MALDI matrix to assist the saccharide compound in analyzing and ionizing, and improves the sensitivity and selectivity of MALDI-MS on saccharide analysis; the invention overcomes the phenomenon that the saccharides are often inhibited by other compounds, obtains very uniform crystals and improves the reproducibility of analysis.

Drawings

FIG. 1 is a schematic diagram of a synthetic PAPAN process;

FIG. 2 is a MALDI mass spectrum of PAPAN;

FIG. 3 is a graph of the ultraviolet absorption spectrum of PAPAN;

FIG. 4 is a MALDI mass spectrum of maltose detecting; FIG. 4a shows PAPAN as the substrate and FIG. 4b shows DHB as the substrate;

FIG. 5 is a schematic view of a MALDI-MS self-assembled microscope obtained by different spotting methods;

FIG. 6 is a MALDI mass spectrum of a mixture of maltohexaose and polypeptide (1:1) being detected; FIG. 6a PAPAN as a matrix assay and FIG. 6b DHB as a matrix;

FIG. 7 is a MALDI mass spectrum of a mixture of maltohexaose and polypeptide (1: 10) being detected; FIG. 7a PAPAN as the substrate assay and FIG. 7b DHB as the substrate;

FIG. 8 is a MALDI mass spectrum of an egg white proteolysis product; FIG. 8a PAPAN as the matrix test and FIG. 8b DHB as the matrix test.

Detailed Description

The present invention will be further illustrated by the following examples, but the present invention is not limited to the following examples.

The containers, reagents and the like used in the following examples are commercially available unless otherwise specified. The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The matrix-assisted laser desorption ionization time-of-flight mass spectrometer used in the following examples was of the type Autoflex speed TOF/TOF (Bruker Daltonics, Germany) and the laser was a 355nm wavelength Nd: YAG laser. Mass spectrometry test parameters: acceleration voltage: 20.000 kv; delayed extraction voltage: 18.000 kv; delay lead-out time: 150 ns; voltage of the reflector: 20.000 kv; lens voltage: 6.000 kv; frequency: 500 Hz.

Example 1: synthesizing and analyzing PAPAN, wherein the synthesis schematic diagram of PAPAN is shown in figure 1, and the specific steps are as follows:

1. first, 20mL of methanol and 0.115g of 0.005mol of metallic sodium were added to a round-bottom flask, and the mixture was stirred until the metallic sodium was completely dissolved and turned into sodium methoxide. Then, 1.17g and 0.01mol of phenylacetonitrile and 1.51g and 0.01mol of p-nitrobenzaldehyde were added, the mixture was stirred at room temperature for 2 hours until the reaction of the raw materials was completed to form a yellow precipitate, and the filtered solid was washed three times with methanol and water to obtain 2.25g of a bright yellow product.

2. A round bottom flask was charged with 2.5g, 0.01mol of the bright yellow product from step 1, 40mL of ethanol and 11.3g, 0.05mol of stannous chloride dihydrate SnCl₂·2H₂O, the mixture was refluxed for 0.5 h. With saturated NaHCO₃Neutralized to weak alkalinity, diluted with 50mL of water, and then extracted with chloroform. The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated using a rotary evaporator to give 2.09g of a yellow solid, i.e., PAPAN.

When the above-obtained PAPAN was subjected to MALDI mass spectrometry and ultraviolet absorption spectrum analysis, it was found that PAPAN was easily ionized in MALDI and strongly absorbed at 355nm, so that PAPAN satisfied the conditions as a matrix.

Example 2: the PAPAN is used as a reactive matrix for MALDI-MS detection of maltohexaose.

1. Preparing 10mM malt hexaose mother liquor (water solution), and storing in a refrigerator at 4 deg.C.

2. 2.5g/L of the PAPAN solution of example 1 was prepared using a 5% acetic acid in methanol. Can be stored in a refrigerator at 4 ℃ in dark.

3. And (3) putting 99 mu L of the PAPAN solution obtained in the step (2) into a 1.5mL centrifuge tube, adding 1 mu L of the maltohexaose mother liquor obtained in the step (1), uniformly mixing, and placing in a water bath at 65 ℃ for reacting for 1 h.

4. 0.5. mu.L of the reaction solution obtained in step 3 was spotted on an anchorchip target plate, and immediately thereafter 0.5. mu.L of pure water was spotted on the reaction solution, followed by natural drying at room temperature.

5. For comparison, the malt hexaose mother liquor in step 1 is diluted to 10 in a gradient manner^-4M, taking 1 mu L of the maltohexaose solution and 1 mu L of a common substrate 2, 5-dihydroxybenzoic acid (DHB, 10 g/L), wherein the solvent is acetonitrile water mixed solution, V_Acetonitrile:V_{Water (W)}1:1), then 1 μ L of the mixture was spotted on an anchorchip target plate and allowed to dry naturally at room temperature.

6. The mixture was analyzed by MALDI-MS. Mass spectral data were acquired in positive ion reflectance mode.

MALDI mass spectrum of maltohexaose analyzed by PAPAN and DHB as matrix in example 2 is shown in FIG. 4, which shows that PAPAN as matrix for detecting maltohexaose has stronger signal than that obtained by conventional matrix DHB

Example 3 comparison of the crystalline morphology formed by the two spotting patterns when PAPAN was used as a substrate.

1. 0.5. mu.L of the reaction solution of example 2 was spotted on an anchorchip target plate, and immediately thereafter 0.5. mu.L of pure water was spotted on the reaction solution, and the reaction solution was spotted and naturally dried at room temperature before drying.

2. 0.5. mu.L of the reaction solution of example 2 was spotted on an anchorchip target plate and allowed to dry naturally at room temperature.

3. The target plate was transferred to MALDI-MS and observed for morphology.

The left side of fig. 5 is the form under MALDI-MS self-prepared microscope obtained by spotting with the method of step 1 in example 3, and the right side of fig. 4 is the form obtained by spotting with the method of step 2 in example 3, which shows that the spotting method of the present invention forms more uniform crystals than the conventional spotting method, which is beneficial to improve the reproducibility of analysis.

Example 4: PAPAN was used as a reactive matrix for MALDI-MS detection of maltohexaose and polypeptide mixtures.

1. Preparing a mixed mother liquor of the maltohexaose and the polypeptide with a molar ratio of 1:1/1:10, wherein the concentration of the maltohexaose is 10mM, and the polypeptide sequence is Ac-EAIYAAPFAKKK.

2. mu.L of the mixed solution obtained in step 1 was added to 99. mu.L of a PAPAN solution (concentration: 2.5g/L, solvent: 5% acetic acid in methanol) and reacted at 65 ℃ for 1 hour.

3. 0.5. mu.L of the reaction solution obtained in step 2 was spotted on an anchorchip target plate, and immediately thereafter 0.5. mu.L of pure water was spotted on the reaction solution, followed by natural drying at room temperature.

4. For comparison, the maltohexaose and the polypeptide mother liquor in step 1 were diluted 100-fold, and 1. mu.L of the diluted solution was mixed with 1. mu.L of a common base, 2, 5-dihydroxybenzoic acid (DHB, 10 g/L) in acetonitrile water, V_Acetonitrile:V_{Water (W)}1:1) and then 1 μ L of the mixture was spotted on an anchorchicip target plate and allowed to dry naturally at room temperature.

5. The mixture was analyzed by MALDI-MS. Mass spectral data were acquired in positive ion reflectance mode.

FIG. 6 shows the measured molar ratio of 1:1, obtaining a MALDI mass spectrogram through the mixture of the maltohexaose and the polypeptide; FIG. 6a shows the detection using PAPAN as the matrix and FIG. 6b shows the detection using DHB as the matrix.

Fig. 7 shows the measured molar ratio of 1:10 obtaining a MALDI mass spectrum diagram of the mixture of the maltohexaose and the polypeptide; FIG. 5a shows the detection with PAPAN as the matrix and FIG. 5b shows the detection with DHB.

In FIGS. 6 and 7, M represents maltohexaose, and M represents polypeptide hydrogenation or alkali metal ion addition. By detecting the mixed test of oligosaccharide and polypeptide, it can be seen that the PAPAN is used as the reactive matrix, has good selectivity to saccharides, and overcomes the phenomenon that the saccharides are inhibited by substances with high proton affinity, such as polypeptide and the like.

Example 5 PAPAN as a derivatizing reagent and matrix for MALDI-MS detection of the N-sugars released by egg white proteolysis.

1. Ovalbumin and trypsin (30:1, W: W) were dissolved in 200uL of 50mM NH4HCO3(pH 7.8) in water (ovalbumin concentration 0.5mg/mL) and incubated at 37 ℃ for 12 hours for enzymatic hydrolysis. Subsequently 1. mu.L of peptide-N-glycosidase (PNGase F) was added and incubated at 37 ℃ for 18h for oligosaccharide release.

2. And (3) taking 100 mu L of the egg white protein enzymolysis product obtained in the step (1), freeze-drying and volatilizing the solvent of the egg white protein enzymolysis product, and storing the egg white protein enzymolysis product in a refrigerator at the temperature of 20 ℃ below zero.

3. Adding 100 μ L PAPAN solution (with concentration of 2.5g/L and solvent of 5% acetic acid in methanol) into the egg white proteolysis product obtained in step 2, and reacting at 65 deg.C for 1 h.

5. For comparison, 1. mu.L of the egg white proteolysis reaction solution obtained in the step was mixed with 1. mu.L of a common substrate, 2, 5-dihydroxybenzoic acid (DHB, 10g/L, solvent used was acetonitrile aqueous solution, V acetonitrile: V water 1:1), and 1. mu.L of the mixture was spotted on an anchorchicip target plate and dried naturally.

6. The mixture was analyzed by MALDI-MS. Mass spectral data were acquired in positive ion linear mode.

FIG. 8 is a MALDI mass spectrum of an egg white proteolysis product; FIG. 8a shows the use of PAPAN as a substrate; FIG. 8b shows the use of DHB as a substrate. The numbers 1-21 in the figure represent the peaks of the reaction product of ovalbumin N-sugar with PAPAN plus Na. The information on the structure of the detected N-sugar is shown in Table 1.

From example 5, it can be seen that PAPAN can selectively detect oligosaccharide from ovalbumin enzymolysis product (trypsin + PNGase F), while DHB only detects peptide fragment. This example further demonstrates that PAPAN has high selectivity for carbohydrate detection.

Table 1: molecular weight of N-sugar in ovalbumin and structural information.

Claims

The application of 2-phenyl-3- (p-aminophenyl) acrylonitrile as a matrix in MALDI-MS analysis of saccharides comprises the following steps:

a. mixing and dissolving PAPAN and sugar to be detected in a methanol solution containing 5% acetic acid, and reacting at 65 ℃ for 1h to obtain a mixed solution; the concentration of the PAPAN is 2-5 mg/mL;

b. and (b) spotting the mixed solution obtained in the step (a) on an anchorchip target plate matched with a MALDI mass spectrum, spotting pure water on the reaction solution, naturally drying to form a uniform sample layer, and performing MALD-MS mass spectrometry on the dried sample, wherein the volume of the mixed solution is 0.5-1 mu L.
2. Use of 2-phenyl-3- (p-aminophenyl) acrylonitrile as a matrix according to claim 1 for MALDI-MS analysis of saccharides, said pure water in step b being 0.5-1 μ L.