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Application of combined matrix dhb/dhbh in detection of reducing sugar in maldi mass spectrometry

A combined, mixed-matrix technology, applied in the field of sugar biochemical analysis, can solve the problems of Schiff base instability, limit wide application, sample signal suppression, etc., to improve crystallization uniformity, improve detection sensitivity, improve accuracy and stability sexual effect

Active Publication Date: 2020-10-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the Girard reagent T itself has a positive charge, and the excess derivatization reagent will inevitably cause the inhibition of the sample signal.
3-Aminoquinoline is a reactive substrate, but its reaction with reducing sugar is relatively slow, and the generated Schiff base is unstable, which limits further extensive application

Method used

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  • Application of combined matrix dhb/dhbh in detection of reducing sugar in maldi mass spectrometry
  • Application of combined matrix dhb/dhbh in detection of reducing sugar in maldi mass spectrometry
  • Application of combined matrix dhb/dhbh in detection of reducing sugar in maldi mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Synthesis and characterization of DHBH

[0078] 1. Synthesis of DHBH

[0079] Add 0.168 g (0.001 mol) of methyl 2,5-dihydroxybenzoate and 10 mL of methanol in turn into a round bottom flask, stir at room temperature for 5 min and mix well. Then slowly add 0.2 mL (about 0.004 mol) of 85% hydrazine hydrate solution dropwise, and stir overnight at room temperature at 25° C. until the raw materials react completely to form a brown precipitate.

[0080] 2. DHBH separation and purification

[0081] The excess solvent and hydrazine hydrate were removed by rotary evaporation at room temperature 25°C to obtain a viscous solid. Then, excess methanol and 1 mL deionized water were added to redissolve the product, and it was recrystallized using a rotary evaporator. The temperature was controlled at room temperature 25°C, and the speed of the solvent rotary evaporation was slowed down until about 3 mL of solvent remained in the flask. The precipitate was washed thre...

Embodiment 2

[0085] Example 2: DHBH itself is used as a MALDI matrix to detect carbohydrates

[0086] (1) Prepare 20pmol / μL G6 and 0.1mg / mL D2000 sugar solution, which can be stored in a refrigerator at 4°C;

[0087] (2) Prepare the DHBH methanol solution in 0.1mol / L embodiment 1, which can be stored in a refrigerator at 4°C;

[0088] (3) Prepare DHB methanol solutions of 0.01, 0.025, 0.05, 0.1, 0.2, 0.3 and 0.4mol / L respectively, which can be stored in a refrigerator at 4°C;

[0089] (4) Take 1 μL of the G6 sugar solution or D2000 sugar solution in step 1 and 1 μL of the DHBH methanol solution in step 2, respectively, and place them on the same well of the polished steel target plate in sequence. Use a pipette gun to pipette directly onto the MALDI target plate 10 times for mixing. Dry naturally at 15°C;

[0090] (5) As a control, take 1 μL of the G6 sugar solution or D2000 sugar solution in step 1 and 1 μL of the 0.1 mol / L DHB methanol solution in step 2, respectively, and spot on the...

Embodiment 3

[0093] Example 3: Combining DHBH and DHB to build a reducing sugar target plate derivatization strategy

[0094] 1. Optimization of the molar concentration ratio of organic acid matrix and DHBH in the mixed matrix

[0095] (1) Prepare DHB / DHBH mixed matrix, use DHB methanol solutions with different concentrations prepared in Example 2 as solvent and DHBH as solute, prepare 0.1M DHBH mixed solutions respectively. That is, the DHB / DHBH mixed matrix solution of 0.01:0.1, 0.025:0.1, 0.05:0.1, 0.1:0.1, 0.2:0.1, 0.3:0.1, 0.4:0.1;

[0096] (2) Prepare CHCA methanol solutions of 0.1, 0.05, 0.025, and 0.01M respectively, which can be stored in a refrigerator at 4°C;

[0097] (3) Prepare CHCA / DHBH mixed matrix, the CHCA / DHBH mixed matrix of 0.01:0.1, 0.025:0.1, 0.05:0.1 and 0.1:0.1 can adopt the preparation method in step (1), that is, use different concentrations in step (2) CHCA methanol solution was used as the solvent, DHBH was used as the solute, and 0.1M DHBH mixed solutions w...

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Abstract

The invention discloses an application of DHBH (2, 5-dihydroxybenzoyl hydrazine) as a novel reactive matrix in combination (DHB / DHBH) with DHB (2, 5-dihydroxybenzoic acid) in high-throughput qualitative and quantitative detection of reducing sugar. In MALDI mass spectrometry, high-throughput qualitative and quantitative analysis of reducing sugar can be achieved based on a DHB / DHBH target plate derivatization strategy. DHBH has hydrazide active groups, and can easily react with reducing sugar hemiacetal as a reactive matrix, so that the ionization efficiency can be remarkably improved; DHBH isused as a matrix and can assist in saccharide ionization, so that excessive DHBH does not need to be separated. DHB is an organic acid, and can greatly catalyze the hydrazone forming reaction; and DHB serving as the most common substrate of saccharides can assist in ionization of the saccharides. DHB / DHBH is used as a matrix and is applicable to detection of acid sugar and neutral sugar.

Description

technical field [0001] The invention relates to the field of sugar biochemical analysis, in particular to the application of a combined matrix of 2,5-dihydroxybenzoylhydrazide (DHBH) and 2,5-dihydroxybenzoic acid (DHB) in the detection of reducing sugars in MALDI mass spectrometry . Background technique [0002] Carbohydrates are the third type of biomacromolecules in living organisms. They have very important biological functions and are mainly used as energy sources, structural substances and functional substances. Carbohydrates play an important role in many life processes, such as fertilization, growth, differentiation, development, signal transduction and immune response. Glycosylation modification is one of the most extensive, complex and important types of protein post-translational modification. More than 50% of proteins will undergo glycosylation modification, and more and more studies have shown that glycosylation modification plays a vital role in the occurrence...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/64G01N1/38G01N1/34G01N1/28
CPCG01N1/28G01N1/34G01N1/38G01N27/64
Inventor 潘远江赵晓勇黄丽丽
Owner ZHEJIANG UNIV
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