CN114460162B

CN114460162B - MALDI detection method of small molecular oligosaccharide

Info

Publication number: CN114460162B
Application number: CN202210027952.7A
Authority: CN
Inventors: 苗露; 李晨; 宋合兴; 周晓光; 李运涛
Original assignee: Guangdong Modiyoupu Biotechnology Co ltd; Bioisland Laboratory
Current assignee: Rongzhi Biotechnology Qingdao Co ltd; Bioisland Laboratory
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2023-08-15
Anticipated expiration: 2042-01-11
Also published as: CN114460162A

Abstract

The application belongs to the field of MALDI detection, and particularly relates to a MALDI detection method of small molecular oligosaccharides, which comprises the following steps: comprising the following steps: incubating the micromolecular oligosaccharide with 4-aminobiphenyl ammonia hydrochloride solution to prepare a reaction solution, then dripping the reaction solution onto a MALDI steel target, and drying at room temperature to perform MALDI detection. The application overcomes the defect that the traditional matrix of MALDI-TOF MS can only analyze macromolecular substances, and adopts 4-aminobiphenyl ammonia hydrochloride as the matrix of MALDI-TOF MS for analyzing oligosaccharides. The 4-aminobiphenyl ammonia hydrochloride has strong ultraviolet absorption, and simultaneously, the structure of aldehyde Cheng Xifu alkali in amino and saccharide molecules improves the ionization efficiency of the oligosaccharide, so that the signal intensity of the oligosaccharide is greatly improved.

Description

MALDI detection method of small molecular oligosaccharide

Technical Field

The application belongs to the field of MALDI detection, and particularly relates to a MALDI detection method of small molecular oligosaccharides.

Background

MALDI-TOF-MS is a novel soft ionization biological mass spectrum, and has been developed as an indispensable analysis tool for analyzing various types of biological molecules due to simple sample preparation, high analysis speed, high throughput, high sensitivity and high salt tolerance, and has been rapidly developed in recent years, thereby playing an important role in analysis and detection. The choice of the matrix is always focused, the perfection and improvement of the matrix performance can enable the detection result of MALDI-TOF-MS to have higher accuracy and sensitivity, smaller background noise and cleaner spectrogram, and the common matrix is generally a plurality of organic small molecules with conjugated structures, forms co-crystallization with the analyte, and absorbs laser energy to transmit to the analyte for ionization. In the case of performing saccharide analysis by MALDI-TOF-MS, MALDI detection of saccharides has a great challenge due to the low ionization efficiency of saccharides.

Disclosure of Invention

The application aims to provide a MALDI detection method of small-molecule oligosaccharides.

In order to achieve the above purpose, the application adopts the following technical scheme:

a method for detecting MALDI of small molecule oligosaccharides, comprising the steps of: incubating the micromolecular oligosaccharide with 4-aminobiphenyl ammonia hydrochloride solution to prepare a reaction solution, then dripping the reaction solution onto a MALDI steel target, and drying at room temperature to perform MALDI detection.

The 4-aminobiphenyl ammonia hydrochloride solution is methanol water solution with the concentration of 2.5mg/ml.

The preparation method of the reaction liquid comprises the following steps: mixing micromolecular oligosaccharide with 4-aminobiphenyl ammonia hydrochloride solution according to the mass ratio of 1:99, and incubating for 1-2 hours at the temperature of 60-65 ℃ in a water bath to prepare a reaction solution.

Compared with the prior art, the application has the beneficial effects that:

the application overcomes the defect that the traditional matrix of MALDI-TOF MS can only analyze macromolecular substances, and adopts 4-aminobiphenyl ammonia hydrochloride as the matrix of MALDI-TOF MS for analyzing oligosaccharides. The 4-aminobiphenyl ammonia hydrochloride has strong ultraviolet absorption, and simultaneously, the structure of aldehyde Cheng Xifu alkali in amino and saccharide molecules improves the ionization efficiency of micromolecular oligosaccharide, so that the signal intensity of the micromolecular oligosaccharide is greatly improved.

Drawings

FIGS. 1, 3 and 5 are MALDI diagrams of maltose, glucose and lactose, respectively, obtained by way of example;

FIGS. 2, 4 and 6 are MALDI diagrams of maltose, glucose and lactose, respectively, obtained by the method of comparative example.

Detailed Description

The present application will be further described in detail with reference to examples for better understanding of the technical scheme of the present application by those skilled in the art.

Example 1, matrix effect test of 4-aminobiphenyl ammonia hydrochloride; the ultraviolet test and MALDI mass spectrum test are carried out on the 4-aminobiphenyl ammonia hydrochloride, the result shows that the compound has good ultraviolet absorption, and the MALDI obtains the peak of the compound, which proves that the compound has the condition of being used as a matrix and meets the requirement of the matrix in MALDI-MS.

The structure of the 4-aminobiphenyl ammonia is shown as a formula (I).

Examples 2-4 MALDI mass spectrometry of oligosaccharides with 4-aminobiphenyl ammonia hydrochloride as matrix were performed, comprising the following steps:

1) Respectively preparing standard substance solutions of glucose, lactose and maltohexaose;

2) Dissolving 4-aminobiphenyl ammonia hydrochloride into methanol water solution to form matrix solution with concentration of 2.5mg/ml; wherein the volume ratio of methanol to water is 1:1;

3) Mixing the standard solution obtained in the step 1) with the matrix solution obtained in the step 2), heating to 60 ℃ and incubating for 1h to obtain a reaction solution, and then dripping 1 mu L of the reaction solution onto a MALDI steel target, and drying at room temperature to perform MALDI detection.

MALDI mass spectrometry was performed on the small molecule oligosaccharides prepared in examples 2 to 4, respectively, and the results are shown in FIGS. 1, 3 and 5.

Comparative examples 1 to 3, which differ from examples 2 to 4 in that 2, 5-dihydroxybenzoic acid DHB was used as a matrix at a concentration of 10g/L in a solvent mixture of acetonitrile and water at a ratio of acetonitrile to water of 1:1 by volume, comprising the following steps: the standard solution and the matrix solution were mixed, and then 1. Mu.L of the mixed solution was dropped onto a MALDI steel target, followed by drying at room temperature for MALDI detection. The results are shown in FIGS. 2, 4 and 6. From the results, it is clear that the use of the 4-aminobiphenyl ammonia hydrochloride of the present application as a matrix is excellent in comparison with conventional DHB as a matrix. Indicating that the matrix is capable of analyzing small molecule oligosaccharides.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims

1. A method for detecting MALDI of small-molecule oligosaccharides, comprising the steps of:

incubating the micromolecular oligosaccharide with 4-aminobiphenyl ammonia hydrochloride solution to prepare a reaction solution, then dripping the reaction solution onto a MALDI steel target, and drying at room temperature to perform MALDI detection;

the 4-aminobiphenyl ammonia hydrochloride solution is a methanol aqueous solution with the concentration of 2.5mg/ml;