Rapid quantitative analysis method and application of n-glycans based on maldi-ms and stable isotope labeling

A stable isotope and quantitative analysis technology, applied in the field of glycoproteomics analysis, can solve the problems of limited glycan quantitative analysis, instrument response value difference, lack of internal standard or external standard, etc., so as to reduce experimental cost, improve quantitative ability, The effect of reducing the complexity of experiments

Active Publication Date: 2020-07-31
EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH +1
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Problems solved by technology

Nowadays, many analytical detection techniques are applied to monitor the changes of glycans, such as capillary electrophoresis, liquid chromatography and mass spectrometry, but these techniques have more or less shortcomings, such as the difference between samples caused by different ionization efficiencies, instrument Differences between response values ​​and lack of internal or external standards, etc.
In addition, although some isotope quantitative techniques have also been applied to the quantitative analysis of glycans, there are still many disadvantages, such as lengthy reaction time (10h-18h), destruction of acidic groups caused by extreme reaction conditions (acidity, high temperature), The post-source and in-source analysis of acidic sugars during mass spectrometry detection severely limit the quantitative analysis of glycans

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  • Rapid quantitative analysis method and application of n-glycans based on maldi-ms and stable isotope labeling
  • Rapid quantitative analysis method and application of n-glycans based on maldi-ms and stable isotope labeling
  • Rapid quantitative analysis method and application of n-glycans based on maldi-ms and stable isotope labeling

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Experimental program
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Embodiment 1

[0032] A kind of N glycan rapid quantitative analysis method based on MALDI-MS and stable isotope labeling, this method comprises the following steps (see figure 1 ):

[0033] (1) Microwave-assisted rapid enzyme digestion: standard glycoprotein (10ug) or serum (5ul) was dissolved in 20ul reaction solution (containing 10mM sodium phosphate, 0.13% sodium lauryl sulfate, 10mM dithiothreitol), Boil for 10min in a water bath at 100°C, add 2.4ul 10% NP-40, mix well, then add 0.5ul PNGase F, and digest under microwave-assisted conditions for 20min (the maximum output power of the microwave oven used in the experiment is 700W, and the reaction is selected lowest gear) to release N-glycans in the form of glycosamines.

[0034] (2) The sugar amine obtained in step (1) is rapidly derivatized with d0 / d5-benzoyl chloride (d0 represents non-isotopic labeling; d5 represents isotopic labeling), and the reaction conditions are: prepare benzoyl chloride solution (because benzoyl chloride is v...

Embodiment 2

[0041] In order to verify the derivatization effect of benzoyl chloride and sugar amine, the pH, temperature and reaction time that will affect the derivatization efficiency were optimized and explored (results in figure 2 ), the experiment was based on the derivatization efficiency of benzoyl chloride and Man5GlcNAc2 (one of the five glycans belonging to the standard glycoprotein-RNase B), figure 2 -A is: the influence of the pH of the reaction environment on the derivation efficiency, it can be seen that when the pH reaches 9.0, the best derivation effect can be achieved; figure 2 -B is: the influence of reaction temperature on derivation efficiency, it can be seen that when the temperature is at 20°C, the best derivation effect can be achieved; figure 2 -C is: the influence of reaction time on derivation efficiency, it can be seen that when the reaction time reaches 40min, the best derivation effect can be achieved.

Embodiment 3

[0043] Three kinds of standard glycoproteins-ribonuclease B, fetal bovine protein Fetuin and immunoglobulin IgG were analyzed respectively (the results are shown in Figure 3), Figure 3-A For: quantitative analysis of ribonuclease B glycans, when d0-benzoyl chloride and d5-benzoyl chloride-derived N glycans are mixed at a molar ratio of 1:1, it can be seen that the five N-glycans in the standard glycoprotein Almost all glycans exist in the form of iso-high ion pairs (the state of iso-high ion pairs is consistent with the molar ratio of 1:1); Figure 3-B For: quantitative analysis of fetal bovine protein Fetuin glycans, when d0-benzoyl chloride and d5-benzoyl chloride-derived N glycans are mixed at a molar ratio of 1:1, it can be seen that the five N-glycans in the standard glycoprotein Almost all glycans exist in the form of equal ion pairs; Figure 3-C For: Quantitative analysis of immunoglobulin IgG glycans, when the N-glycans derived from d0-benzoyl chloride and d5-benzoyl...

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Abstract

The invention discloses an MALDI-MS based and stable isotope labeled N-glycan rapid quantitative analysis method and application of the MALDI-MS based and stable isotope labeled N-glycan rapid quantitative analysis method, and belongs to the field of glycoproteomics analysis. The method comprises the following steps that (1) under the assistance of microwave, PNGase F makes N-glycans released in a form of osamine, (2) d0 / d5-benzoyl chloride has a nucleophilic reaction with osamine respectively and then purified, (3)d0-benzoyl chloride and d5-benzoyl chloride-derived glycans are mixed at a molar ratio of 1: 1, (4) mixed d0 / d5-benzoyl chloride derived N-glycans are subjected to a methylamine reaction, (5) MALDI-MS detection is performed, (6) data are analyzed; the MALDI-MS based and stable isotope labeled N-glycan rapid quantitative analysis method and the application of the MALDI-MS based and stable isotope labeled N-glycan rapid quantitative analysis method disclosed by the invention realize simultaneous quantitative analysis of neutral sugar and acid sugar, reduce the experimental cost, improve the experimental efficiency and shorten the reaction time, and have a wider linear quantitative range, can be used for serum glycan analysis, and have the medical application values.

Description

technical field [0001] The invention belongs to the field of glycoproteomics analysis, in particular to a rapid quantitative analysis method for N-glycans based on MALDI-MS and stable isotope labeling. Background technique [0002] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) technology was born in the late 1980s. It is a "soft ion" mass spectrometry technology, which has the characteristics of difficult cracking of samples, strong molecular ion peaks, and high sensitivity. Thermally unstable and non-volatile biological samples provide a new way, and gradually become the preferred method for analyzing protein samples, peptides, and nucleic acids. [0003] Stable isotope labeling technology is mainly based on the characteristics of stable isotopes and their compounds. In nature, the chemical and biological properties of stable isotopes and their compounds are the same as those of corresponding ordinary elements and compounds, but they have differen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/34G01N1/38G01N27/64
CPCG01N1/34G01N1/38G01N27/64
Inventor 刘欣王畅吴亦可刘胜张亮
Owner EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH
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