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A method for the relative quantification of n-glycan chains based on mass spectrometry

A relatively quantitative, sugar chain technology, applied in the field of biochemical analysis, can solve the problems of low abundance, microscopic inhomogeneity, low ionization efficiency, etc., and achieve the effect of improving fragmentation, improving ionization efficiency, and high derivatization efficiency.

Active Publication Date: 2022-03-18
FUDAN UNIV
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Problems solved by technology

Studies have reported that N-glycans may become a serum marker for cancer. Therefore, analyzing the structure of N-glycans and further quantitative analysis is a necessary prerequisite for in-depth understanding of the role played by N-glycans in biological processes. However, practice shows that The above research is facing great difficulties. First, although there are many types of proteins containing glycosylation modifications, their abundance is very low. Second, since N-glycan chains are synthesized by non-template, they have microscopic heterogeneity, and ionic The conversion efficiency is low, and there are still many bottlenecks in the analysis of mass spectrometry. Third, although there are many methods to achieve the quantification of N-glycans, such as metabolic labeling, 18 O labeling, chemical labeling, etc., but these quantitative methods still have the following defects, all of which are based on isotopes 2 D, 13 C, 15 N and 18 O's tagging implements the introduction of poor-quality tags, etc.

Method used

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  • A method for the relative quantification of n-glycan chains based on mass spectrometry
  • A method for the relative quantification of n-glycan chains based on mass spectrometry
  • A method for the relative quantification of n-glycan chains based on mass spectrometry

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 Reductive amination and the investigation experiment of metal chelation reaction derivation efficiency

[0033] 100 μg of DP7 (10 μg / μL) was mixed with 232 μg of aniline-functionalized DOTA (5.8 μg / μL) and lyophilized, dissolved in 40 μL of methanol solution (acetic acid / methanol: v / v 15:85), at 37°C After 1 hour of reaction, the reducing agent sodium cyanoborohydride (acetic acid / methanol: v / v 15:85) was added so that the final concentration was 5 mg / mL, and the total system was 50 μL, and the reaction was continued at 37°C for 2 hours. MALDI mass spectrometry was performed on the DP7 before the reaction and the DP7 after the reaction. DP7 was almost completely converted into DOTA-Lu-labeled DP7, and it was almost invisible before underrivatization. The results were as follows image 3 (a) and 3(b);

[0034] After lyophilizing 75 μL of the DOTA-derivatized DP, add 20 μL of HoCl 3 (4.82μg / μL, 10mM NH 4 AC,pH5.6) or LuCl 3 (5.00μg / μL, 10mM NH 4 AC, pH 5...

Embodiment 2

[0035] Example 2 Investigation of the ability of aniline-functionalized DOTA-labeled sugar chains to rechelate rare earth metal ions to improve the sugar chain signal

[0036] 100 μg of DP7 (10 μg / μL) was mixed with 232 μg of aniline-functionalized DOTA (5.8 μg / μL) and lyophilized, dissolved in 40 μL of methanol solution (acetic acid / methanol: v / v 15:85), at 37°C After reacting for 1 hour, add the reducing agent sodium cyanoborohydride (acetic acid / methanol: v / v 15:85) to make the final concentration 5 mg / mL, and the total system is 50 μL. After continuing to react for 2 hours at 37 ° C, take out 5 μL frozen After drying, add 20 μL of LuCl 3 (5.00μg / μL, 10mM NH 4 AC, pH 5.6), react at 37°C for 1 hour, add 10 μg of DP7 and mix well, take out 1 μL and dilute 10 times with ultrapure water for MALDI mass spectrometry analysis, the results are as follows Figure 4 (a) and 4(b).

Embodiment 3

[0037] Example 3 Investigating the accuracy and repeatability of DOTA-labeled sugar chain rechelation of rare earth metal ions relative quantitative method

[0038] According to the method of Example 1, after derivatizing DP7 with DOTA-Lu and DOTA-Ho respectively, take equimolar DOTA-Lu-labeled DP7 and DOTA-Ho-labeled DP7 and mix them for MALDI mass spectrometry analysis. The results are as follows Figure 5 As shown in (a); the signal intensity of DOTA-Lu-labeled DP7 is close to the theoretical ratio of DOTA-Ho-labeled DP7 signal intensity, and different molar ratios (1:10,1:5,1:2,1:1,2: 1,5:1,10:1) DOTA-Lu-labeled DP7 and DOTA-Ho-labeled DP7 were mixed, analyzed by MALDI mass spectrometry, and the intensity ratio obtained from the experiment was compared to the theoretical graph to obtain Figure 5 (b), the results show that the method of the present invention has good quantitative accuracy (R=0.9995), the experiment was repeated three times, and the coefficient of variation...

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Abstract

The invention belongs to the technical field of biochemical analysis, and relates to a method for the relative quantification of N-sugar chains based on mass spectrometry. The method uses aniline-functionalized macrocyclic compounds to mark N-sugar chains and then chelates rare earth metal ions for relative quantification, wherein aniline is used Functionalized DOTA derivatized with N‑glycan chains in chelating rare earth metal ions Ho 3+ / Lu 3+ , the two-step reaction has a high derivatization efficiency; the test results show that the ionization efficiency of sugar chains in MALDI-MS negative ion reflection mode can be improved after derivatization of DOTA chelated with rare earth metal ions, and the concentration of sugar chains can be improved. Fragmentation in mass spectrometry is helpful for sugar chain structure analysis. The present invention is used in the difference analysis of N-sugar chain expression levels in the serum of patients with colorectal cancer and normal people. The results show that this method can achieve multi-label quantification by chelating different rare earth metal ions, and has good quantitative accuracy. and repeatability.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, and relates to N-glycomics analysis, in particular to a method for the relative quantification of N-sugar chains based on mass spectrometry, in particular to a method for relabeling N-sugar chains using aniline functionalized macrocyclic compounds. A new method for the relative quantification of chelated rare earth metal ions. This method can improve the ionization efficiency of sugar chains in negative ion reflection mode of MALDI-MS after derivatizing DOTA chelated with rare earth metal ions, and improve the fragmentation of sugar chains in mass spectrometry, which is helpful for the analysis of sugar chain structures ; By chelating different rare earth metal ions, multi-labeling and quantification can be realized, and it has good quantitative accuracy and repeatability. Background technique [0002] The prior art discloses that N-glycosylation is the most ubiquitous post-translat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/64G01N1/38G01N1/42
CPCG01N1/38G01N1/42G01N27/64
Inventor 陆豪杰张莹杨丽君
Owner FUDAN UNIV
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