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A method for the enrichment and separation of glycopeptides based on two-dimensional porous crystalline nitrogen-doped carbon materials

A technology of nitrogen-doped carbon and crystals, which is applied in the fields of material analytical chemistry and post-translational modification proteomics, can solve the problems of cumbersome hydrazide chemical method, low glycopeptide ionization efficiency, and only targeting specific glycoforms. Achieve the effect of simple and controllable operation, high enrichment selectivity and high selectivity

Active Publication Date: 2020-08-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At present, the mass spectrometry analysis of the glycopeptide structure in the glycoprotein hydrolyzate is the main means to obtain protein glycosylation information. The problem it faces is: due to the low ionization efficiency of glycopeptides, the number only accounts for all peptides after enzymatic hydrolysis. 2% to 5%[6], its mass spectrometry response is easily suppressed by high-abundance non-glycopeptides
The existing glycopeptide enrichment methods mainly include lectin affinity method, hydrazide chemical method, hydrophilic interaction chromatography, boron affinity method, etc. [7-10], each method has its own shortcomings and limitations, such as agglutination Glycopeptides only target specific glycoforms, and the coverage of glycopeptides is small; hydrazide chemical method is cumbersome to operate; hydrophilic interaction chromatography has the problem of insufficient selectivity of glycopeptides of hydrophilic materials, etc.

Method used

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  • A method for the enrichment and separation of glycopeptides based on two-dimensional porous crystalline nitrogen-doped carbon materials
  • A method for the enrichment and separation of glycopeptides based on two-dimensional porous crystalline nitrogen-doped carbon materials
  • A method for the enrichment and separation of glycopeptides based on two-dimensional porous crystalline nitrogen-doped carbon materials

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Experimental program
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Effect test

Embodiment 1

[0060] Using C2N as the enrichment material, glycopeptides were enriched in SPE mode. Put 2 mg of material into a centrifuge tube, redissolve 5 μL of spin-dried fetuin hydrolyzate (1 mg / mL) in 100 μL of 85% acetonitrile / 0.1% formic acid solution, after loading, use 80% of the volume of 200 μL respectively Wash with acetonitrile / 0.1% formic acid, 70% acetonitrile / 0.1% formic acid and 60% acetonitrile / 0.1% formic acid solution, and finally elute with 30 μL of 50% acetonitrile / 0.1% formic acid solution. The eluate is analyzed directly on the mass spectrometer. The result is as figure 1 As shown, the signals of glycopeptides are all in the mass spectrum, indicating that the materials used have high selectivity for glycopeptides.

Embodiment 2

[0062] Using C2N as the enrichment material, glycopeptides were enriched in dSPE mode. Put 2 mg of material into a centrifuge tube, redissolve 5 μL of spin-dried fetuin hydrolyzate (1 mg / mL) in 100 μL of 85% acetonitrile / 1% formic acid solution, after loading, use 80% of the volume of 200 μL respectively Wash with acetonitrile / 1% formic acid, 70% acetonitrile / 1% formic acid and 60% acetonitrile / 1% formic acid solution, and finally elute with 30 μL of 50% acetonitrile / 1% formic acid solution. The eluate is analyzed directly on the mass spectrometer. The result is as figure 2 As shown, the signals of glycopeptides are all in the mass spectrum, indicating that the materials used have high selectivity for glycopeptides.

Embodiment 3

[0064] Using C2N as the enrichment material, the schematic diagram of the sheet structure and layer spacing of the material used, as well as the schematic diagram of the pore size and pore structure are as follows image 3 As shown, glycopeptides were enriched in SPE mode. Put 1 mg of the material into the tip, redissolve 5 μL of the spin-dried fetuin hydrolyzate (1 mg / mL) in 30 μL of 85% acetonitrile / 0.1% formic acid solution, after loading the sample, use 30 μL of 80% acetonitrile / 1% formic acid, 70% acetonitrile / 1% formic acid and 60% acetonitrile / 1% formic acid solution, and finally eluted with 30 μL of 50% acetonitrile / 1% formic acid solution. The eluate is analyzed directly on the mass spectrometer.

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Abstract

The present invention relates to the fields of material analysis chemistry, post-translational modification proteomics and the like, and provides a method for separating and enriching glycopeptides. According to the method, a highly nitrogen-doped carbon material having a two-dimensional porous crystal structure contacts a proteolysate, the hydrophilic interaction, the electrostatic interaction, CH3-pi and other actions between the material and glycopeptides are used, glycopeptides are separated and enriched by using a solid-phase extraction (SPE) or dispersive solid-phase extraction (dSPE) separation mode, and the sample is analyzed through mass spectrometry. According to the present invention, by optimizing the compositions, the pH values, the buffer salts and other parameters of the sample solution, the leaching solution and the eluent, the selective enrichment of glycopeptides is achieved; and the method has advantages of good stability, high selectivity, large adsorption capacity,strong flexibility, easy and controllable operation and the like, and can be used for the glycopeptide enrichment of standard products and complex samples.

Description

technical field [0001] The invention relates to the fields of material analysis chemistry and post-translation modification proteomics, in particular to a method for enriching and separating glycopeptides. [0002] technical background [0003] Protein glycosylation is one of the most important and common post-translational modifications in organisms, and is closely related to biological processes and physiological functions such as cell adhesion, signal transduction, apoptosis, and immune response [2-4]. As an important biomarker, glycoprotein has been widely used in disease diagnosis, early warning and drug efficacy evaluation [5]. At present, the mass spectrometry analysis of the glycopeptide structure in the glycoprotein hydrolyzate is the main means to obtain protein glycosylation information. The problem it faces is: due to the low ionization efficiency of glycopeptides, the number only accounts for all peptides after enzymatic hydrolysis. 2% to 5% [6], its mass spectr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/40
CPCG01N1/405
Inventor 梁鑫淼李秀玲张小菲
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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