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Absolute quantitative analysis of IgG glycopeptide in serum

A quantitative analysis and glycopeptide technology, applied in the field of proteomics, can solve the problems of long cycle, complex chemical synthesis, expensive glycosyltransferase, etc.

Active Publication Date: 2019-06-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis method is complicated and the cycle is very long; the glycosyltransferase used in the enzymatic synthesis method is very expensive; due to the complex sugar chain, many connection methods and branches, and the existence of isomers, it is difficult to separate and purify

Method used

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  • Absolute quantitative analysis of IgG glycopeptide in serum
  • Absolute quantitative analysis of IgG glycopeptide in serum
  • Absolute quantitative analysis of IgG glycopeptide in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Simultaneous absolute quantification of glycopeptides in IgG1, IgG2, and the sum of IgG3 and IgG4 (IgG3 / 4).

[0059] (1) Trypsin hydrolysis of standard IgG and serum: Add 50 μL of 50 mM (pH=7.4) ammonium bicarbonate solution to 5 mg IgG / 50 μL serum each, then add 10 μL of 0.3M dithiothreitol, and reduce for 180 min at 37°C; Add 20 μL of 0.3M iodoacetic acid to the reaction product, and store in the dark at 20°C for 60 minutes; add 60 μg of trypsin to the reaction solution, and perform enzymatic hydrolysis reaction at 40°C for 72 hours. After the enzymatic hydrolysis, 1% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0060] (2) Enrichment of standard IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase XAqua C18 (4.6*150mm, 5μ) chromatographic column for glycopeptide enrichment. The mobile phase uses methanol and water, and the condition is 90% (v / v) water isocratic elution; the detection wavelength is 1...

Embodiment 2

[0068] Absolute quantification of glycopeptides of the sum of IgG1, 2 and 3, 4, respectively.

[0069] (1) Trypsin hydrolysis of serum: Add 100 μL of 100 mM (pH=7.8) ammonium bicarbonate solution to 5 mg IgG / 50 μL serum, then add 10 μL of 0.5M dithiothreitol, and reduce for 120 min at 37 ° C; Add 20 μL of 0.5M iodoacetic acid, and store in the dark at 25°C for 40 minutes; add 75 μg of trypsin to the reaction solution, and perform enzymatic hydrolysis reaction at 37°C for 36 hours. After the enzymatic hydrolysis, 2% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0070] (2) Enrichment of IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase XAqua C18 (4.6*150mm, 5μ) chromatographic column for glycopeptide enrichment. The mobile phase is acetonitrile and water, the condition is 80% (v / v) water isocratic elution; detection wavelength is 280nm; TOF mass spectrometry analysis, the 5-12min fraction is IgG1, the 13-15...

Embodiment 3

[0078] (1) Trypsin hydrolysis of serum: Add 150 μL of 100 mM (pH=7.8) ammonium bicarbonate solution to 5 mg IgG / 50 μL serum, then add 10 μL of 0.6M dithiothreitol, and reduce for 90 min at 37°C; Add 20 μL of 0.6M iodoacetic acid, and store in the dark at 30°C for 30 minutes; add 150 μg of trypsin to the reaction solution, and perform enzymatic hydrolysis reaction at 35°C for 18 hours. After the enzymatic hydrolysis, 5% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0079] (2) Enrichment of IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase XAqua C18 (4.6*150mm, 5μ) chromatographic column for glycopeptide enrichment. The mobile phase was acetonitrile and water, containing 0.1% (v / v) formic acid. The condition is that the water phase changes from 98% (v / v) to 70% (v / v) linear gradient for 5-60 minutes to elute; the detection wavelength is 254nm; the column temperature is 35°C, and the eluent flow rate is 1.2mL...

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Abstract

The invention provides an absolute quantitative analysis of immune globulin G (IgG) glycopeptide in serum. The technical process comprises two parts: on the one hand, performing two-dimensional hydrophilic chromatography purification preparation of an IgG glycopeptide standard product and quantitative analysis thereof; on the other hand, performing the IgG glycopeptide absolute quantitative analysis in a serum sample by adopting a standard adding way. The preparation of the glycopeptide standard is performed by adopting the standard IgG, the preparation comprises: releasing of the glycopeptide, enriching of IgG glycopeptide through a reversed-phase chromatography, and separation purification and mass spectrum representation of the two-dimensional hydrophilic chromatography; the quantitative analysis performs PNGase F carbohydrate chain releasing on the glycopeptide standard, performing MRM quantitative analysis on the deglycosylated IgG glycopeptide fragment by adopting an internal standard curve, thereby acquiring molar concentration of each purified IgG glycopeptide; the IgG glycopeptide content determination in the serum is realized through a standard adding method, that is, onepart is added with the IgG glycopeptide standard with the known concentration, and the other part is free from adding the IgG glycopeptide standard, and then the MRM analysis is performed, the content of the IgG corresponding glycoform glycopeptide in the serum is computed according to the peak area difference and the adding standard glycopeptide concentration. On this basis, the glycopeptide absolute corresponding factor can be computed.

Description

technical field [0001] The invention relates to the development of an absolute quantitative analysis method for immunoglobulin G glycopeptides in human serum, and belongs to the technical field of proteomics. Background technique [0002] Glycosylation modification of proteins is ubiquitous in the human body, and about 50% of human proteins are modified by glycosylation. Immunoglobulin (Ig) is a class of serum glycoproteins with antibody activity. Includes IgG, IgM, IgA, IgE and IgD. Among them, IgG accounts for about 75% of the total serum immunoglobulins. IgG is further divided into four subtypes: IgG1 accounts for 60-70%, IgG2 accounts for 15-20%, IgG3 accounts for 5-10%, and IgG4 accounts for 1-7%. IgG consists of Fc (crystalline fragment) and Fab (antigen-binding fragment), both of which contain conservative N-glycosylation modifications. Many studies have shown that abnormal changes in N-glycosylation of IgG Fc segment are closely related to the occurrence and deve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 梁鑫淼曹翠岩于龙付冬梅
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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