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Absolute quantitative analysis method of immunoglobulin G glycopeptide in serum

A technology of immunoglobulin and glycopeptide, applied in the field of proteomics, can solve the problems of lack of N-glycan chain standard products, limited production optimization of MRM technology, etc., and achieve the effect of high sensitivity, good accuracy and high repetition rate

Inactive Publication Date: 2018-12-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with the standardized quantitative research of metabolomics and proteomics, the quantitative research of MRM technology in glycomics is still in its infancy. Although the above case shows that MRM technology has potential in its clinical application, due to the lack of available N-glycans Application of standard, MRM techniques in monitoring production optimization of diagnostic and therapeutic antibodies remains limited

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  • Absolute quantitative analysis method of immunoglobulin G glycopeptide in serum
  • Absolute quantitative analysis method of immunoglobulin G glycopeptide in serum
  • Absolute quantitative analysis method of immunoglobulin G glycopeptide in serum

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Simultaneous absolute quantification of glycopeptides in IgG1, IgG2, and the sum of IgG3 and IgG4 (IgG3 / 4).

[0056] (1) Trypsin hydrolysis of serum: Add 50 μL of 50 mM (pH=7.4) ammonium bicarbonate solution to 50 μL of serum, then add 10 μL of 0.3M dithiothreitol, and reduce for 180 min at 37°C; add 0.3M iodine to the reaction product 20 μL of acetic acid, stored at 20°C in the dark for 60 minutes; 60 μg of trypsin was added to the reaction solution, and the enzymatic reaction was carried out at 40°C for 72 hours. After the enzymatic hydrolysis, 1% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0057] (2) Enrichment of IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase 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 190nm; the column temperature is 25°C...

Embodiment 2

[0062] Absolute quantification of glycopeptides of the sum of IgG1, 2 and 3, 4, respectively.

[0063] (1) Trypsin hydrolysis of serum: add 100 μL of 100 mM (pH=7.8) ammonium bicarbonate solution to 50 μL of serum, then add 10 μL of 0.5M dithiothreitol, and reduce for 120 min at 37°C; add 0.5M iodine to the reaction product 20 μL of acetic acid was stored at 25°C in the dark for 40 minutes; 75 μg of trypsin was added to the reaction solution, and the enzyme hydrolysis reaction was carried out at 37°C for 36 hours. After the enzymatic hydrolysis, 2% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0064] (2) Enrichment of IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase 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...

Embodiment 3

[0070] (1) Trypsin hydrolysis of serum: Add 150 μL of 100 mM (pH=7.8) ammonium bicarbonate solution to 50 μL of serum, then add 10 μL of 0.6M dithiothreitol, and reduce for 90 minutes at 37°C; add 0.6M iodine to the reaction product 20 μL of acetic acid was stored at 30°C in the dark for 30 minutes; 150 μg of trypsin was added to the reaction solution, and the enzyme hydrolysis reaction was carried out at 35°C for 18 hours. After the enzymatic hydrolysis, 5% (v / v) formic acid was added to terminate the enzymatic hydrolysis reaction.

[0071] (2) Enrichment of IgG glycopeptides: the enzymatic hydrolysis solution was passed through a reversed-phase 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,...

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Abstract

The invention provides an absolute quantitative analysis method of immunoglobulin G (IgG) glycopeptide in serum. The process includes the release of IgG glycopeptides in serum and the enrichment of IgG glycopeptides by chromatography. the collected IgG glycopeptide is divided into two parts, one part is subjected to profiling analysis of various glycoforms in the IgG glycopeptides, and another IgGglycopeptide is subjected to PNGase F enzymatic hydrolysis, and MRM quantitative analysis is performed on deglycosylated IgG peptides; and the content of glycopeptide corresponding to various glycoforms of IgG is calculated by combining the results of Profiling. The method of the invention can measure the molar concentration of each glycopeptide on each subtype of IgG in serum, and has the advantages of high sensitivity, good precision, high accuracy, high recovery rate, and good operability, and is suitable for high-flux absolute quantitative research of IgG glycopeptide in serum.

Description

technical field [0001] The invention relates to the development of a mass spectrometry absolute quantitative method for quantifying immunoglobulin G glycopeptides in human serum, and belongs to the technical field of proteomics. Background technique [0002] Immunoglobulin (Ig) is a class of soluble serum glycoproteins with antibody activity. According to the difference in the properties of the heavy chain, it can be divided into five types, including IgG, IgM, IgA, IgE and IgD. Among them, IgG is the main antibody component of serum, accounting for about 75% of the total serum immunoglobulin. IgG includes four subtypes: IgG1-4, IgG1 accounts for 60-70%, IgG2 accounts for 15-20%, IgG3 accounts for 5-10%, and IgG4 accounts for 1-7% (Mathiesen T, Hammarstrom L, Fridell E, et al. Aberrant IgG subclass distribution to measles in healthy seropositive individuals, in patients with SSPE and in immunoglobulin-deficient patients [J]. Clin Exp Immunol, 1990, 80(2): 202-5). IgG cons...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
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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