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A method for pretreatment of glycosylated protein samples based on homogeneous reaction system

A glycosylated protein and sample pretreatment technology, which is applied in the field of glycosylated protein sample pretreatment, can solve the problems of enrichment efficiency to be improved, slow mass transfer rate, etc., achieve excellent enrichment and treatment, and improve pretreatment Speed, the effect of shortening the time of the processing

Active Publication Date: 2021-08-27
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the enrichment methods currently used are all based on heterogeneous interaction systems
Due to the inherently slow mass transfer rate of heterogeneous systems, the enrichment efficiency of the existing enrichment methods needs to be improved

Method used

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  • A method for pretreatment of glycosylated protein samples based on homogeneous reaction system
  • A method for pretreatment of glycosylated protein samples based on homogeneous reaction system
  • A method for pretreatment of glycosylated protein samples based on homogeneous reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Glycosylated protein pretreatment method based on homogeneous interaction system for analysis of standard glycoprotein samples

[0028] Preparation of glycoprotein sample enzymatic hydrolysis solution: Add 1mg human serum immunoglobulin (IgG) to 1mL 0.1M bicarbonate / 8M urea solution (pH 8.2), 20μL 1M dithiothreitol (DTT), mix well Then react at 37°C for 2h. Add 7.2 mg of iodoacetamide (IAA) to the above solution, and react for 40 min at room temperature under the condition of avoiding light. After adding 40 μL (40 μg) of trypsin solution, the system was reacted at 37° C. for 16 h. The resulting solution was desalted through a SPE column. The obtained enzymatic hydrolyzate of glycoprotein samples was stored in a -30°C refrigerator for future use.

[0029] Glycosylated protein pretreatment and MALDI-TOF MS analysis based on homogeneous interaction system:

[0030] 1. Add 0.3mg / mL aqueous solution of gold nanocluster material to the centrifuge tube;

[0031] 2. Ultras...

Embodiment 2

[0041] Glycosylated protein pretreatment method based on homogeneous interaction system for analysis of standard glycoprotein samples

[0042] Preparation of glycoprotein samples: Add 1 mg of human serum immunoglobulin (IgG) to 1 mL of 0.1 M bicarbonate / 8 M urea solution (pH 8.2), 20 μL of 1 M dithiothreitol (DTT), and mix well at 37 ℃ reaction 2h. Add 7.2 mg of iodoacetamide (IAA) to the above solution, and react for 40 min at room temperature under the condition of avoiding light. After adding 40 μL (40 μg) of trypsin solution, the system was reacted at 37° C. for 16 h. The resulting solution was desalted through a SPE column. The obtained enzymatic hydrolyzate of glycoprotein samples was stored in a -30°C refrigerator for future use.

[0043] Glycosylated protein pretreatment and MALDI-TOF MS analysis based on homogeneous interaction system:

[0044] 1. Add 0.5mg / mL aqueous solution of gold nanocluster material to the centrifuge tube;

[0045] 2. Ultrasonic the above m...

Embodiment 3

[0055] Glycosylated protein pretreatment method based on homogeneous interaction system for analysis of standard glycoprotein samples

[0056] Preparation of glycoprotein samples: Add 1mg horseradish peroxidase (HRP) to 1mL 0.1M ammonium bicarbonate / 8M urea solution (pH 8.2), 20μL 1M dithiothreitol (DTT), mix well at 37 ℃ reaction 2h. Add 7.2 mg of iodoacetamide (IAA) to the above solution, and react for 40 min at room temperature under the condition of avoiding light. After adding 40 μL (40 μg) of trypsin solution, the system was reacted at 37° C. for 16 h. The resulting solution was desalted through a SPE column. The obtained enzymatic hydrolyzate of glycoprotein samples was stored in a -30°C refrigerator for future use.

[0057] Glycosylated protein pretreatment and MALDI-TOF MS analysis based on homogeneous interaction system:

[0058] 1. Add 0.6 mg / mL aqueous solution of gold nanocluster material to the centrifuge tube;

[0059] 2. Ultrasonic the above mixed system a...

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Abstract

The invention provides a glycosylated protein sample pretreatment method based on a homogeneous reaction system. In the method, the water-soluble gold nanocluster material is dissolved in water, and acetonitrile is used for separation and enrichment of glycosylated polypeptides. The method is based on the enrichment of glycosylated polypeptides by a homogeneous system of water-soluble gold nanocluster materials. The method has high sensitivity and is suitable for pretreatment of post-translationally modified protein samples in biological samples.

Description

technical field [0001] The invention relates to sample pretreatment of glycosylated protein, in particular to a method for pretreatment of glycosylated protein sample based on a homogeneous reaction system. Background technique [0002] Protein post-translational modification (PTMs) is a hotspot in proteomics research in recent years. Protein glycosylation modification is the most common and one of the most important protein post-translational modifications. It plays a key regulatory role in many important life activities. In addition, many disease diagnostic markers and therapeutic targets are derived from glycosylated proteins. Therefore, the study of protein glycosylation modification is of great significance for the diagnosis and treatment of diseases. [0003] At present, the study of protein glycosylation and glycosylation sites mainly relies on mass spectrometry (MS). However, due to the low abundance of glycosylated proteins, the mass spectrometry signal of glyco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/08
CPCG01N30/06G01N30/08G01N2030/067
Inventor 王方军李吉楠
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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