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Antibody Protein Disulfide Bond Pairing Analysis Method

An analysis method and disulfide bond technology, applied in the field of antibody disulfide bond pairing analysis, to achieve the effect of abundant fragment ions and fewer enzyme cleavage methods

Active Publication Date: 2020-11-10
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to provide a method for analyzing antibody protein disulfide bond pairing, which overcomes the steric hindrance and the effect on peptides when the N-sugar pair is specifically enzymatically hydrolyzed in the existing disulfide bond pairing analysis technology. Inhibition of ionization efficiency, as well as the disadvantages of using multiple enzyme digestion methods, can accurately confirm and locate disulfide bond peptides with only a few enzyme digestion methods

Method used

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  • Antibody Protein Disulfide Bond Pairing Analysis Method
  • Antibody Protein Disulfide Bond Pairing Analysis Method
  • Antibody Protein Disulfide Bond Pairing Analysis Method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Analysis of antibody protein disulfide bond pairing (trypsin combined with Lys-C digestion)

[0047] Antibody protein disulfide bond pairing analysis steps:

[0048] (1) Removal of N-Glycosylations

[0049] Take 1mg antibody protein sample, add 50mM NH 4 HCO 3 (pH 8.0) solution to a volume of 20-50 μL, add 1 μL of peptide N-glycosidase PNGase F (NEB, 500000u / mL), mix well, and incubate at 37°C for 24 hours.

[0050] (2) Protein denaturation treatment

[0051] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0052] (3) Enzymolysis

[0053] Take 100 μg of the protein in (2), add Trypsin at 1:20 (wt / wt), add Lys-C at 1:200, combine with enzymatic hydrolysis of antibody protein, and incubate at 37°C for 4 hours.

[0054] (4) Red...

Embodiment 2

[0070] Example 2: Analysis of the complete molecular weight of antibody protein (trypsin digestion)

[0071] Antibody protein disulfide bond pairing analysis steps:

[0072] (1) Removal of N-Glycosylations

[0073] Take 1mg antibody protein sample, add 50mM NH 4 HCO 3 (pH 8.0) solution to a volume of 20-50 μL, add 1 μL of peptide N-glycosidase PNGase F (NEB, 500000u / mL), mix well, and incubate at 37°C for 24 hours.

[0074] (2) Protein denaturation treatment

[0075] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0076] (3) Enzymolysis

[0077] Take 100 μg of the protein in (2), add Trypsin to digest the antibody protein at 1:20 (wt / wt), and incubate at 37°C for 4 hours.

[0078] (4) Reduction and termination of enzymatic hydrolysis

[007...

Embodiment 3

[0084] Example 3: Analysis of the complete molecular weight of antibody protein (Lys-C digestion)

[0085] Antibody protein disulfide bond pairing analysis steps:

[0086] (1) Removal of N-Glycosylations

[0087] Take 1mg antibody protein sample, add 50mM NH 4 HCO 3 (pH 8.0) solution to a volume of 20-50 μL, add 1 μL of peptide N-glycosidase PNGase F (NEB, 500000u / mL), mix well, and incubate at 37°C for 24 hours.

[0088] (2) Protein denaturation treatment

[0089] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0090] (3) Enzymolysis

[0091] Take 100 μg of the protein in (2), add Lys-C to digest the antibody protein at a ratio of 1:200, and incubate at 37°C for 4 hours.

[0092] (4) Reduction and termination of enzymatic hydrolysis

[0093]...

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Abstract

The invention relates to an antibody protein disulfide bond paired analysis method. The method sequentially comprises the following steps that 1, N-saccharide removing treatment is conducted on protein; 2, denaturation treatment is conducted on the protein subjected to N-saccharide removing; 3, enzymolysis is conducted on the protein subjected to denaturation treatment through a specific enzyme under the non-reductive condition; 4, the protein subjected to enzymolysis is divided into two samples, reduction is conducted on one sample, the other sample is not subjected to reduction, and the two samples are both subjected to enzymolysis termination; 5, mass peptide spectrum analysis is conducted on disulfide bond peptide segments of the samples subjected to enzymolysis termination. According to the antibody protein disulfide bond paired analysis method, the spatial steric hindrance of N-saccharide to protein enzymolysis conducted through the specific enzyme and inhibition to peptide segment ionization efficiency in the prior art are overcome, enzyme digestion ways are reduced, and the disulfide bond peptide segments can be confirmed and positioned accurately.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody disulfide bond pairing analysis method. Background technique [0002] The disulfide bond is the S-S bond, which is a covalent bond between sulfur atoms in the form of -S-S- formed by the oxidation of two sulfhydryl groups. Cysteine ​​plays a very important role in forming a stable protein spatial structure, maintaining the correct spatial conformation, and regulating biological activity. The correct pairing of disulfide bonds is conducive to the rapid folding of the peptide chain and the formation of a tight and stable spatial structure, forming a local hydrophobic center, which can prevent water molecules from entering the interior of the peptide to destroy hydrogen bonds and form a stable higher-order structure region. [0003] The disulfide bond is only relatively stable, and its covalent bond is easily reduced and broken. When the disulfide bond is broken and reduced...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 吕锋华黄黎明环民霞刘周阳谭青乔
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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