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Method for determining composition of disulfide bond of recombinant human granulocyte colony stimulating factor

A colony-stimulating factor and disulfide bond technology, applied in the field of proteomics, can solve problems such as poor repeatability, high requirements for experimental equipment, and complicated processing procedures, and achieve high vigor, low data analysis difficulty, and simple operation.

Inactive Publication Date: 2018-07-27
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The pretreatment process of this method is complicated, the repeatability is poor, and the requirements for experimental equipment are high, so it is not suitable for use as a routine method.

Method used

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  • Method for determining composition of disulfide bond of recombinant human granulocyte colony stimulating factor
  • Method for determining composition of disulfide bond of recombinant human granulocyte colony stimulating factor
  • Method for determining composition of disulfide bond of recombinant human granulocyte colony stimulating factor

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Embodiment 1

[0047] 1. Pepsin hydrolysis of rhG-CSF samples

[0048] Pepsin (purchased from Sigma, source: pig gastric mucosa) was prepared into a 1 mg / ml solution with ultrapure water, and each 100 μl tube was frozen and stored for future use. Prepare 50 mM citric acid solution, adjust to pH 5.0 with 0.1 mol / L sodium hydroxide solution, and filter with 0.45 μm microporous membrane. Use this solution to dilute the rhG-CSF stock solution to 1 mg / ml, add 1 mg / ml pepsin according to the volume ratio of 40:1, vortex and mix three times, each time for 5 seconds, and then place it in a 37°C water bath for 16 hours after mixing. Take 100 μL of the above enzymatic hydrolysis product and place it in a liquid chromatography sampling bottle, which is the sample before reduction. Take out 100 μL of the enzymatic hydrolysis product and put it in an EP tube, add 1 μL of TCEP solution (concentration: 1mol / L, prepared with ultrapure water), vortex and mix three times, each time for 5 seconds, mix well an...

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Abstract

The invention relates to a method for determining composition of a disulfide bond of a recombinant human granulocyte colony stimulating factor, which belongs to the technical field of proteomics. Themethod combines a LC-Q-Q-TOF combination technology and a post-column on-line reduction method for determining an rhG-CSF disulfide bond and a disulfide bond mismatching proportion. The test step comprises the following steps: performing enzyme digestion on rhG-CSF under condition of pH 5.0 of a buffer solution by pepsin, performing LC-MS detection of a reduced sample and an unreduced sample, andperforming on-line post-column on-line reduction liquid chromatogram-mass spectrometry detection on the unreduced sample. The method can perform direct enzyme digestion on rhG-CSF under acidic condition, and omits denaturation and alkylation steps, so that operation is concise, the reaction condition is mild, repeatability is good, and the sensitivity is high; the post-column on-line reduction method is matched to obviously reduce the data analysis difficulty, the identification of the disulfide bond of rhG-CSF and calculating of the disulfide bond mismatching proportion result are more reliable, the experiment process is concise, and the data analysis is efficient.

Description

technical field [0001] The invention belongs to the technical field of proteomics, and relates to a method for determining disulfide bonds and disulfide bond errors in recombinant human granulocyte colony-stimulating factor (rhG-CSF) based on high performance liquid chromatography mass spectrometry technology and online post-column reduction technology. Proportional method. Background technique [0002] Disulfide bonds are a common post-translational modification of proteins, which play a very important role in stabilizing the spatial structure of proteins, maintaining conformation and regulating their biological activities. Therefore, exploring how disulfide bonds connect in proteins is of great significance for a comprehensive understanding of protein chemical structures. [0003] At present, the main methods for locating disulfide bonds are divided into non-fragment method and fragment method: non-fragment method is to measure on the basis of maintaining the original con...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88G01N2030/067G01N2030/8831
Inventor 刘海龙王英武刘斌孙莹莹
Owner JILIN UNIV
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