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Positioning method of polypeptide disulfide bond

A positioning method and disulfide bond technology, applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve the problems of long detection cycle, low accuracy, and low throughput, and achieve long detection cycle and high cost long effect

Active Publication Date: 2016-11-23
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has the problems of high false positives and low accuracy, especially when there are two or more adjacent cysteines, it is impossible to determine the position of the disulfide bond
[0006] The method of using partial reduction method combined with MS / MS to analyze the disulfide bond position of polypeptide has also been reported (Chinese invention patent CN 102721734 B), but the sample processing process of this method is still a relatively complicated part. The quantity requirement is relatively large (2 mg), the sample purity requirement is high, after the reduction, it needs to be separated and purified by high performance liquid chromatography (HPLC), the partial reduction alkylation needs to be carried out under high temperature conditions, the mass spectrometry data quality is poor, manual analysis factors Very large, resulting in poor controllability and repeatability of the results, and only qualitative results can be obtained but quantitative results are difficult to obtain, and it is not easy to perform high-throughput analysis on a large number of different samples at the same time
In short, the existing methods based on partial reduction combined with MS / MS to analyze polypeptide disulfide bond positioning have large sample size requirements, high purity requirements, complex operations, low reproducibility, difficult analysis, low throughput, and high accuracy of results. Problems of low cost, high cost and long detection cycle

Method used

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  • Positioning method of polypeptide disulfide bond
  • Positioning method of polypeptide disulfide bond
  • Positioning method of polypeptide disulfide bond

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Simple and quick alkylation of partial reducing agent and subsequent total reduction

[0081] In this embodiment, a conotoxin polypeptide sample containing three pairs of disulfide bonds (molecular weight 1769.69Da, sequence: RCCVHPACHDCICCIT, MALDI-TOF detection mass spectrum is as follows figure 1 ) as an example to illustrate the positioning method of polypeptide disulfide bonds.

[0082] 1. Partial restoration

[0083] Weigh 0.1mg of conotoxin polypeptide sample containing three pairs of disulfide bonds, dissolve in 1ml ddH 2 In O, DTT (sigma) was added so that the final concentration was 10 mM, and the partial reduction was performed at room temperature for 10 min. After the reaction, use a C18 extraction column (strata TM -X, phenomenonex) to remove the remaining DTT (to avoid the disulfide bond being continuously reduced during subsequent alkylation), and the eluate was freeze-dried;

[0084] 2. Alkylation of IAM

[0085] The freeze-dried sample w...

Embodiment 2

[0088] Example 2. MALDI-TOF detection of partial reductive alkylation effect

[0089] Take 1 μL of the sample after partial reductive alkylation and subsequent complete reduction, and spot it on the MALDI-TOF (Bruker) sample target. After the sample is dried, add 0.3 μL matrix (CHCA, English full name α-Cyano-4-hydroxy-cinnamic acid, the full name in Chinese is α-cyano-4-hydroxycinnamic acid), after drying, add 2.5μL 0.1% TFA aqueous solution, let it stand for 5 seconds and then quickly suck it away, so as to play the role of simple desalination. Then put the sample target into the mass spectrometer, select the primary mass spectrometry reflectance mode detection method to detect the effect of partial reductive alkylation, the results are as follows figure 2 As shown, the mass spectrum peaks corresponding to one pair, two pairs and three pairs of disulfide bonds respectively appear obviously, and the effect of partial reductive alkylation is better.

Embodiment 3

[0090] Embodiment three, LC-MS / MS mass spectrometry detection

[0091] LC-MS / MS (using ThermoFisher Scientific's Q-Exactive mass spectrometer) mass spectrometry detection of partially reduced alkylated polypeptide samples that have passed the MALDI-TOF mass spectrometry detection, the total detection time is 65min, and the core mass spectrometry parameters are as follows:

[0092] MS resolution: 70,000; scan range: 350-1800m / z; resolution of MS: 7,500; data dependent acquisition is the top 20 parent ions of MS for MS acquisition; fragmentation energy of MS is 27%; the dynamic exclusion time is set to 8 seconds;

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Abstract

The invention discloses a positioning method of a polypeptide disulfide bond. The comprises the steps that to-be-tested polypeptide is partially reduced in the solution environment of a disulfide bond containing reducing agent; the partially reduced polypeptide is alkylated in the solution environment of a polypeptide containing denaturing agent and an alkylated reagent; the alkylated polypeptide is completely reduced in the solution environment of the polypeptide containing denaturing agent and the alkylated reagent; a matrix auxiliary laser desorption ionization-flight time mass spectrum is used for performing molecular weight on products; samples qualified through detection are subjected to hygroplasm combination tandem mass spectrum detection, and mass spectrometric data is obtained; the spectrometric data is converted into a data format compatible with the mass spectrum identification software, then, the mass spectrum identification software is used for performing search comparison on the spectrum data and a reference polypeptide sequence database, and the proportion of the connecting position of the polypeptide disulfide bond and different connection modes is calculated. The method is small in sample amount requirement, low in purity requirement, easy to implement, high in repeatability, small in analysis difficulty, high in result accuracy, high in flux and low in cost.

Description

technical field [0001] The invention relates to the technical field of polypeptide structure determination, in particular to a method for locating polypeptide disulfide bonds. Background technique [0002] Disulfide bond is a common post-translational modification widely present in natural proteins and peptides, covalently cross-linked between two cysteines (Qiu Xiaoyan, Cui Meng, Liu Zhiqiang, Liu Shuying. Localization of disulfide bonds in proteins and its mass spectrometric analysis. Advances in Chemistry. Vol. 20, No. 6, 975-983). The formation of disulfide bonds is important for the spatial structure of proteins and polypeptides (Wedemeyer W J, Welker E, Narayan M, Scheraga HA. ​​Biochemistry.2000, 39: 4207-4216), maintenance of correct folding conformation (Pitt J J, da SilvaE, Gorman J J.J.Bio1.Chem., 2000, 275:6469-6478), maintaining and regulating its biological activity (Abkevich V I, Shakhnovich E I.J.Mo1.Bio1., 2000, 300:975-985), etc. all play a vital role . ...

Claims

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Application Information

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IPC IPC(8): G01N27/64
Inventor 林志龙刘杰闻博冯强
Owner SHENZHEN HUADA GENE INST
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