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Method for simultaneous quantification of protein abundance and the cysteine oxidation level and application of method

A cysteine ​​and protein technology, which is applied in the field of simultaneous quantification of protein abundance and cysteine ​​oxidation level, can solve the problems of inability to accurately determine changes in protein redox levels, and inability to simultaneously quantify protein abundance, etc. Conducive to detection and quantification, improved efficiency, and rapid identification by mass spectrometry

Inactive Publication Date: 2019-01-04
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to quickly identify small amounts of substances called cysteine or histidylate residue proteins through their specific chemical structure. It also includes methods like measuring these quantities over time to make sure they are consistently high quality. By analyzing this data we aim to discover new ways to better study them and develop future treatments based on those findings.

Problems solved by technology

This patented technical problem addressed in this patent relates to developing accurate tools for measuring both intracellular and extracelluar radical shifts during chemical reaction between specific types of compounds called reactants. Current analysis systems require complicated operations like liquid chromatography and gas phase ionization spectroscopy which may result in errors due to nonlinear responses caused by various factors involved in chemistry. Quantitationally analyzablequantum dyes have become increasingly popular because their ability to distinguish small amounts of certain substances allows for precise measurements without complex calculations.

Method used

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  • Method for simultaneous quantification of protein abundance and the cysteine oxidation level and application of method
  • Method for simultaneous quantification of protein abundance and the cysteine oxidation level and application of method
  • Method for simultaneous quantification of protein abundance and the cysteine oxidation level and application of method

Examples

Experimental program
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Embodiment 1

[0046] Example 1. Redox Proteomic Analysis of Spinach Leaf High Temperature Response

[0047] attached figure 1 An experimental flow diagram of the method for simultaneous quantification of protein abundance and cysteine ​​oxidation levels is shown. Include the following steps:

[0048] 1. Apply the extract that has been added with NEM to extract the spinach (Spinacia oleracea L.) leaf protein treated at 37°C for 0h, 2h and 4h, then use acetone to precipitate the protein, and then apply HES buffer (50mM HEPES, 1mM EDTA, 0.1%SDS, pH 8.0) to reconstitute the protein.

[0049] 2. Add 5mM TCEP reduced protein to the protein solution and incubate at 50°C for 70min.

[0050] 3. Desalt the protein sample by referring to the Zeba desalting spin column desalting instructions for protein samples.

[0051] 4. Apply iodoTMT tags 126, 127 and 128 to label leaf protein samples treated at 37°C for 0h, 2h and 4h, respectively. After the reaction was completed, 20 mM DTT was added to term...

Embodiment 2

[0072] Example 2. Redox proteomics analysis of chloroplast oxidative stress response of star grass

[0073] 1. The analysis process is the same as in Example 1, except that the sample analyzed is that Puccinellia tenuiflora responds to 0mM, 5mM and 10mM H 2 o 2 Stressed chloroplast protein samples. 0 mM, 5 mM and 10 mM H were labeled with iodoTMT tags 126 and 129, 127 and 130, and 128 and 131, respectively 2 o 2 Stressed chloroplast protein samples; 0mM, 5mM and 10mM H 2 o 2 Stressed chloroplast protein samples.

[0074] 2. When using Waters nanoAcquity UPLC and Thermal Orbitrap Fusion high-resolution mass spectrometer to identify polypeptide samples, except that the setting parameters of HCD collision energy are inconsistent with those in Example 1, other parameters are the same as in Example 1. image 3 Example of a secondary spectrum for the peptide "QINVTCEVQQQLLGNNR" with HCD collision energy set to 32%, Figure 4 Example of a secondary spectrum for the peptide "QI...

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Abstract

The invention discloses a method for simultaneous quantification of protein abundance and the cysteine oxidation level. By means of different quality report group molecular weights of an iTRAQ reagentand an iodo TMT reagent and according to different characteristics of polypeptide positions, the iodo TMT reagent is used for labeling protein cysteine, meanwhile, the iTRAQ reagent is used for marking polypeptide amino terminals, changes of the cysteine oxido-reduction level and the protein expressive abundance are simultaneously detected, and therefore the changes of the protein oxido-reductionlevel can be accurately judged. The invention further discloses application of the method in quantitatively analyzing the protein abundance and the cysteine oxido-reduction level in samples such as cells, tissue and body fluid.

Description

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Claims

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Application Information

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Owner SHANGHAI NORMAL UNIVERSITY
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