Methylsulfonyl miazines isotope labelling reagent, synthesis method and uses thereof

An isotope labeling, sulfone-based pyrimidine technology, applied in biological testing, material testing products, measuring devices, etc., can solve the problems of quantitative error of labeling efficiency, high cost of isotope labeling reagents, etc., to achieve stable properties, improve reliability and accuracy Sex, the effect of reducing complexity

Inactive Publication Date: 2009-01-07
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still many imperfections in these technologies, such as the high cost of isotope-labeled reagents, limited labeling efficiency, and quantitative errors introduced by subsequent separation and detection processes.

Method used

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  • Methylsulfonyl miazines isotope labelling reagent, synthesis method and uses thereof
  • Methylsulfonyl miazines isotope labelling reagent, synthesis method and uses thereof
  • Methylsulfonyl miazines isotope labelling reagent, synthesis method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: [d 6 ]-4,6-dimethoxy-2-thiamphenicol pyrimidine ([d 6 ]-DMMSP) synthesis

[0037] The operation steps are as follows:

[0038] 4,6-Dichloro-2-methylthiopyrimidine (500mg, 2.5mmol) was dissolved in 10mL of anhydrous deuterated methanol, and gradually added dropwise to 20mL of deuterated sodium methoxide solution (200mg of sodium metal was dissolved in 20mL of anhydrous deuterated instead of methanol). After the reaction solution was stirred overnight at 25°C, it was concentrated by filtration, then dissolved in 10 mL of deionized water, and extracted with dichloromethane (3×5 mL). The organic phases were combined, dried over sodium sulfate, and spin-dried to obtain [d 6 ]-4,6-dimethoxy-2-methylthiopyrimidine as a crude product.

[0039] will [d 6 ] - The crude product (400 mg, 2.1 mmol) was dissolved in 40 mL of anhydrous dichloromethane, and 3-chloroperoxybenzoic acid (mCPBA, 1.4 g, 8 mmol) was added. After the mixture was stirred at 30 °C for 5 hours,...

Embodiment 2

[0043] Example 2: Labeling reaction of mixed polypeptides

[0044] The operation steps are as follows:

[0045] 1. Polypeptide mixture: His-Cys-Lys-Phe-Trp-Trp (HW-6), angiotensin I human acetate hydrate (DL-10), [Glu 1 1 μmol each of ]-fibrinopeptide B human (ER-14) and neurotensin (EL-13), dissolved in 500 μL of 0.03 M ammonium bicarbonate buffer solution (pH 8.0-8.2), and 2 μmol of dithiothreitol (DTT) was added , adjust the pH to 6.5 with acetic acid solution.

[0046] 2. Add [d 0 ]-DMMSP (5 μmol), the modification reaction was carried out in a constant temperature and humidity box at 37° C. for 2 hours.

[0047] 3. MALDI-TOF MS analysis conditions are: matrix α-cyano-4-hydroxycinnamic acid (α-CHCA), dissolved in 1:1 acetonitrile / water solution containing 0.1% trifluoroacetic acid, the concentration is 6mg / mL; Data acquisition was performed in positive ion, reflectance mode.

[0048] MALDI-TOF MS analysis results are as follows:

[0049] 1. [M+H] of only cysteine ​​r...

Embodiment 3

[0051] Example 3: Comparative Analysis of Proteins

[0052] The experimental steps are attached figure 1 shown.

[0053] The operation steps are as follows:

[0054] 1. Two lysozyme standard products, 0.9mg and 1mg, were respectively dissolved in 500μL 30mmol / L ammonium bicarbonate buffer solution (pH 8.0-8.2, containing 8M urea), then respectively added 2μmol of DTT, and placed at 37℃ with constant temperature and humidity Keep warm in the box for 6 hours.

[0055] 2. After cooling, dilute the two lysozyme solutions with 30mmol / L ammonium bicarbonate buffer solution (pH 8.0-8.2) to 1mL respectively, and add trypsin according to the ratio of trypsin / lysozyme 1:50 (w / w) The solution was placed in a 37°C constant temperature and humidity box for enzymatic hydrolysis overnight.

[0056] 3. Dilute the two parts of lysozyme hydrolyzate to 2mL with 30mmol / L ammonium bicarbonate buffer solution (pH 8.0-8.2), adjust the pH to 6.5 with acetic acid solution, then add [d 0 ]-DMMSP (...

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Abstract

The invention relates to a method for designing and synthesizing a stable isotope labelling reagent for methylsulphonyl miazines and the application in protein comparative analysis. The isotope labelling reagent of the invention has a chemical name of (d6)-4, 6 dimethoxy-2 methylsulphonyl metadiazine. The reagent can be applied to qualitative and relatively quantitative analysis of cysteine, polypeptide containing cysteine, and protein. The method includes the following methods: a) denaturation and reduction as well as zymohydrolysis of target analyte; b) reaction of the target analyte and the light or heavy isotope labelling reagent; c) direct MALDI-TOF MS analysis of sample solution labeled.

Description

technical field [0001] The present invention relates to the design and synthesis method of a thiamphenyl pyrimidine stable isotope labeling reagent and its application in protein comparative analysis, especially in combination with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method, Enable fast, efficient, and sensitive differential proteomics analysis. technical background [0002] Proteomics is one of the most promising emerging disciplines in the field of life sciences after genomics. The initial goal of its research is to find methods to separate and identify proteins on a large scale. Relatively mature technology and effective research methods. With the deepening of work, people are no longer satisfied with the qualitative analysis of proteins in a mixed system, and require more accurate quantitative analysis. One of the key content is to compare the protein expression levels in normal and abnormal cells or tissues diffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/52G01N33/68G01N27/64
Inventor 郭寅龙张菁
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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