Use of piperazinopyrimidine isotope labeling reagent

A technology of isotope labeling and base pyrimidine, which is used in measurement devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of weak mass spectral response, difficult qualitative and quantitative detection, weak content, etc., to avoid signal interference and improve product quality. The effect of mass spectral response intensity and product background noise is low

Inactive Publication Date: 2013-02-27
SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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Problems solved by technology

In diagnostic medical research, many peptides are difficult to detect qualitativel

Method used

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  • Use of piperazinopyrimidine isotope labeling reagent
  • Use of piperazinopyrimidine isotope labeling reagent
  • Use of piperazinopyrimidine isotope labeling reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: [d 0 ]- or [d 6 ]-2,4-dimethoxy-6-piperazinyl pyrimidine, (abbreviated as [d 0 ]- / [d 6 ]-DMPP) synthesis.

[0045] The operation steps are as follows:

[0046] Boc-protected piperazine (373mg, 2mmol) and potassium carbonate (304mg, 2.2mmol) were dissolved in an appropriate amount of 20ml of anhydrous DMF, and 2,4,6-trichloropyrimidine (0.23ml ,2mmol), and stirred gently. After reacting at room temperature for 10 hours, the reaction was quenched by pouring into water, and extracted with dichloromethane to obtain crude product 2,4-dichloro-6-Boc-piperazinylpyrimidine (466mg, 1.4mmol). Dissolve 150mg of sodium metal in 15mL of anhydrous methanol to form a sodium methoxide solution. Dissolve 2,4-dichloro-6-Boc-piperazinylpyrimidine in 10 mL of anhydrous methanol, then gradually add it dropwise into the prepared sodium methoxide solution, stir overnight, and place the reaction solution at 25°C and stir overnight, Then it was concentrated by filtration, then...

Embodiment 2

[0066] Embodiment 2: comparative analysis of polypeptide

[0067] The operation steps are as follows:

[0068] 1. Place bovine serum albumin in an aqueous solution containing 0.04-0.05M ammonium bicarbonate and pH 8.0-8.5 for heat denaturation for 10 minutes.

[0069] 2. After cooling to room temperature, add trypsin to the solution at a weight ratio of 50:1, and enzymolyze it at 37°C for 12-16 hours.

[0070]3. Divide the enzymatic hydrolysis product and standard polypeptide a and n into two parts, and react the two parts with the light and heavy labeling reagent DMPP respectively, the solvent is acetonitrile, perform the same post-processing steps as in Example 1, but without resin adsorption, etc. Subsequent steps, followed directly by mass spectrometry.

[0071] 4. The analysis conditions of MALDI-TOF MS are: all the acquisition conditions are carried out in the positive ion mode, and the sample matrix CHCA solvent is 0.1% trifluoroacetic acid and 50% acetonitrile water....

Embodiment 3

[0078] Example 3: Labeling of proteins

[0079] The operation steps are as follows:

[0080] 1. As in Example 1, undenatured proteins (lysozyme, bovine serum albumin, BSA) were reacted with light labeling reagents, and the same post-processing steps were performed.

[0081] 2. Mix the unlabeled protein with the labeled protein at the same concentration.

[0082] 3. The analysis conditions of MALDI-TOF MS are: all acquisition conditions are carried out in the positive ion mode, the laser energy is appropriately increased by 20%, the detection mode is linear, and the spotting matrix SA solvent is 50% of 0.1% trifluoroacetic acid Acetonitrile in water.

[0083] The analysis results of MALDI-TOF MS are as follows:

[0084] 1. If Figure 9 As shown, both bovine serum albumin and lysozyme are completely labeled, and compared with the unlabeled original protein, the peak area is increased by more than five times, which is very beneficial for the detection of low kurtosis protein....

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Abstract

The invention relates to a use of a piperazinopyrimidine isotope labeling reagent. The piperazinopyrimidine isotope labeling reagent is a light/heavy isotope labeling reagent of [d0]-/[d6]-2,4-dimethoxy-6-piperazinopyrimidine. The piperazinopyrimidine isotope labeling reagent can be used for comparison analysis of carboxyl compounds such as fatty acid, polypeptides or proteins. Fatty acid analysis comprises the following steps of a) fatty acid reacts respectively with light and heavy labeling reagents; after after-treatment, an LC-MS/MS analysis process is carried out; and product ions m/z 225 and m/z 231 are selected as quantitative ions; and b) through observation of pairs of mass spectrum peaks of the labeled fatty acid, identification of a plurality of carboxyl fatty acids is realized and quantification is realized according to relative peak areas. Polypeptide/protein comparison analysis comprises the following steps of a) a protein needing to be analyzed is subjected to thermal denaturation and enzymolysis and then reacts with the light/heavy labeling reagent; and b) the reaction solution is subjected to after-treatment; and a MALDI-TOF MS comparison analysis process is carried out.

Description

technical field [0001] The invention relates to a design and synthesis method of a piperazinyl pyrimidine isotope labeling reagent and its application in mass spectrometry analysis of carboxyl-containing compounds. In particular, the combination of liquid chromatography mass spectrometry and matrix-assisted laser desorption mass spectrometry can achieve fast, efficient and accurate mass spectrometry analysis, which provides a good means for high-throughput research on carboxyl compounds, and is even more important for mass spectrometry of biomarkers in diagnostic medicine. Analysis provides a strategy. technical background [0002] One of the important topics in diagnostic medical research is to discover and monitor new parameters of human diseases—biomarkers (see J.Clin.Pharmacol.2003,43,329.). For disease research, biomarkers generally refer to certain characteristic biochemical indicators in a common physiological or pathological or therapeutic process that can be object...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N30/02
Inventor 郭寅龙冷嘉鹏张立张菁王昊阳张芳康文昱许楚
Owner SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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