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Isotope labeling reagent as well as preparation method and application thereof

A technology of isotope labeling and labeling reagents, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as wrong results, low labeling efficiency, and product mass spectrometry signal decline, so as to improve signal strength, signal strength and stability, The effect of reducing complexity

Active Publication Date: 2012-09-26
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

However, there are still some defects in the previous methods, such as: the mass spectrometry signal of the product after labeling decreases, the labeling efficiency is low, and some peptides can not produce clear N-terminal ions, resulting in wrong results.
In addition, N-terminal isomeric residues (leucine, isoleucine) cannot be easily identified

Method used

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  • Isotope labeling reagent as well as preparation method and application thereof
  • Isotope labeling reagent as well as preparation method and application thereof
  • Isotope labeling reagent as well as preparation method and application thereof

Examples

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Embodiment 1

[0036] Embodiment 1: Isotope labeling reagent of the present invention ([d 0 ] / [d 6 ]-DMPITC)

[0037] Dissolve 200mg of sodium metal in 20mL of anhydrous methanol / deuterated methanol to prepare a 20mL sodium methoxide / deuterated sodium methoxide solution;

[0038] Dissolve 500mg of 4,6-dichloro-2-aminopyrimidine in 10mL of anhydrous methanol / deuterated methanol, then add dropwise to the existing 20mL sodium methoxide / deuterated sodium methoxide solution, and place at 25°C stir overnight;

[0039] Concentrate by filtration, then dissolve in 10mL deionized water, extract 3 times with dichloromethane, 5mL each time, combine the organic phases, dry with sodium sulfate and spin dry to obtain [d 0 ] / [d 6 ]-4,6-dimethoxy-2-aminopyrimidine crude product;

[0040] 745 mg [d 0 ] / [d 6 The crude product of ]-4,6-dimethoxy-2-aminopyrimidine was dissolved in 20 mL of anhydrous dichloromethane, and thiophosgene (thiophosgene, 0.36 ml, 4.8 mmol) and sodium bicarbonate (1 g, 12 mmol) w...

Embodiment 2

[0044] Embodiment 2: Isotope labeling reagent of the present invention ([d 0 ] / [d 6 ]-DMPITC)

[0045] The principle of application is as follows:

[0046]

[0047] 1. Comparative labeling reaction of standard peptide (DRVYIHPF)

[0048] 1. Configure a mixed solution of ethanol / pyridine / water with a volume ratio of 2:2:1, and use this solvent as a reaction solvent;

[0049] 2. Add 400-600 μL of the above-mentioned reaction solvent to the two solutions of light-labeled reagents DMPITC and PITC (both 50 nmol / μL) dissolved in 100-200 μL of ethanol, and mix according to the molar ratio of labeling reagent and peptide segment of 500:1. Proportionally add the peptide solution, mix and react at 55-60°C for 90-120 minutes;

[0050] 3. After the reaction solution is cooled, blow it dry with nitrogen, then extract with dichloromethane to remove excess labeling reagent, then dry it with nitrogen, and dissolve the labeled peptide with 50-100 μL of acetonitrile-water solution with a...

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Abstract

The invention discloses an isotope labeling reagent which is an isothiocyanopyrimidine isotope labeling reagent with a chemical name of [d0] / [d6]-4,6-dimethoxine-2-isothiocyanate. The preparation method of the reagent comprises the following steps of: firstly, reacting 4,6-dichloro-2-amiopyrimidine with sodium methoxide / deuterosodium methoxide to obtain a [d0] / [d6]-4,6-dimethoxy-2-amiopyrimidine crude product; carrying out reflux reaction on the obtained crude product, thiophosgene and sodium bicarbonate to obtain a final crude product; and then carrying out column chromatography to obtain the isotope labeling reagent disclosed by the invention. The isotope labeling reagent disclosed by the invention can be applied to the identification analysis of polypeptide N-end residues and the quantitative analysis of protein, can speed up the labeling reaction rate and improve a signal of the labeled peptide section, is beneficial to strengthening the accuracy and the credibility of the identified peptide section and has the advantages of less molecular weight of the labelled part, relative simple structure, stable performance, simpleness and convenience for operation, and the like.

Description

technical field [0001] The invention relates to an isotope labeling reagent and its preparation method and application, in particular, to an isothiocyanatopyrimidine isotope labeling reagent and its preparation method, and the identification of the isotope labeling reagent at N-terminal residues of polypeptides and protein Applications in Comparative Analysis. Background technique [0002] Translational Medicine (see Clin.Sci., 2007, 112, 217) is a new research strategy proposed in response to the increasing complexity of medical research and the serious separation from basic clinical practice. It is a new medical research model that is continuously circulating, rising, and deepening. It shortens the distance from the laboratory to the hospital bed and is a new driving force for the development of medicine in the 21st century. One of the important topics in translational medical research is to discover and monitor new parameters of human diseases—biomarkers (see J. Clin. Ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D239/52G01N30/00
Inventor 郭寅龙冷嘉鹏张立张菁王昊阳
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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