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Method for screening cathepsin B inhibitor by adopting ultra performance liquid chromatography and mass spectrometry

A cathepsin, ultra-high performance liquid phase technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc. High accuracy, rapid sample detection, and the effect of avoiding false positive and false negative results

Active Publication Date: 2010-12-08
CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Screening of cathepsin B inhibitors generally uses spectroscopic methods, and its disadvantage is that spectroscopic methods are susceptible to background interference and may obtain false positive or false negative results (Arjen R.de Boer, Henk Lingeman, Wilfried M.A.Niessen, Hubertus Irth.Trends in Analytical Chemistry, Vol.26, No.9, 2007.)

Method used

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  • Method for screening cathepsin B inhibitor by adopting ultra performance liquid chromatography and mass spectrometry
  • Method for screening cathepsin B inhibitor by adopting ultra performance liquid chromatography and mass spectrometry
  • Method for screening cathepsin B inhibitor by adopting ultra performance liquid chromatography and mass spectrometry

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Effect test

Embodiment 1

[0053] The method for screening cathepsin B inhibitors by ultra-high performance liquid chromatography and mass spectrometry provided by the invention comprises the following steps:

[0054] (1) Preparation of enzymatic reaction buffer

[0055] The buffer solution includes 0.4 mol / L sodium acetate, 4 mmol / L EDTA, 8 mmol / L dithiothreitol, and the pH value is adjusted to 5.5 with acetic acid;

[0056] Preparation of 7-amino-4-methylcoumarin standard substance: use methanol to make 7-amino-4-methylcoumarin standard substance into concentrations of 100 nanomoles / liter, 200 nanomoles / liter, 500 nanomoles / liter respectively Nanomol / L, 1μmol / L, 2μmol / L, 5μmol / L, 10μmol / L, 20μmol / L standard solutions, and each concentration of the standard contains 1μmol / liter of 7-amino-4-methoxymethyl coumarin as internal standard;

[0057] (2) Preparation of standard products

[0058] Use methanol to prepare the standard products with concentrations of 100 nanomoles / liter, 200 nanomoles / liter,...

Embodiment 2

[0090] The sample is as follows: the total reaction volume is 100 microliters, the final concentration of cathepsin B is 8 nanomoles / liter, and the standard product of ligustrazine with a final concentration of 100 micromoles / liter is incubated at 37 degrees Celsius for 30 minutes, and a final concentration of 20 micromoles / liter is added. Substrate, react at 37 degrees Celsius for 20 minutes, add 1% trifluoroacetic acid to terminate the reaction, and add 1 micromol / liter internal standard at the same time, as a sample for ultra-high performance liquid chromatography / mass spectrometry;

[0091] All the other are with embodiment 1;

[0092] According to the detection steps of Example 1, the inhibition rate of cathepsin by 1 μmol / L ligustrazine standard substance was detected and calculated to be 9%. Figure 6 It is the extraction chromatogram of sample in embodiment 2.

Embodiment 3

[0094] The sample is as follows: the total reaction volume is 100 microliters, the final concentration of cathepsin B is 8 nanomoles / liter, and the final concentration of 100 micromoles / liter tanshinone IIA standard is incubated at 37 degrees Celsius for 30 minutes, and the final concentration of 20 micromoles / liter is added. Substrate, react at 37 degrees Celsius for 20 minutes, add 1% trifluoroacetic acid to terminate the reaction, and add 1 micromol / liter internal standard at the same time, as a sample for ultra-high performance liquid chromatography / mass spectrometry;

[0095] All the other are with embodiment 1;

[0096] According to the detection procedure of Example 1, the inhibition rate of cathepsin by 1 micromole / liter tanshinone IIA standard substance was detected and calculated to be 1%. Figure 7 It is the extraction chromatogram of sample in embodiment 3.

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Abstract

The invention provides a method for screening cathepsin B inhibitor by adopting ultra performance liquid chromatography and mass spectrometry. A cathepsin B inhibitor is a natural product extract or a monomer. The ultra performance liquid chromatography and mass spectrometry is used for analyzing the in vitro inhibition ratio of the natural product extract or monomer to a cathepsin B and the catalytic activity of the cathepsin B. The invention applies the ultra performance liquid chromatography and mass spectrometry to the screening of enzyme inhibitors, ensures rapid and accurate sample detection and the related coefficient of a linear equation of 0.997, adopts the mass spectrometry to detect a compound mass-charge ratio, has high accuracy and excellent specificity, avoids the false position and false negative results in a spectrometry screening, and can be used for the screening of the cathepsin B inhibitor and the kinetic study of the cathepsin B.

Description

field of invention [0001] The invention belongs to the field of analytical chemistry, and relates to a method for screening cathepsin B inhibitors by ultra-high performance liquid chromatography and mass spectrometry, in particular, it relates to the in vitro inhibition rate of cathepsin B by natural product extracts or monomers and the catalytic activity of cathepsin A method of ultra-high performance liquid chromatography coupled with mass spectrometry. technical background [0002] Cathepsin B is a cysteine ​​proteolytic enzyme in lysosomes, belonging to the papain family, and has catalytic activity at pH 3.0 to 7.0, and its specific substrate is Z-Arg-Arg-7-amino-4-methanol Cathepsin B catalyzes the cleavage of the substrate peptide bond to generate 7-amino-4-methylcoumarin. In recent years, studies have shown that cathepsin B is a key enzyme that plays a role in the development of tumors. Cathepsin B promotes angiogenesis through various mechanisms, thereby promoting t...

Claims

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Application Information

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IPC IPC(8): G01N30/72C12Q1/37
Inventor 刘淑莹牛俊刘志强宋凤瑞
Owner CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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