Application of a maldi-tof mass spectrometry probe in the preparation of γ-glutamyl transpeptidase detection kit

A technology of glutamyl transpeptidase and detection kit, which is applied in the field of analysis and testing, can solve the problems of autofluorescence interference of biological samples, high requirements for excitation conditions, photobleaching of molecular probes, etc., and achieve easy promotion and application, Compositing simple, good sensitivity effects

Active Publication Date: 2021-11-16
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most GGT detection is based on colorimetric and fluorescence methods. Spectroscopic methods are accompanied by certain limitations, such as the need for fluorescent or chromophore labels, relatively difficult synthesis, high requirements for excitation conditions, autofluorescence interference of biological samples, and Photobleaching and Quenching Phenomena of Molecular Probes

Method used

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  • Application of a maldi-tof mass spectrometry probe in the preparation of γ-glutamyl transpeptidase detection kit
  • Application of a maldi-tof mass spectrometry probe in the preparation of γ-glutamyl transpeptidase detection kit
  • Application of a maldi-tof mass spectrometry probe in the preparation of γ-glutamyl transpeptidase detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of molecular probes and internal standard molecules of the present invention.

[0044] At room temperature, 4 mmol glutathione (ECG) and 4 mmol tripeptide (ECA) were dissolved in 5 mL of 50% acetonitrile / water (v / v), respectively, and then 4 mmol anthraquinone was added, stirred for 3 h, and evaporated under reduced pressure. After the solvent, use column chromatography to separate and purify to obtain the final product of brown yellow solid, which is formula I and formula II, and its structure confirmation results are as follows:

[0045] Formula I: 1 H NMR(400MHz,298K,DMSO-d6):δ:8.68(m,1H),8.29(m,1H),7.79(m,1H),7.02(s,1H),4.68(m,1H),3.80 (d, 2H, J=6.0Hz), 3.36(d, 2H, J=6.0Hz), 3.15(m, 1H), 2.38(m, 2H), 1.97(t, 2H).ESI-MS: m / z 516.443;

[0046] Formula II: 1 H NMR(400MHz,298K,DMSO-d6):δ:8.68(m,1H),8.29(m,1H),7.80(m,1H),7.02(s,1H),4.07(m,1H),4.24 (m,2H),3.32(d,2H,J=6.0Hz),3.14(m,1H),2.36(m,2H),1.98(t,2H),1.31(d,3H,J=6.0Hz) .ESI-MS: m / z 530....

Embodiment 2

[0047] Example 2: MALDI mass spectrometry analysis of glutathione and the molecular probe of formula I.

[0048] Prepare aqueous solutions of different concentrations of glutathione and molecular probes of formula I, take 1 μL of sample solution and an equal volume of CHCA matrix (5 mg / mL, dissolved in 50% acetonitrile / trifluoroacetic acid aqueous solution (V / V)) or DHB The matrix (20 mg / mL, dissolved in 50% acetonitrile / trifluoroacetic acid aqueous solution (V / V)) was directly mixed on the ground steel target plate, dried at room temperature and analyzed by MALDI mass spectrometry.

[0049] Such as figure 2 Shown are comparison spectra of MALDI assays for 50 μM glutathione and 50 μM Molecular Probe. The peak of glutathione cannot be detected at this concentration, and there are a large number of self-interfering peaks in the traditional matrix, which makes it difficult to identify the peaks and cannot achieve the purpose of accurate characterization. Through the analysis o...

Embodiment 3

[0050] Example 3: Quantitative analysis of different concentrations of GGTase and drawing a standard curve.

[0051] Configure 500 μM MALDI formula I molecular probe and internal standard aqueous solution as stock solution;

[0052] Configure 180 μL of GGT enzyme standard PBS buffer solution (155.2mM NaCl, 2.97mM, Na 2 HPO 4 ,1.05mM KH 2 PO 4 ; pH=7.4), 20 μL of the stock solution of molecular probes was added, and the digestion reaction was carried out in a water bath at 37° C. for 1.5 h. Add 0.5 μL of formic acid solution and 8 μL of internal standard solution, mix well, take 1 μL of sample solution and 1 μL of DHB matrix solution and mix directly on the ground steel target plate, dry at room temperature and perform MALDI mass spectrometry analysis. Value the abscissa with the different concentrations of GGT enzyme, account for the ratio (I II / I I +I II ) is the ordinate, and the standard curve is drawn.

[0053] From Figure 4 It can be seen that the molecular pro...

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Abstract

The invention relates to the application of a MALDI-TOF mass spectrometry probe in the preparation of a gamma-glutamyl transpeptidase detection kit, which is used for the quantitative analysis of GGTase and the screening of related inhibitor drugs. The cost and labor of molecular probe synthesis avoid the interference of substrate light signal, light scattering and photobleaching in the spectrum, and realize the rapid and accurate analysis of GGT enzyme in cell lysate and human serum. Simultaneous analysis of hundreds of samples greatly improves the analysis speed and efficiency.

Description

technical field [0001] The invention belongs to the technical field of analysis and testing, and in particular relates to the application of a MALDI-TOF mass spectrometry probe in the preparation of a gamma-glutamyl transpeptidase detection kit. Background technique [0002] γ-Glutamyl transpeptidase (GGT) plays an important role in maintaining the metabolism of glutathione and cysteine ​​in the body. As an extracellular enzyme with an active site towards the extracellular surface, GGT can specifically catalyze Cleavage of the γ-glutamyl group in extracellular glutathione yields cysteine-glycine, which is then further hydrolyzed to cysteine ​​and glycine by dipeptidases. Due to the strong dependence of malignant cells on cysteine, this GGT-triggered process potentially provides favorable conditions for the survival and proliferation of tumor cells. Therefore, GGTase is an important indicator for the diagnosis of liver and gallbladder diseases in clinical practice, and is wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/037C12Q1/48G01N27/64
CPCC07K5/0215C12Q1/48G01N27/62G01N2333/9108
Inventor 国新华王晟郭黎明玲玲
Owner JILIN UNIV
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