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Application of mass spectrometry probe in SGK1 (serum glucocorticoid-ind ucible kinase 1) protein activity detection

A protein activity and probe technology, which is used in measurement devices, peptide preparation methods, and microorganism determination/inspection to achieve accurate reactions and high accuracy.

Inactive Publication Date: 2020-01-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the detection of SGK1 activity by mass spectrometry probes. Mass spectrometry probes have the advantages of high specificity and high sensitivity.

Method used

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  • Application of mass spectrometry probe in SGK1 (serum glucocorticoid-ind ucible kinase 1) protein activity detection
  • Application of mass spectrometry probe in SGK1 (serum glucocorticoid-ind ucible kinase 1) protein activity detection
  • Application of mass spectrometry probe in SGK1 (serum glucocorticoid-ind ucible kinase 1) protein activity detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the synthesis of SGK1 mass spectrometry probe

[0025] The synthesis method of the SGK1 mass spectrometry probe in this embodiment mainly includes the following steps:

[0026] 1. Resin swelling

[0027] Weigh 0.6g of 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.4mmol / g, put the resin into a reaction tube, add DCM (dichloromethane) (15ml / g), and shake for 30min.

[0028] 2. Take the first amino acid

[0029] Filter off the solvent through a sand core, add a 3-fold molar excess of Fmoc-L-Glu-OH amino acid, then add a 5-fold molar excess of DIEA, finally add a small amount of DMF to dissolve, and shake for 1 hour. Alternately wash 6 times with DMF and DCM.

[0030] 3. Deprotection

[0031] Add 15ml of 20% piperidine DMF solution (15ml / g) for 5min, remove and add 15ml of 20% piperidine DMF solution (15ml / g) for 15min.

[0032] Four. Detection

[0033] Take out the piperidine solution, take more than a dozen resins, wash them th...

Embodiment 2

[0042] Example 2: Application of SGK1 mass spectrometry probe in the detection of SGK1 protein activity

[0043] Buffer: Tris-HCl (50mM, pH 7.5, containing 10mM MgCl2, 400μM ATP, 2mM DTT, 5mMβ-Glycerophosphate) made up to 100μL

[0044] Sample group 1: add 50 μL CKRPRAASFAE (50 μM), make up to 100ul with buffer;

[0045] Sample group 2: add 25 μL SGK1 (0.1 mg / mL), 50 μL CKRPRAASFAE (50 μM), make up to 100ul with buffer;

[0046] Sample group 3: add 25 μL of SGK1 (0.1 mg / mL), 50 μL of CKRPRAASFAE (50 μM), inhibitor EMD638683 (10 μM), and make up to 100ul with buffer;

[0047] The three sample groups were incubated at 30° C. for 1 hour, and then 200 μL of methanol (containing 0.1% TFA) was added to terminate the reaction. The solution was vortexed and centrifuged, and the supernatant was taken for injection analysis.

[0048] The analysis conditions are: Agient 1100 liquid phase-1946D single quadrupole mass spectrometer; chromatographic column, Zorbax SBC18 (4.6mm×250mm, 5μm);...

Embodiment 3

[0050] Example 3: Investigation of the detection sensitivity of SGK1 mass spectrometry probes to SGK1

[0051] Take 50 μL of mass spectrometry probe CKRPRAASFAE (final concentration is 25 μM), different concentrations of SGK1 (final concentrations are 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL), and 50 mM Tris-HCl (pH=7.5, containing 10 mM MgCl2 , 400 μM ATP, 2 mM DTT, 5 mM β-Glycerophosphate) to 100 μL, incubated at 30°C for 1 hour, then added 200 μL methanol (containing 0.1% TFA) to stop the reaction, the solution was vortexed and centrifuged, and the supernatant was taken for injection analysis. Analyze according to the analytical method described in embodiment 2, the detection result sees Figure 4 .

[0052] Depend on Figure 4 It can be seen that the detection limit of mass spectrometry probe CKRPRAASFAE for SGK1 is 0.05 μg / mL, and the linear range is 0.05 μg / mL-0.5 μg / mL (R 2 >0.97).

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Abstract

The invention discloses application of a mass spectrometry probe in SGK1 (serum glucocorticoid-ind ucible kinase 1) protein activity detection. The mass spectrometry probe is a peptide of which the amino acid sequences are Cys-Lys-Arg-Pro-Arg-Ala-Ala-Ser-Phe-Ala-Glu. The mass spectrometry probe provided by the invention not only can be phosphorylated by SGK1 to generate phosphorylation products, but also has high mass spectrometry response, is high in mass spectrometry detect accuracy, is capable of accurately reflecting the activity of SGK1, and can be used to detect SGK1 protein activity, and can also be used to screen SGK1 inhibitors.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to the application of a mass spectrometry probe in the detection of SGK1 protein activity. Background technique [0002] Serum and glucocorticoid-induced protein kinase 1 (serum glucocorticoid-induciblekinase1, SGK1), one of the members of the serine / threonine kinase family, is an isoform of AKT. SGK1 is expressed in many species, and almost all tissues in mammals express SGK1, but the expression level varies greatly. The expression level of SGK1 is low in most cells, but under certain pathophysiological conditions, such as excessive glucocorticoid or mineralocorticoid, inflammation caused by TGF-β release, hyperglycemia, cell shrinkage and ischemia, etc., the expression of SGK1 expression was significantly increased. [0003] SGK1 stimulates a variety of renal tubular ion channels and transporters, participates in the regulation of renal electrolyt...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C07K7/06C07K1/06C07K1/04
CPCC12Q1/485C07K7/06G01N2333/91215
Inventor 王毅程翼宇张舒静
Owner ZHEJIANG UNIV
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