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Kinase enrichment detection method based on chemical proteomics

A proteomics and detection method technology, applied in the field of kinase enrichment detection based on chemical proteomics, can solve the problem of undetectable kinases

Pending Publication Date: 2022-01-18
上海中科新生命生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antibody chip-based method can measure dozens to hundreds of kinases at a time, but it can only be used for the research of kinases with antibodies, and cannot detect new kinases

Method used

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  • Kinase enrichment detection method based on chemical proteomics
  • Kinase enrichment detection method based on chemical proteomics
  • Kinase enrichment detection method based on chemical proteomics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Synthesis of Kinobeads, enrichment of kinases and liquid-mass analysis.

[0028] Iodoacetamide (IAA), dimethylsulfoxide (DMSO), N-hydroxysuccinimidyl-sepharose, ethanolamine, triethylamine, glycerol, dithiothreose used in this example Alcohol (DTT), Igepal CA-630, phosphatase inhibitor cocktail, magnesium chloride, sodium vanadate were all purchased from Sigma-Aldrich; five small molecules were purchased from MCE; trypsin (Trypsin) was purchased from Promega; protease inhibitor cocktail purchased from Roche; NP-40 lysate was purchased from Biyuntian; water (H 2 O), acetonitrile (ACN) were purchased from Fisher scientific; formic acid (FA) was purchased from Fluka.

[0029] A kinase enrichment detection method based on chemical proteomics, which firstly couples 5 small molecule inhibitors VI16832, PP58, Purvalanol B, Bisindolylmaleimide X and CTX-0294885 to a solid support to synthesize 5 different chemical affinity Probes (Kinobeads), and then the kinases i...

Embodiment 2

[0039] Example 2: Competition experiment and phosphorylation pathway analysis.

[0040] Water used in this example (H 2 O), acetonitrile (ACN) were purchased from Fisher scientific; formic acid (FA) was purchased from Fluka; phosphatase inhibitor mixture, iodoacetamide (IAA), dithiothreitol (DTT) were purchased from Sigma-Aldrich; Protease (Trypsin) was purchased from Promega; protease inhibitor cocktail was purchased from Roche; hydroxymethylaminomethane (Tris) was purchased from Bio-Rad.

[0041] Cells were incubated with 1M small molecule tinib compound for 30 min. Subsequently, the cells were washed twice with PBS, and lysed by adding lysis solution (40mM Tris-HCl pH 7.6, 8M urea, EDTA-free protease inhibitor complete mini and phosphatase inhibitor cocktail), and the resulting protein solution was centrifuged at 4°C, 21,000rcf for 1 hour , transfer the supernatant to a new centrifuge tube, measure the concentration, take 2 mg for subsequent phosphorylation experiments, a...

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Abstract

The invention provides a kinase enrichment detection method based on chemical proteomics, and belongs to the technical field of kinase detection. A plurality of small molecule inhibitors are respectively coupled on a solid carrier to carry out affinity enrichment on kinase in cells or tissues, and the abundance and phosphorylation level of the kinase are researched in a physiological state. Kinase identification is realized through high performance liquid chromatography separation, mass spectrometry detection and database search, and large-scale unbiased analysis can be carried out on kinase.

Description

technical field [0001] The invention belongs to the technical field of kinase detection, and in particular relates to a kinase enrichment detection method based on chemical proteomics. Background technique [0002] Phosphorylation of proteins catalyzed by kinases is the basis of intracellular signal transduction in eukaryotes. Kinases frequently exhibit dysregulation of activity in cancer, inflammation, and diabetes and are considered important drug targets because they can be inhibited by small molecules. There are 518 kinds of kinases encoded by human genes, which can be divided into: TKL (tyrosine kinase-like), STE (kinases derived from yeast Sterile 7 (MAP2K), Sterile 11 (MAP3K) and Sterile 20 (MAP4K)), CK1 ( Casein kinase1), AGC (including PKA, PKG and PKC family), CAMK (calcium / calmodulin-regulated kinase), CMGC (including CDK, MAPK, GSK3 and CLK family), TK (tyrosine kinase), SARS family of kinases. So far, at least 62 kinase inhibitor drugs have been approved for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08
CPCG01N30/08
Inventor 阮宏强薛冰冰刘泰驿
Owner 上海中科新生命生物科技有限公司