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Method and application for enriching phosphorylation peptide fragment from two-dimensional metal organic skeleton nano-sheets

A metal-organic framework, phosphorylated peptide technology, applied in the fields of peptide preparation, organic chemistry, chemical instruments and methods, etc., can solve the problems of low detection limit, low enrichment efficiency of affinity materials, etc. The effect of many surface active sites and high anti-interference ability

Inactive Publication Date: 2019-06-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method and application of two-dimensional metal-organic framework nanosheets for enriching phosphorylated peptides. The method uses two-dimensional MOFs nanosheets to enrich phosphorylated peptides in a sample, and the nanosheet material is synthesized The process is simple, the yield is high, and the enrichment method is simple, which solves the shortcomings of low enrichment efficiency of commercial affinity materials in the prior art, and demonstrates the ability to enrich efficiently (phosphorylated peptide: non-phosphorylated peptide = 1:10000, The phosphorylated peptide concentration is 4 pmol μL -1 ) and extremely low detection limits (1×10 -10 M), which is conducive to the promotion and use of

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  • Method and application for enriching phosphorylation peptide fragment from two-dimensional metal organic skeleton nano-sheets
  • Method and application for enriching phosphorylation peptide fragment from two-dimensional metal organic skeleton nano-sheets
  • Method and application for enriching phosphorylation peptide fragment from two-dimensional metal organic skeleton nano-sheets

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Experimental program
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Effect test

Embodiment 1

[0030] In the present invention, the specific process of using two-dimensional MOFs nanosheets to enrich phosphorylated peptides hydrolyzed by standard phosphorylated proteases is as follows:

[0031] First prepare the enrichment solution, washing solution and eluting solution used in the experiment. The enrichment solution is a mixed solution prepared from acetonitrile, trifluoroacetic acid and ultrapure water, wherein the contents of acetonitrile and trifluoroacetic acid are respectively 40vol%. and 3vol%; the washing solution 1 is a mixed solution prepared from acetonitrile, trifluoroacetic acid and ultrapure water, wherein the content of acetonitrile and trifluoroacetic acid is respectively 50vol% and 4vol%; the washing solution 2 is acetonitrile, A mixed solution prepared from trifluoroacetic acid and ultrapure water, wherein the contents of acetonitrile and trifluoroacetic acid are 30vol% and 0.1vol% respectively, and the eluting solution is 10wt% NH 3 ·H 2 O.

[0032]...

Embodiment 2

[0034] The same enrichment solution, washing solution and eluting solution as in Example 1 were used.

[0035] Draw 150 μL of the enrichment solution and mix it with 0.5 mg two-dimensional MOFs nanosheets, and then ultrasonically disperse it for 15 min to make it uniform, and then draw 0.2 μL of the enzymatically hydrolyzed standard peptide mixture (from bovine serum β-casein, Sigma-Aldrich China) Add to the above dispersed suspension, vortex for 30 min, centrifuge to remove the supernatant, then absorb 100 μL of washing solution 1, vortex for 2 min, centrifuge, remove the supernatant, then absorb 100 μL of washing solution 2 and vortex Centrifuge for 2 min and remove the supernatant. The purpose of these two steps is to wash off non-phosphorylated peptides. After the washing step, pipette 10 μL of 10 wt% NH 3 ·H 2 O was added to the washed material, sonicated for 8 min, and vortexed for 10 min for elution to obtain enriched phosphorylated peptides.

Embodiment 3

[0037] The same enrichment solution, washing solution and eluting solution as in Example 1 were used.

[0038] 200 μL of the enrichment solution was mixed with 0.75 mg of two-dimensional MOFs nanosheets, and then ultrasonically dispersed for 15 min to make them uniform, and then 0.6 μL of enzymatically hydrolyzed standard peptide mixture (from bovine serum β-casein, Sigma-Aldrich China) Add to the above dispersed suspension, vortex for 30 min, centrifuge to remove the supernatant, then absorb 100 μL of washing solution 1, vortex for 2 min, centrifuge, remove the supernatant, then absorb 100 μL of washing solution 2 and vortex Centrifuge for 2 min and remove the supernatant. The purpose of these two steps is to wash off non-phosphorylated peptides. After the washing step, pipette 10 μL of 10 wt% NH 3 ·H 2 O was added to the washed material, sonicated for 8 min, and vortexed for 10 min for elution to obtain enriched phosphorylated peptides.

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Abstract

The invention discloses a method and application for enriching a phosphorylation peptide fragment from two-dimensional metal organic skeleton nano-sheets. The method comprises the following steps thatthe two-dimensional metal organic skeleton nano-sheets and an enrichment solution are mixed and dispersed, a phosphorylation peptide sample is added for performing enrichment, centrifuging is performed after enrichment, a supernate is removed, a washing solution is added, vortex centrifuging is performed, and a supernate is removed; finally, an elution solution is added, elution is performed after ultrasonic vortex, and the enriched phosphorylation peptide fragment is obtained. The nano-sheets are used for enriching the phosphorylation peptide fragment, the efficient enriching capability, theextreme detection limit and the quite high interference resistance are shown, and compared with commercial immobilized metal ion affinity chromatography and metal oxide affinity chromatography, the enrichment efficiency is greatly improved; meanwhile, the good effect is shown for detecting complex samples, the nano-sheets have the excellent performance of wide application range, good selectivity,high sensitivity and the like, and large-scale application and popularization are facilitated.

Description

technical field [0001] The invention relates to the technical field of enrichment and application of phosphorylated peptides, in particular to a method and application of two-dimensional metal-organic framework nanosheets for enrichment of phosphorylated peptides. Background technique [0002] Metal-organic frameworks (Meatal-Organic Frameworks, MOFs) are a class of porous crystalline materials, which have some unique properties, such as ultra-high specific surface area and large pore volume, and easy functional modification of the pore surface. For gas adsorption and separation, catalysis, energy storage and carbon dioxide capture. Two-dimensional MOFs nanosheets are currently a research hotspot, but their applications are mainly focused on energy storage, membrane separation and sensing. The main advantages of two-dimensional MOFs nanosheets include well-formed two-dimensional sheet structure, large active surface area and good dispersion, so they have the potential to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14G01N27/62
Inventor 沈健古志远杨世庶
Owner NANJING NORMAL UNIVERSITY
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