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Synthesis method of nano-material with surface rich in phosphate groups and fixed with Ti < 4 + > carbon spheres and application of nano-material in phosphorylated proteomics

A technology of nanomaterials and synthesis methods, applied in the field of high-throughput MALDI-TOFMS analysis, can solve the problems of complicated synthesis steps, harmful organic reagents, time-consuming and labor-intensive, etc., to achieve the best performance, optimize the reaction time, and improve the enrichment ability. Effect

Pending Publication Date: 2022-07-19
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But traditional IMAC-Ti 4+ The material preparation method is time-consuming and laborious, often accompanied by complicated synthesis steps, and a large amount of toxic and harmful organic reagents are used in the preparation process, which is not in line with the design of chemicals and processes to reduce or eliminate the use and generation of hazardous substances in green chemistry

Method used

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  • Synthesis method of nano-material with surface rich in phosphate groups and fixed with Ti &lt; 4 + &gt; carbon spheres and application of nano-material in phosphorylated proteomics
  • Synthesis method of nano-material with surface rich in phosphate groups and fixed with Ti &lt; 4 + &gt; carbon spheres and application of nano-material in phosphorylated proteomics
  • Synthesis method of nano-material with surface rich in phosphate groups and fixed with Ti &lt; 4 + &gt; carbon spheres and application of nano-material in phosphorylated proteomics

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Carbon spheres rich in phosphate groups on the surface and immobilized Ti 4+ synthesis of nanomaterials;

[0036] (1) 3.04g of glucose-6-phosphate disodium (G6PNa 2 ) dissolved in 10 mL of deionized water and configured into a solution with a concentration of 1 mol / L;

[0037] (2) taking out the stainless steel reaction kettle of the polytetrafluoroethylene lining with a capacity of 25mL, and pouring the solution obtained in step (1) into it;

[0038] (3) put the reactor of step (2) into oven and react 6 hours at 180 DEG C;

[0039] (4) fully wash the product obtained in step (3) with deionized water and ethanol, remove impurities and raw material monomers on the surface of the product, and place it in a vacuum drying oven at 50 ° C to dry overnight to obtain carbon rich in phosphoric acid groups on the surface. ball material;

[0040] (5) Disperse the product obtained in step (4) into 10 mL, 100 mmol / L of Ti(SO 4 ) 2 In the solution, shake at a constant temperatu...

Embodiment 2

[0044] Application of carbon spheres rich in phosphate groups on the surface and immobilized Ti ions for phosphorylated peptides enriched in β-casein:

[0045] (1) Preparation of sample: β-casein (β-casein) in 25mmol / L NH 4 HCO 3 The solution was enzymatically hydrolyzed at 37°C for 16h.

[0046](2) Enrichment: 0.5 mg of nanomaterials were dispersed into 200 μL of 0.1% by volume TFA 50% by volume ACN centrifuge tube, then 2 μL of the sample prepared in step (1) was added, and enriched at 37°C for 30 min; Wash with a buffer ratio of TFA and 50% ACN by volume and centrifuge for 3 times; elute with 10 μL of 0.4M ammonia water for 10 min, and centrifuge to obtain the supernatant.

[0047] (3) Mass spectrometry analysis: take 0.5 μL of the supernatant target obtained in step (2), and conduct mass spectrometry analysis. The mass spectrogram is as follows Figure 4 shown, Figure 5 It is the detection limit (1fmol) of phosphorylated peptides in β-casein enriched with carbon spher...

Embodiment 3

[0049] Application of carbon spheres enriched with phosphate groups on the surface and Ti ions immobilized nanomaterials for enrichment of phosphorylated peptides in mixed proteins.

[0050] (1) Preparation of sample: bovine serum albumin (BSA) was firstly reductively alkylated with dithiothreitol and iodoacetamide, and then enzymatically hydrolyzed at 37°C for 16h. β-casein in 25mM NH 4 HCO 3 The solution was enzymatically hydrolyzed at 37°C for 16h. Bovine serum albumin (BSA) and β-casein were added in a 1000:1 molar ratio to a 50% ACN centrifuge tube containing 0.1% TFA.

[0051] (2) Enrichment: 0.5 mg of the nanomaterial obtained in Example 1 was dispersed into 200 μL of 0.1% by volume TFA 50% by volume ACN centrifuge tube containing the phosphorylated peptide of step (1), and enriched at 37°C for 30 min; The buffer solution of % TFA and 50% ACN by volume was washed and centrifuged 3 times; 10 μL of 0.4M ammonia water was washed for 15 min.

[0052] (3) Mass spectromet...

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Abstract

The invention belongs to the field of nanotechnology, G6PNa2 is used as a carbon source, a carbon sphere material with the surface rich in phosphate groups is prepared in one step through a hydrothermal carbonization method, and then metal cations Ti < 4 + > (named as CS-Ti < 4 + >) are fixed through chelation. The preparation method comprises the following steps: firstly, adding a G6PNa2 aqueous solution with a certain concentration into a reaction kettle, putting the reaction kettle into a drying oven, reacting at 180 DEG C for 6 hours, then washing a reaction product with water and ethanol for multiple times, and obtaining a carbon sphere material rich in phosphate groups on the surface under a centrifugal condition; and then dispersing the material into a 100 mmol / L Ti (SO4) 2 solution, oscillating for 24 hours at a constant temperature at room temperature, carrying out water treatment, centrifuging, and drying at 50 DEG C to obtain a final product. The preparation process is simple, a material preparation process with an extremely short synthesis period is adopted, and a large amount of time and labor are saved; the reaction raw materials are common and no organic reagent is used.

Description

technical field [0001] The invention belongs to the category of advanced nanomaterials and nanotechnology, and specifically relates to a carbon ball material whose surface is rich in phosphoric acid groups and which fixes Ti 4+ A technique for the separation and analysis of phosphorylated peptides using high-throughput MALDI-TOFMS. Background technique [0002] Reversible phosphorylation of proteins is one of the most common and important post-translational modifications. A large number of reports indicate that phosphorylation plays an irreplaceable role in biological processes, such as intercellular signaling, neural activity, etc. Studies have shown that some physiological abnormalities and pathologies are considered to be related to abnormal phosphorylation, and some phosphorylated proteins have become biomarkers for some diseases. Therefore, understanding the reaction mechanism and phosphorylation sites of protein phosphorylation plays an important role in understandin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/20B01J20/28B01J20/30G01N27/62G01N33/68
CPCB01J20/0248B01J20/0281B01J20/205B01J20/28019G01N33/6848G01N27/62
Inventor 闫迎华刘彬唐科奇丁传凡
Owner NINGBO UNIV
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