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N-phosphorylated peptide enrichment method

A phosphorylated peptide, phosphorylation technology, applied in the preparation method of peptide, chemical instrument and method, peptide and other directions

Pending Publication Date: 2017-03-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the phosphorylation modification (N-phosphorylation modification) on the amino group of histidine, arginine, and lysine, whose P-N amide bond will be hydrolyzed rapidly at pH less than 3, resulting in dephosphorylation, so the present Some methods of enriching O-phosphorylated peptides under strong acid conditions are applied to the enrichment of N-phosphorylated peptides

Method used

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Experimental program
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Embodiment 1

[0023] 1. β-elimination of O-phosphorylated peptides: Dissolve 40ug of peptides 1 and 2 (see Table 1) in barium hydroxide alkaline solution with pH = 10, and react in a water bath at 37°C for 1 h. figure 1 (a)(b) are MALDI-TOF of peptides before and after β-elimination picture Spectrum, picture The asterisks in the middle are N-phosphorylated peptides, and the circles are O-phosphorylated peptides.

[0024] 2. Magnetic mesoporous titania nanocomposites (Fe 3 o 4 @mTiO 2 ) preparation:

[0025] Fe3O4 nanomaterials were synthesized by a solvent method. Ultrasonic disperse 1g of iron oxide hexahydrate and 1.06g of trisodium citrate in 20mL of ethylene glycol, add 1.2g of anhydrous sodium ethylate, stir vigorously at room temperature for 0.5h, pour into the reactor at 200°C for 10h, and wait for the reactor to cool After reaching room temperature, the prepared ferroferric oxide magnetic nanometer material was washed with water, and freeze-dried at 45° C. for use. figure 2...

Embodiment 2

[0038] 1. β-elimination of O-phosphorylated peptides: Dissolve 40ug of peptides 1 and 2 (see Table 1) in a sodium hydroxide alkaline solution with pH = 12, and react in a water bath at 25°C for 48h.

[0039] 2. Magnetic mesoporous titania nanocomposites (Fe 3 o 4 @mTiO 2 ) preparation:

[0040] Fe3O4 nanomaterials were synthesized by a solvent method. Ultrasonically disperse 1g of iron oxide hexahydrate and 3.6g of hexamethylenediamine in 50mL of ethylene glycol, add 4g of anhydrous sodium ethylate, stir vigorously at room temperature for 1h, pour into the reaction kettle at 200°C for 6h, and wait for the reaction kettle to cool to room temperature. After the prepared ferroferromagnetic nanometer material is washed, it is freeze-dried at 45° C. for use.

[0041] Titanium dioxide, as an effective enrichment material for phosphorylation modification, is coated on ferroferromagnetic nanomaterials. Disperse the ferroferric oxide magnetic nanomaterial prepared in a mixed solut...

Embodiment 3

[0050] 1. β-elimination of O-phosphorylated peptides: Dissolve 40ug of peptides 1 and 2 (see Table 1) in a lithium hydroxide alkaline solution with a pH greater than 14, and react in a water bath at 52°C for 0.5h.

[0051] 2. Magnetic mesoporous titania nanocomposites (Fe 3 o 4 @mTiO 2 ) preparation:

[0052] Fe3O4 nanomaterials were synthesized by a solvent method. Ultrasonically disperse 1.06g of iron oxide hexahydrate and 0.2g of sodium citrate in 20mL of ethylene glycol, add 1.2g of anhydrous sodium ethylate, stir vigorously at room temperature for 0.5h, pour it into the reaction kettle at 250°C for 20h, and wait for the reaction kettle to cool After reaching room temperature, the prepared ferroferric oxide magnetic nanometer material was washed with water, and freeze-dried at 45° C. for use.

[0053] Titanium dioxide, as an effective enrichment material for phosphorylation modification, is coated on ferroferromagnetic nanomaterials. Disperse the prepared ferroferric ...

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Abstract

The present invention relates to an N-phosphorylated peptide enrichment method. According to the method, O-phosphorylated peptide interference in a peptide sample is removed through beta-elimination, and the N-phosphorylated modified peptide in the sample is enriched by using a magnetic mesoporous titania composite nanometer material; the O-phosphorylated modification on serine and threonine is removed through a beta-elimination method under an alkaline condition so as to form stable dehydroalanine and beta-methyl dehydroalanine; a magnetic Fe3O4 nanometer material is synthesized through a solvent method, titanium dioxide is wrapped on the surface through a sol-gel method, and hole amplification is performed through a hydrothermal method to obtain the magnetic mesoporous titania composite nanometer material; and the magnetic mesoporous titania composite nanometer material can specifically enrich the N-phosphorylated peptide under a weak acid buffer system. According to the present invention, the beta-elimination and specific enrichment combined method is used for enriching the N-phosphorylated peptide in the sample.

Description

technical field [0001] The invention relates to a method for specifically enriching N-phosphorylated peptides in combination with a magnetic mesoporous titanium dioxide nanocomposite material after β-elimination to remove O-phosphorylated peptides of serine and threonine. Background technique [0002] Protein phosphorylation modification is involved in almost all life activities such as cell proliferation and apoptosis, development and differentiation, signal transduction (Nat. Biotechnol, 2005, 23, 94-101). In particular, the N-phosphorylation modification of proteins plays a pivotal role in core life processes such as life evolution, nucleosome assembly, cell transcription, and signal transduction. Therefore, it is of great biological significance to study the phosphorylation modification of proteins. Among them, the phosphorylation modification (O-phosphorylation modification) on the hydroxyl group of serine, threonine and tyrosine, its P-O phosphate bond has good stabil...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 张丽华胡晔晨陈远波江波张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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