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Method for specifically separating and enriching phosphorylated peptide and glycosylated peptide

A technology for separation, enrichment, and phosphorylation of peptides, which is applied to peptide preparation methods, chemical instruments and methods, and peptides. The effect of large specific surface area, good enrichment capacity, good magnetic response and hydrophilicity

Active Publication Date: 2018-08-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many nanomaterials have been synthesized for the separation and enrichment of glycosylated peptides or phosphorylated peptides, but due to the shortcomings of small specific surface area and low functional molecule loading, they cannot show better enrichment performance. Materials that can simultaneously enrich glycosylated peptides and phosphorylated peptides for a small number of samples are also very rare

Method used

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  • Method for specifically separating and enriching phosphorylated peptide and glycosylated peptide
  • Method for specifically separating and enriching phosphorylated peptide and glycosylated peptide
  • Method for specifically separating and enriching phosphorylated peptide and glycosylated peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Synthesis of hydrophilic magnetic mesoporous titanium dioxide material

[0032] (1) Add 1.35 g FeCl 3 ·6H 2 After O is magnetically stirred in 75 mL of ethylene glycol until the solution is clear, add 3.6 g of NaAc, then transfer to a hydrothermal reactor after fully stirring and sonicating, heating at 200°C for 12 hours, and deionizing after the reactor is cooled Wash the product with water and ethanol three times, and dry it in vacuum at 50℃;

[0033] (2) Disperse 50 mg of the product obtained in (1) ultrasonically in 400 ml ethanol containing 4 ml ammonia, add 2.5 ml n-butyl titanate dropwise, and react the resulting mixed solution at 30 ℃ for 24 h. Wash thoroughly with deionized water and absolute ethanol;

[0034] (3) Transfer the product obtained in (2) to a mixed solution containing 60 ml of ethanol and 30 ml of deionized water after ultrasonic dispersion, then transfer it to a hydrothermal reactor and heat it at 160°C for 12 h;

[0035] (4) The product obtai...

Embodiment 2

[0040] Example 2: The hydrophilic magnetic mesoporous titanium dioxide material obtained in Example 1 was used as a solid-phase adsorbent for the separation and enrichment of glycopeptides in glycoprotein HRP enzymatic hydrolysis products

[0041] (1) Sample preparation: 1 mg HRP in 50 mM NH 4 HCO 3 Enzymatic hydrolysis in the solution at 37°C for 16 h.

[0042] (2) 150 μg of hydrophilic magnetic mesoporous titanium dioxide material was dispersed in 100 μL of loading buffer containing 100 fmol / μL of the HRP digestion product of step (1), and incubated at 37°C for 20 min. Rinse the sample three times with 200 μL of loading buffer. Use 8 μL ACN / H 2 O / TFA (50 / 49 / 1, v / v / v) was eluted for 30 min.

[0043] The loading buffer is a buffer solution containing 85% by volume of acetonitrile and 5% by volume of trifluoroacetic acid.

[0044] (3) Mass spectrometry analysis: Take 1 μL of the eluent in step (2) and spot the target, dry it naturally, and perform mass spectrometry analysis. The mass ...

Embodiment 3

[0046] Example 3: The hydrophilic magnetic mesoporous titanium dioxide material obtained in Example 1 is used as a solid-phase adsorbent for the separation and enrichment of phosphorylated peptides in the hydrolyzed product of phosphorylated protein β-casein

[0047] (1) Sample preparation: 1 mg β-casein in 50 mM NH 4 HCO 3 Enzymatic hydrolysis in the solution at 37°C for 16 h.

[0048] (2) 150 μg of hydrophilic magnetic mesoporous titanium dioxide material was dispersed in 100 μL of loading buffer containing 100 fmol / μL of the β-casein enzymatic hydrolysis product of step (1), and incubated at 37°C for 20 min. Rinse the sample three times with 200 μL of loading buffer. Elute with 8 μL 0.4 M ammonia water for 30 min.

[0049] (3) The loading buffer is a buffer solution containing 95% by volume of acetonitrile and 0.1% by volume of trifluoroacetic acid.

[0050] (4) Mass spectrometry analysis: Take 1 μL of the eluent in step (2) and spot the target, dry it naturally and perform mass s...

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Abstract

The invention provides a method for specifically separating and enriching a phosphorylated peptide and a glycosylated peptide. The method concretely comprises the following steps: preparing a dispersion from a hydrophilic magnetic meso-porous titanium dioxide material, adding the dispersion and a target phosphorylated peptide and glycosylated peptide solution into a sample introduction buffer solution, performing incubation at 37 DEG C for 30-60 min, washing a material with the sample introduction buffer solution, using ammonia water with the volume ratio of 5-20% as an elution buffer solution, performing dot targeting on the obtained eluate, and performing mass spectrometry. The method realizes the simultaneous enrichment of the low-abundance phosphorylated peptide and glycosylated peptide by controlling enriching and eluting conditions, can realize simultaneous large-scale identification of a phosphorylated protein and a glycosylated protein by MALDI-TOF MS or nano-LC-MS / MS, and hasa broad application prospect in post-translational modification proteomics.

Description

Technical field [0001] The invention belongs to the preparation of a novel nanometer mesoporous adsorption material, and specifically relates to a method for specifically separating and enriching phosphorylated peptides and glycosylated peptides, and in particular to a selective enrichment based on hydrophilic magnetic mesoporous titanium dioxide nanomaterials Method for purifying glycosylated peptides and phosphorylated peptides. Background technique [0002] Protein glycosylation and phosphorylation are one of the most important ways of protein post-translational modification. They play an extremely important role in many biological processes such as protein folding, cell signal transduction, and cell differentiation. Wide attention. Studies have shown that the degree of protein glycosylation and the abnormal changes in sugar chain composition and structure are closely related to many cancers and other diseases, and glycosylated proteins are commonly found in the extracellular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22
CPCC07K1/22
Inventor 邓春晖王嘉雯姚继宗孙念荣
Owner FUDAN UNIV
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