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Method for enriching glycopeptide and simultaneously enriching glycopeptide and phosphorylated peptide by using metal oxide

A technology for phosphorylating peptides and oxides, applied in the direction of peptides, etc., to achieve the effect of strong universality, no activation time, and convenient and easy-to-obtain raw materials

Inactive Publication Date: 2013-08-14
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 use of metal oxides to simultaneously obtain glycopeptides and phosphorylated peptides in one operation has not been reported

Method used

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  • Method for enriching glycopeptide and simultaneously enriching glycopeptide and phosphorylated peptide by using metal oxide
  • Method for enriching glycopeptide and simultaneously enriching glycopeptide and phosphorylated peptide by using metal oxide
  • Method for enriching glycopeptide and simultaneously enriching glycopeptide and phosphorylated peptide by using metal oxide

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Experimental program
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preparation example Construction

[0033]Preparation of sample solution: 1mg of α-casein was dissolved in 1mL of 50mM ammonium bicarbonate solution, and trypsin was added according to the mass ratio of α-casein and trypsin at a ratio of 1:40 (w / w), and then digested at 37°C. After reacting for 16 hours, formic acid with a final concentration of 0.5% (v / v) was added to terminate the enzymatic hydrolysis.

[0034] Dissolve 1 mg of human serum immunoglobulin and 1 mg of horseradish peroxidase in 100 μl of 50 mM ammonium bicarbonate containing 8 mol / L urea and shake for 3 hours. Thiothreitol (Dithiothreitol; DTT for short) solution was placed at 37°C for 2 h, and then 10 μL of iodoacetic acid (IAA) with a concentration of 50 mmol / L was added to the protein solution respectively, and stood at room temperature for 30 minutes in the dark. Dilute this solution ten times with ammonium bicarbonate, add trypsin, enzymolyze according to the mass ratio of protein to trypsin 1:40 (w / w), react at 37°C for 16 hours, and then a...

Embodiment 1

[0036] Titanium oxide enrichment of glycopeptides: 1 mg of titanium oxide (purchased from GL Science) was homogenized into a GELoader tip tube, and 3.3 μL (83.3 pmol) of horseradish peroxidase that had been neutralized to neutral with ammonia water The solution solution was loaded and washed three times with 30 μL of 50 mM ammonium bicarbonate solution, 30 μL of 0.1% formic acid solution for three times, and three times for 30 μL of acetonitrile aqueous solution containing 0.5% formic acid. 50% acetonitrile, and finally eluted with 10 μL of acetonitrile aqueous solution containing 5% formic acid in volume concentration, wherein the acetonitrile aqueous solution contains 50% acetonitrile in volume concentration, and the eluate was collected and analyzed by nanoESI-Q-Tof injection.

[0037] Comparison of spectra before and after enrichment figure 1 (c) and 1(a), it can be seen that the glycopeptides hydrolyzed by the standard glycoprotein horseradish peroxidase are effectively e...

Embodiment 2

[0039] The difference from Example 1 is that the titanium oxide used is self-made titanium oxide by sol-gel method [Patent No. 200810202077.1].

[0040] The preparation method of titanium oxide is as follows:

[0041] Step (1) Add 0.0024mol of dodecylamine, 100ml of absolute ethanol, 0.006mol of acetylacetone, 0.012mol of tetrabutyl titanate into a 200ml round bottom flask, stir well at 20°C, add 0.3mol of deionized water, and the solution After turbidity, stop stirring, let stand for 45 minutes, filter, wash with absolute ethanol for 2 to 4 times, and dry at 25°C to 30°C to obtain a solid.

[0042] Step (2) Add 1 g of the solid obtained in step (1), 0.2 g of urea, add 16 ml of absolute ethanol, and 4 ml of deionized water, place it in an autoclave lined with polytetrafluoroethylene, let it stand at 130 ° C for 8 hours, and after cooling After filtering, washing with acetone and methanol for 2-3 times, drying in vacuum at high temperature for 6 hours, and sintering in a muffl...

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Abstract

The invention discloses a method for enriching glycopeptide and simultaneously enriching glycopeptide and phosphorylated peptide by using a metal oxide. The method is characterized in that: the metal oxide is used as a solid-phase extraction material for separating and enriching the glycopeptide from protein enzymolysis solution, and simultaneous enrichment of the phosphorylated peptide and the glycopeptide can be realized in a sample by using simple operation. Specifically, the method comprises the following steps of: mixing the metal oxide and the sample under a neutral or weak acid condition, eluting the mixture under an acid condition to obtain the glycopeptide, and eluting the mixture under an alkali condition to obtain the phosphorylated peptide. The method can realize high-efficiency and high-selectivity enrichment of the phosphorylated peptide and the glycopeptide in the same sample.

Description

technical field [0001] The invention relates to the separation and purification of glycopeptides and phosphorylated peptides, in particular to a method for enriching glycopeptides and simultaneously enriching phosphorylated peptides and glycosylated peptides by using metal oxides. technical background [0002] Post-translational protein modification is a hot research topic in proteomics, especially protein phosphorylation and glycosylation, which are the two most common and most extensively studied post-translational modification processes. Phosphorylation of proteins almost regulates the entire process of life, including cell proliferation, development and differentiation, and is also one of the main transmission methods of signals known so far. At the same time, understanding the glycosylation process of proteins is of great significance to the diagnosis and treatment of some diseases such as cancer inflammation. At present, a very important method for the study of protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K2/00C07K1/14C07K1/16C07K1/34
Inventor 梁鑫淼闫竞宇柯燕雄李秀玲张秀莉薛兴亚
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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