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Trace phosphorylated peptide desalting column

A phosphorylated polypeptide and micro-quantity technology, which is applied in the direction of measuring devices, instruments, and material analysis by electromagnetic means, can solve the problems of inability to effectively retain phosphorylated modified polypeptides, and achieve simple and easy control of the process, high yield, and application wide range of effects

Active Publication Date: 2014-12-10
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ZipTip C18 The disadvantage is that it cannot effectively retain phosphorylated modified peptides, or some small peptides with strong hydrophilicity

Method used

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  • Trace phosphorylated peptide desalting column
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  • Trace phosphorylated peptide desalting column

Examples

Experimental program
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Effect test

preparation example Construction

[0036] The preparation method of micro-volume desalting small column comprises the following steps:

[0037] 1) Fill the polypyrrole-modified quartz wool stored in ethanol solution into the micro ZipTip containing C18 reversed-phase separation material C18 Pipette, fill it tightly with a pipette;

[0038] 2) Seal the upper end of the micropipette to prevent the quartz wool from being sucked out during use.

[0039] The small column preservation and cleaning method comprises the following steps:

[0040] 1), the prepared micro desalting column is stored in ethanol solution;

[0041] 2) Wash three times with 80 wt% acetonitrile solution containing 0.1 wt% TFA before use, and then wash three times with aqueous solution containing 0.1 wt% TFA.

[0042] Sample loading method, sample washing method and sample elution method, including the following steps:

[0043] 1) Use 0.1wt% TFA to adjust the acidity of the trypsin hydrolyzate (~pH 8) to pH 2~3, then pipette back and forth se...

Embodiment 1

[0047] Preparation of micro phosphorylated polypeptide desalting cartridge and sample analysis.

[0048] The preparation steps are as follows:

[0049] 1) Weigh 0.01 g of quartz wool into a centrifuge tube, add 1 ml of deionized water and 25 μl of pyrrole, and sonicate for 10 minutes;

[0050]2) Quickly add 100 microliters of ammonium persulfate aqueous solution with a concentration of 20 micrograms / microliter to the mixture obtained in step 1), vortex to mix quickly, and then sonicate for 10 minutes;

[0051] 3), take step 2) to obtain the polypyrrole-modified quartz wool, wash it with ethanol, and store it in an ethanol solution;

[0052] 4), before analyzing the sample, load the product obtained in step 3) into a micro ZipTip C18 The upper part of the pipette was washed successively three times with 80 wt% acetonitrile solution containing 0.1 wt% TFA and 0.1 wt% TFA aqueous solution. Trypsin hydrolyzate (~pH8) was first adjusted to acidity with 0.1wt% TFA, pH 2~3, or dir...

Embodiment 2

[0055] The trace phosphorylated polypeptide desalting column prepared in Example 1 was used to analyze zebrafish egg phosphorylated proteins

[0056] 1. Preparation of TMTIP PPY-C18 a batch;

[0057] 2. Washing the desalting small column of trace phosphorylated polypeptide obtained in step 1 with 80wt% acetonitrile solution and aqueous solution containing 0.1wt% trifluoroethylene (TFA) successively;

[0058] 3. Use a pipette to directly load the trypsin lysate of zebrafish eggs, or adjust the pH to 2-3 before loading;

[0059] 4. Wash three times with an aqueous solution containing 0.1% TFA;

[0060] 5. Elution with 50% acetonitrile containing 0.1% TFA;

[0061] 6. Mass spectrometry experiment, mix the eluent and the matrix and drop it on the sample target, put it into the mass spectrometer (SYNAPT G2HDMS, WATERS, USA), adjust the frequency of the ultraviolet laser to 200HZ, the obtained mass spectrogram is as follows Figure 4 and 5 shown.

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Abstract

The invention relates to a trace phosphorylated peptide desalting column, which takes a reversed-phase C18 and polypyrrole composite as a separation material, makes use of electrostatic interaction between positive charges on polypyrrole nitrogen atoms and electronegative phosphate groups and pi-pi stacking interaction of polypyrrole unsaturated double bonds and aromatic amino acid residues to compensate insufficient of C18 stationary phase single hydrophobic interaction and enhance interactions of separation materials and target molecules. Pyrrole and ammonium persulfate are used as raw materials, processed by ultrasound at room temperature, then washed, filled, and sealed to get the trace phosphopeptide peptide desalting column. The polypyrrole-modified quartz material prepared by the invention is high in separation efficiency, purity and stability. The material can be used in acidic conditions and alkaline conditions, so peptides which cannot be detected by conventional C18 stationary phases can be detected, signal to noise ratio is high, and interference is small. The method is simple, does not require sophisticated equipments, can effectively gather phosphorylated peptides of low abundance, is simple in sample analysis operation, and has no complex sample preparation.

Description

technical field [0001] The invention relates to a small amount of phosphorylated polypeptide desalting small column TMTip PPY-C18 , this small column packs polypyrrole and C18 stationary phase in series in a micropipette tip (Tandem Polypyrrole-C18 packed micropipette tip, referred to as TMTip PPY-C18 ). The invention is suitable for analyzing protein phosphorylation sites in trace biological samples such as cells and tissues, and is a separation method integrating electrostatic interaction, Π-Π stacking interaction and hydrophobic interaction. Background technique [0002] A large number of proteins in cells undergo post-translational phosphorylation modification, which directly participates in a variety of signal transduction pathways, and its protein regulatory network is the target of many drugs. The difficulty in analyzing phosphorylated modifications is that the abundance of such modified proteins is very low, so it is necessary to develop new materials for the enric...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/34G01N27/62
Inventor 钟鸿英郑石付洁英王晓丽黄璐璐
Owner HUAZHONG NORMAL UNIV
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