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Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method

A measurement method and glycosylation technology, which is applied in the field of site determination, can solve the problem that the hydrophilic properties of phosphopeptides are washed out, etc.

Inactive Publication Date: 2010-09-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Third, the hydrophilic nature of phosphopeptides may lead to wash-out in RP-HPLC
[0008] Fourth, it is difficult to comprehensively sequence specific large phosphorylated proteolytic fragments

Method used

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  • Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method
  • Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method
  • Phosphorylated and/or glycosylated protein or peptide one-step enrichment modification determination method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the terminal alkyne label compound shown in the synthetic formula II structural formula

[0081]

[0082] Add 585 mg of Fmoc-Cys(Trt)-OH (fluorenylmethoxycarbonyl cysteine ​​mercaptotrityl) and 151 μL of 3-butyn-1-ol into the flask, then add dry acetonitrile, add 50 mg of DMAP (4 - dimethylaminopyridine). After the system was completely clarified, an appropriate amount of 2.2 g of DCC (1,3-dicyclohexylcarbodiimide) was added, and the temperature was naturally raised to room temperature. After reacting for 8 hours, the insoluble matter was removed by filtration, separated by a silica gel column, and the filtered solution was rotary evaporated with an oil pump, and the obtained product was drained by an oil pump, and weighed.

[0083] Separation was carried out on a silica gel column.

[0084] After the obtained product was dissolved in ethyl acetate, TFA (trifluoroacetic acid) was added and shaken for 2 h under the protection of argon. The material wa...

Embodiment 2

[0085] Embodiment 2, the terminal alkyne group label compound shown in the synthetic formula IV structural formula

[0086]

[0087] Add 210 μL of thioglycolic acid to 10 mL of 2N sodium hydroxide solution, slowly add 2.15 mL of p-toluenesulfonyl chloride dropwise while stirring in an ice-water bath, measure the pH value of the system after the dropwise addition, and add sodium hydroxide to control the pH value to 9 ~10. After stirring and reacting for 1 hour, wash off excess p-toluenesulfonyl chloride with ether, repeat the ether washing step twice, dilute with 15 mL ethyl acetate, acidify with 6M hydrochloric acid to pH 1, separate the organic phase and the aqueous phase Extract with ethyl acetate. The organic phases were combined, washed with 5% (wt) sodium chloride solution until neutral, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a viscous liquid.

[0088] Add 10 mL of dry acetonitrile to the flask from the previous step, and add 50 mg of DM...

Embodiment 3

[0090] Example 3. Synthesis of the alkyne-terminated label compound SS-01 shown in the structural formula II

[0091]

[0092] 1. Methylbenzenesulfonyl protection of 3-butyn-1-ol

[0093] Add 4.55g of toluenesulfonyl chloride and 10mL of chloroform into a three-necked flask, stir well and cool to 3°C with an ice-water bath; drop 0.701g of 3-butyn-1-ol and 2.06g of pyridine into the flask, pass Ice-water bath and control the rate of addition to keep the reaction temperature at 6-8°C. After the dropwise addition, keep stirring at 8-10°C for 5 hours; pour the reaction mixture into 40mL ice water and wash it with stirring, then wash it twice with cold water, and adjust the pH value 8-9 sodium carbonate aqueous solution (temperature below 3°C) was fully stirred and washed twice, and finally washed with distilled water until the pH value was stabilized at 6.57-6.70; the water layer was separated by standing, and the organic layer was dried with anhydrous sodium sulfate. Atmosphe...

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Abstract

The invention discloses a phosphorylated and / or glycosylated protein or peptide one-step enrichment modification determination method. The method includes the following steps: 1) modification on end alkynyl or triazon of protein or peptide; 2) resin capturing of protein or peptide with modified end alkynyl; 3) cutting of the peptide / protein with modified alkynyl on resin. The protein or peptide enrichment modification determination method provided by the invention not only can enrich and purify the modified protein or peptide after translation but also can add one molecule segment on the protein or peptide, and the molecule segment has the functions of mass spectrum label and mass spectrum signal gain. The method has wide application prospect in the science analysis field.

Description

technical field [0001] The invention relates to a separation and enrichment method and site determination of phosphorylated and / or glycosylated proteins and polypeptides. Background technique [0002] Phosphorylation is the most common covalent protein modification, present in one-third of eukaryotic gene expression products. It regulates fundamental cellular behaviors: cell division, growth, differentiation. Since phosphorylation and other post-translational modifications cannot be predicted using gene sequences, researchers have developed other methods. [0003] The traditional method for determining the phosphorylation site generally includes: radioactive labeling of the phosphorylation site in the culture growth medium of the strain, and then immunoprecipitation or SDS-PAGE method, enzyme digestion, and Edman degradation separation containing 32P isotopically labeled proteins. This method, in addition to the potential hazards to the operator's health, the low abundanc...

Claims

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Application Information

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IPC IPC(8): G01N27/62C07K1/13C07K1/107
Inventor 李艳梅魏玮程智慧卢小利冯玉萍黄智华胡笳蔡辉赵镭刘冲张凌锋王芹珠林天舒
Owner TSINGHUA UNIV
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