Data processing method for automatically rapid identifying protein phosphorylation site

A data processing and phosphorylation technology, applied in the detection and identification of protein phosphorylation sites, to achieve the effect of improving reliability, avoiding human factors, and high reliability

Inactive Publication Date: 2008-10-22
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

By actively matching the identification results of the secondary spectrum and the corresponding tertiary spectrum, the method effectively reduces the number of random matches and improves the reliability of identification; On the whole, it overcomes the problem that the lack of skeleton fragment

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  • Data processing method for automatically rapid identifying protein phosphorylation site
  • Data processing method for automatically rapid identifying protein phosphorylation site
  • Data processing method for automatically rapid identifying protein phosphorylation site

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[0030] The sample preparation and data collection are the same in Example 1 and Example 2, and the specific process is as follows:

[0031] Enzymatic hydrolysis conditions for protein: the buffer is phosphate buffer at pH 8.5, and the concentration of urea is 8 mol / L. After the protein sample was dissolved in the buffer, the disulfide bonds were first reduced with dithiothreitol at 37°C for a reaction time of 2 hours. Then use iodoacetamide to block the sulfhydryl group, and the reaction is to block the light for 2h. Then trypsin was used to digest the reaction at 1:50 overnight at 37°C.

[0032] Mass spectrometry detection conditions for phosphopeptides: The phosphopeptide mixture is analyzed by capillary liquid chromatography-ion trap mass spectrometry, wherein the capillary liquid chromatography adopts a C18 capillary column with an inner diameter of 75 μm, and the mobile phase is (A) 0.1% formic acid aqueous solution (B) 0.1 % formic acid aqueous solution / acetonitrile, b...

Example Embodiment

[0040] Example 2 Applied to the Identification of Phosphorylation Sites in Proteomic Samples

[0041] Extract the liver paracancerous tissue protein from liver cancer patients, and the extracted protein concentration is 2 μg / μL by Coomassie Brilliant Blue Quantitative Method. After 1 mg is digested with trypsin at 37°C for 16 hours, the enzymatic hydrolyzed peptide is directly loaded on the SAX chromatogram For column enrichment, solvent A (40 mM NH4Cl / 30% acetonitrile, pH 4.0) was equilibrated for 5 min, and then eluted by gradient. The elution solvents are solvent A and solvent B (1M NH4Cl / 30% acetonitrile, pH 4.0); the gradient setting is: 0-10min, and the composition of the eluent increases linearly from 100% solvent A to 100% solvent B, and then uses 100% Solvent B eluted isocratically. A fraction was collected every 2 minutes, divided into 8 fractions and collected separately. After the sample was desalted, freeze-dried and reconstituted, 1 / 4 of each fraction was analyz...

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Abstract

The invention relates to detection and identification of protein phosphorylation sites, in particular to a data processing method for automatically and quickly identifying the protein phosphorylation sites, which identifies the protein phosphorylation sites in a biological sample through the high flux of a nano-spray capillary liquid chromatogram, namely a multiple mass spectrum. The method can utilize the multiple spectrometry to perform triple spectrum analysis on fragment ions according to the fact that osphorylating peptide fragment ions in the spectrum are easy to lose phosphoric acids and form characteristic neutral missed fragment ions, combine evaluation of false positive rate, utilize matching of MS3 and a search result of a corresponding secondary spectrum database, and conveniently realize identification of the protein phosphorylation sites under special reliability through set of threshold values. Compared with the prior commonly used manual calibration method, the data processing method has the advantages of no manual calibration, simplicity and quick acquisition of the identification result of high-reliability osphorylating peptide fragments, and is suitable for high-flux analysis and large-scale identification.

Description

technical field [0001] The invention relates to the detection and identification of protein phosphorylation sites, in particular to the automatic and rapid identification of highly reliable phosphorylation sites using liquid chromatography-multistage tandem mass spectrometry in the regulation of biological life activities and the study of disease pathogenesis Methods. Background technique [0002] Protein phosphorylation is the most common and important post-translational modification of proteins in the biological world. In the life cycle of mammalian cells, about 1 / 3 of the proteins undergo hyperphosphorylation modification. In the human genome, about 2% of the genes encode 500 kinds of kinases and 100 kinds of phosphatases. Protein phosphorylation and dephosphorylation are key links in the expression regulation of prokaryotic and eukaryotic cells, and they play a role in switching and regulating the cellular functions of many organisms, which is a common and important reg...

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Application Information

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IPC IPC(8): G01N30/00C12Q1/68
Inventor 邹汉法江新宁叶明亮韩广辉
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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