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Tag peptide of casein phosphopeptide and application of tag peptide

A casein phosphopeptide and protein phosphopeptide technology, applied in the field of casein phosphopeptide tag peptide, can solve the problems of high separation efficiency, low sensitivity, difficult to find without interference, etc., and achieves high recovery rate, good accuracy, good linear effect

Active Publication Date: 2017-05-31
杭州帕匹德检测技术服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HPLC has high separation efficiency but low sensitivity
Dairy products are complex matrix samples rich in proteins and peptides, and casein phosphopeptides are a series of peptide mixtures with different molecular weight distributions. It is difficult to find undisturbed characteristic peaks under ultraviolet or fluorescence detectors, and accurate and effective detection cannot be achieved. Quantitative detection analysis

Method used

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  • Tag peptide of casein phosphopeptide and application of tag peptide
  • Tag peptide of casein phosphopeptide and application of tag peptide
  • Tag peptide of casein phosphopeptide and application of tag peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Search and determination of CPPs tag peptides.

[0036] Since CPPs is a mixture of polypeptides. Firstly, multiple batches of CPPs raw materials were dissolved and diluted to 100 μg / mL for reversed-phase chromatographic separation and mass spectrometry full scan. According to the Proteome Discoverer 2.1 software and the protein database, find out as much as possible the characteristic peptides that are contained in all batches and are relatively stable (Table 1), and a total of the following 7 peptides were found (Table 1).

[0037] Table 1 Basic information of the seven characteristic peptides of casein phosphopeptides screened

[0038]

[0039] Note: The average hydrophilicity is 0~2, the polypeptide has good hydrophilicity in water; the average hydrophilicity=0, the solubility of the polypeptide in water is average; the average hydrophilicity is -2~0, the solubility of the polypeptide in water is relatively low Difference.

[0040] On the premise of ensuring the ...

Embodiment 2

[0052] Determination of isotopic internal standards.

[0053] One of the biggest problems in mass spectrometry quantitative detection is matrix interference. The use of internal standards to eliminate the influence of matrix interference on the results is a common and ideal method, especially isotope internal standards are the best choice. In view of the fact that the object detected by the method of the present invention is a polypeptide, what is produced in the electrospray ion (ESI+) mode is a multiply charged ion. In addition, carbon (1.11% 13 C), nitrogen (0.37% 15 The natural isotope abundance of N) elements is relatively high, and the response is more significant in sensitive mass spectrometer detectors. If the number of isotope labels is too small, there will be interference. Therefore, two amino acids L and S are selected for isotope labeling, and isotope labeling peptides is (LSQSKVL*PV*PQK). L* and V* are all isotopic labels of carbon and nitrogen.

[0054] After...

Embodiment 3

[0058] (1) Optimization of sample pretreatment methods.

[0059] The present invention optimizes the sample extraction (dissolution) and purification (precipitation protein).

[0060] a. Dissolution of the sample

[0061] It can be seen from Table 1 that the LSQSKVLPVPQK polypeptide is generally hydrophilic, so it is necessary to select a suitable solution to dissolve the selected peptide. The experimental design compares the peak areas of CPPs-labeled peptides after the samples are dissolved in different concentrations of acetonitrile solutions. The specific operation is as follows:

[0062] Accurately weigh 21 parts of 5.0 g samples, and dissolve the samples with pure water, 10%, 15%, 20%, 25%, 30%, and 40% acetonitrile solutions respectively, and parallelize 3 samples for each concentration. Dilute to 100mL after fully dissolved. Take 80 μL respectively, dilute to 1 mL with 10 mmoL ammonium acetate solution (pH=4.6), dispense into centrifuge tubes, centrifuge at 6000 r / ...

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Abstract

The invention discloses a tag peptide of casein phosphopeptide. An amino acid sequence of the tag peptide is LSQSKVLPVPQK. The invention also discloses an internal standard peptide of the casein phosphopeptide, and an amino acid sequence of the internal standard peptide is LSQSKVL*PV*PQK, wherein L* and V* are carbon and nitrogen all-isotope marked amino acid residues. According to the tag peptide and the internal standard peptide disclosed by the invention, one characteristic peptide in the casein phosphopeptide is selected as a detecting object for quantitatively detecting the casein phosphopeptide in a milk product; the characteristic peptide is finally determined by screening and a most representative peptide in the casein phosphopeptide; the content of the characteristic peptide in a casein phosphopeptide raw material is relatively stable; then the internal standard peptide marked by isotope is added for correcting a result; the method has the advantages of better linearity, high recovery rate and good accuracy.

Description

technical field [0001] The invention belongs to the technical field of food detection, and in particular relates to a casein phosphopeptide label peptide and its application. Background technique [0002] Casein Phosphopeptides (hereinafter referred to as: CPPs) are hydrolyzed bovine casein by a single enzyme or a complex enzyme system, and the products are distributed in α s1 、α s2 , β, and different regions in κ casein. It can be used in various nutritional and health foods, and can effectively increase the intake, absorption and utilization of calcium, iron, zinc and other divalent minerals by the human body. There are a large number of infant formula milk powders added with CPPs on the market. Due to different processing techniques and other reasons, the quality of the products varies, but no accurate and effective evaluation and detection methods have been established. [0003] Currently, ethanol precipitation, electrophoresis, high performance liquid chromatography,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47G01N30/02G01N30/06
CPCC07K14/4732G01N30/02G01N30/06
Inventor 黄棣华
Owner 杭州帕匹德检测技术服务有限公司