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Method for screening feature peptide fragment for quantitatively detecting protein in food

A screening method and a technology of characteristic peptides, which are applied in the field of screening of characteristic peptides for quantitative detection of proteins in food

Active Publication Date: 2017-08-29
杭州璞湃科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a screening method for characteristic peptides for quantitative detection of protein in food, to overcome the problem that existing screening methods have certain limitations in quantitative detection of protein in food

Method used

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  • Method for screening feature peptide fragment for quantitatively detecting protein in food
  • Method for screening feature peptide fragment for quantitatively detecting protein in food
  • Method for screening feature peptide fragment for quantitatively detecting protein in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: The method for optimizing the quantitative characteristic peptides of bovine serum albumin in raw milk.

[0101] 1. Peptide specificity evaluation: use the BLAST test in proteomics (https: / / blast.ncbi.nlm.nih.gov) to analyze and determine the matching of each theoretical enzymatic peptide in bovine serum albumin Score and number of interferences. Among them, the matching score is determined by the length of the peptide and the matching degree of the database, and the number of interference is the main parameter to determine the specificity of the peptide. Searching in the whole proteomics database, the more the number of interfering peptides that match 100% of the target peptide, the weaker the specificity of the peptide.

[0102]As shown in Table 2, according to the test results of each peptide, 16 candidate target peptides with strong specificity were finally selected, namely LVNELTEFAK, TCVADESHAGCEK, NECFLSHK, LKPDPNTLDEFK, AEFVEVTK, YICDNQDTISSK, SHCIAE...

Embodiment 2

[0131] Example 2: Methodological verification of the quantitative characteristic peptides of bovine serum albumin in raw milk.

[0132] The two peptides LVNELTEFAK and LVVSTQTALA screened in Example 1 were validated in the quantitative experiment of bovine serum albumin in milk.

[0133] Method validation includes four parts: linearity and range, detection limit and quantitation limit, recovery rate and precision.

[0134] According to the method verification results, the linearity of the two quantitative characteristic peptides is good (R 2 >0.999), the range is 1ppm-100ppm; the limit of detection is 0.21-0.22mg / kg, the limit of quantification is 0.70-0.74mg / kg; the recovery rate is 99.0%-111.1%, the precision is 2.20%-6.67%, in line with ISO / IEC 17025:2005 standard.

Embodiment 3

[0135] Example 3: Method for optimizing the quantitative characteristic peptides of α-lactalbumin in infant formula powder.

[0136] Refer to Example 1 for the preferred method of characteristic peptides, and the results are shown in Table 3.

[0137] Table 3 Characteristic parameters of quantitative characteristic peptides of α-lactalbumin

[0138]

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Abstract

The invention discloses a method for screening a feature peptide fragment for quantitatively detecting protein in food, and belongs to the technical field of protein quantitative detection. The screening method comprises the following steps: (1) comparing and analyzing matching score and interference number corresponding to a to-be-detected protein theoretical enzymolysis fragment by utilizing BLAST; (2) analyzing a mass spectrum response value and fragment abundance of a target peptide fragment through high performance liquid chromatography-tandem mass spectrometry; (3) calculating the enzymolysis rate of the target peptide fragment when the enzymolysis is conducted for 1 to 3 hours by utilizing a fitted curve; (4) calculating the recovery rate and the precision degree of the target peptide fragment. The feature peptide fragment suitable for protein accurate quantification is comprehensively screened from the aspects of specificity, mass spectrum characteristic, enzymolysis characteristic, accuracy, stability and the like and by utilizing various methods in a combined way; the screened feature peptide fragment can meet the quantitative detection requirement of high performance liquid chromatography-tandem mass spectrometry on protein in food matrix, and realizes protein quantitative detection requirement with high sensitivity, high precision degree and excellent reproducibility.

Description

technical field [0001] The invention relates to the technical field of protein quantitative detection, in particular to a method for screening characteristic peptides for quantitative detection of protein in food. Background technique [0002] Accurate quantification of protein is an important content in the research fields of biology, pharmacy, food, etc. The traditional quantitative methods are mainly divided into total protein content detection methods such as Kjeldahl method and Coomassie brilliant blue method and electrophoresis method, enzyme Linked immunosorbent assay as an example of a single target protein content detection method. [0003] Quantitative proteomics is a new type of accurate protein quantification technology, based on the research ideas of genomics and proteomics, as well as the technical means of traditional electrophoresis and high-performance liquid chromatography-tandem mass spectrometry, to achieve accurate quantification of various proteins in t...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 任一平蒋易蓉
Owner 杭州璞湃科技有限公司
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