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A kind of osteopontin characteristic peptide and its application

A technology of osteopontin and characteristic peptides, which is applied in the direction of specific peptides, animal/human proteins, instruments, etc., to achieve the effect of ensuring accuracy and good linearity

Active Publication Date: 2021-07-20
杭州璞湃科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no report on the quantitative detection of osteopontin in dairy products using the characteristic peptide of osteopontin

Method used

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  • A kind of osteopontin characteristic peptide and its application
  • A kind of osteopontin characteristic peptide and its application
  • A kind of osteopontin characteristic peptide and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Search and determination of characteristic peptides

[0049] The selection of specific peptides is a key step in targeted proteomics detection technology, which is directly related to the accuracy of quantitative methods. In the process of selecting specific peptides, the theoretical database is usually combined with the actual sample detection data for analysis, and bioinformatics software is used to assist in the prediction and calculation of protein hydrolysis products.

[0050] The present invention selects Uniprot database and Peptide Mass tool to osteopontin OPN (as in cow, yak, buffalo, sheep, goat milk) figure 1 shown) for prediction and calculation of possible trypsin-digested peptides.

[0051] In order to verify whether there are theoretically determined peptides in the actual samples, the present invention pre-treats five milk products (i.e. samples), respectively, and directly adds formic acid to terminate the reaction after enzymatic hydrolysis, and th...

Embodiment 2

[0106] 1. Standard curve, recovery rate and precision for internal standard peptide 1

[0107] (1) Establishment of standard curve

[0108] Accurately pipette 10 μL, 25 μL, 50 μL, 75 μL, 100 μL of osteopontin-labeled peptide solution (1 μg / mL), add 50 μL of internal standard peptide 1 solution (1 μg / mL), and then add 940 μL, 925 μL, 900 μL of 0.1% formic acid aqueous solution , 875 μL, and 850 μL by vortex mixing.

[0109] Gained solution detects and analyzes by the liquid chromatography described in the 5th part of embodiment 1 and the mass spectrometry condition, each sample is detected in parallel 6 times, and the gained result makes standard curve as follows Figure 5 As shown, the standard curve formula: y=0.024985x-0.041725, R 2 =0.999, where x is the concentration (ng / mL) of the osteopontin tag peptide, and y is the ratio of the peak area of ​​the sample to the peak area of ​​the internal standard peptide 1.

[0110] (2) Recovery rate and precision

[0111] The accu...

Embodiment 3

[0123] Example 3 Detection of Osteopontin Content in Milk and Dairy Products Using Internal Standard Peptide 1

[0124] Cow's milk, buffalo's milk, yak's milk, goat's milk, sheep's milk and formula milk powder were selected as the research objects to determine the content of osteopontin in milk and dairy products. The specific method is as follows:

[0125] (1) Liquid milk: Take 50 μL of liquid milk in a 2 mL plastic tube, add 950 μL of ultrapure water, and vortex to mix.

[0126] Milk powder sample: Weigh 2.5g of milk powder sample to dissolve, transfer to a volumetric flask and dilute to 50mL. Take 100 μL of liquid milk in a 2 mL plastic tube, add 900 μL of ultrapure water, and vortex to mix.

[0127] (2) Accurately draw 300 μL of sample solution, add 4.2 mL of sodium bicarbonate (100 mmol / L) solution, vortex and mix well, add 60 μL of DTT solution (500 mmol / L), react at a constant temperature of 70 °C for 30 minutes, take it out and cool to room temperature, add 180 μL of ...

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Abstract

The invention discloses a characteristic peptide of osteopontin and application thereof. The amino acid sequence of the characteristic peptide is GDSVAYGLK. The present invention obtains the internal standard peptide corresponding to the characteristic peptide by screening the characteristic peptide of osteopontin, and realizes the quantitative detection of osteopontin by using the analysis technology of high-performance liquid chromatography and mass spectrometry. The method has better Linearity, sensitivity, recovery and precision.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to an osteopontin characteristic peptide and its application. Background technique [0002] Osteopontin (OPN) is a highly phosphorylated and negatively charged non-collagenous bone matrix glycoprotein, containing about 300 amino acid residues, of which aspartic acid, serine and glutamic acid residues occupy a high proportion, accounting for about half of the total amino acid content. Osteopontin (OPN) contains an arginine-glycine-aspartic acid (RGD) sequence, which is ubiquitous in osteopontin (OPN) of different species and plays an important role in the adhesion of osteopontin (OPN) Features play an important role. [0003] Osteopontin is widely distributed in a variety of tissues and cells, and plays an important role: (1) Osteopontin (OPN) plays a certain role in nonspecific immunity and autoimmunity against infection, and helps to control the progression of inflammatory...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/52G01N30/02G01N30/06
CPCC07K14/52G01N30/02G01N30/06G01N2030/045G01N2030/062
Inventor 任一平胡蓓蒋易蓉赖世云
Owner 杭州璞湃科技有限公司
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