Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof

A technology for the quantitative detection of lactalbumin, applied in measuring devices, instruments, scientific instruments, etc.

Inactive Publication Date: 2013-06-05
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After inquiry, no method has been found so far that uses the isotopic internal standard peptide dilution method combined with RPLC-ESI-MS / MS to determine heat-denatured and non-denatured bovine α-lactalbumin in milk and dairy products

Method used

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  • Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof
  • Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof
  • Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] 1. Design and determination of the isotope-specific peptide of the present invention:

[0046] According to the amino acid sequence of bovine α-lactalbumin-specific peptide determined by experiments, fully considering its related physical and chemical properties, interference and ionization efficiency in the process of mass spectrometry detection, and taking into account the cost and feasibility of actual production and application, stable isotope labeling was introduced Isoleucine (I * ) and leucine (L * ) designed and synthesized the isotope specific peptide of bovine α-lactalbumin specific peptide, its amino acid sequence is VGI * NYWL * AHK.

[0047] Elements such as retention time, linearity, and matrix effect can reflect the chromatographic separation and mass spectrometric ionization efficiency of the measured substance to a certain extent. Whether the behavior in the ionization process of mass spectrometry is similar to the greatest extent, the experimental...

Embodiment 2

[0052] Embodiment 2: kit preparation and instructions for use

[0053] 1. Reagent preparation:

[0054] 1. Preparation of bovine α-lactalbumin-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add it to the standard substance 1 tube, and ultrasonically dissolve (30s), the resulting solution is 500μmol / L α-lactalbumin-specific peptide Standard stock solution;

[0055] 2. Preparation of isotope-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add it to standard substance 2 tubes, and ultrasonically dissolve (30s), the resulting solution is 500μmol / L isotope-specific peptide standard stock solution;

[0056]3. Preparation of the isotope internal standard standard stock solution: Accurately pipette 5mL of ultrapure water, add it to the standard substance 3 tubes, and ultrasonically dissolve (30s), the resulting solution is the 500μmol / L isotope internal standard standard stock solution;

[0057] 4. Preparatio...

Embodiment 3

[0069] Sample type: Commercial infant formula milk powder.

[0070] Weigh 0.5g of the sample into a 100mL volumetric flask, add warm water to dissolve, wait to cool to room temperature, add water to the mark, accurately draw 500μL, add 100μL 20μmol / L isotope internal standard and 400μL 500mmol / L NH 4 HCO 3 solution, add 10μL 500mmol / L DTT solution, react at a constant temperature of 50°C for 30 minutes, take it out and cool to room temperature, add 30μL 500mmol / L IAA solution, let it stand in the dark for 30 minutes, add 10μL 100mmol / L CaCl 2 Solution and 40 μL of 200 μg / mL trypsin solution, 37 ℃ constant temperature for overnight enzymolysis, take out the next day and add 20 μL of pure formic acid, let stand at room temperature for 1 hour, finally dilute the enzymolysis solution to 5mL, take the obtained sample solution according to the method in Example 2 Detecting and analyzing, and using the internal standard method to calculate the results, the content of bovine α-lactal...

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Abstract

The invention provides a cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box which mainly comprises isotope labeling cattle 2, 2-bipyridine-lactalbumin specific peptide, isotope labeling cattle 2, 2-bipyridine-lactalbumin internal standard substance and cattle 2, 2-bipyridine-lactalbumin specific peptide standard substance. The amino acid sequence of the cattle 2, 2-bipyridine-lactalbumin specific peptide is VGI*NYWL*AHK, wherein I* and L* are amino acid of carbon nitrogen whole isotope labeling. The amino acid sequence of the cattle 2, 2-bipyridine-lactalbumin internal standard substance is VKKILDKVGI*NYWL*AHKALCSEKL, wherein I* and L* are amino acid of carbon nitrogen whole isotope labeling. The amino acid sequence of the cattle 2, 2-bipyridine-lactalbumin specific peptide is VGINYWLAHK. The isotope labeling internal standard substance synthesized by studies and design can accurately quantify cattle 2, 2-bipyridine-lactalbumin in milk of cows, formular powder and natural raw materials, and reliability of results is ensured.

Description

(1) Technical field [0001] The invention relates to a bovine α-lactalbumin quantitative detection kit and application thereof. (2) Background technology [0002] Bovine α-Lactalbumin (Bovine α-Lactalbumin) is a compact monomeric globulin protein composed of 123 amino acid residues, containing 4 disulfide bonds, relatively stable in structure, with an average molecular weight of 14186.1243. Bovine α-lactalbumin is an excellent source of essential and branched-chain amino acids, and it has a 76% amino acid composition similarity to α-lactalbumin in human milk. Its main physiological function is that it has a better binding ability with metal ions, and it is also rich in tryptophan, which helps to increase the release of serotonin in the blood, promotes nerve development, and has good nutritional value. At present, there are a large number of infant formula milk powder supplemented with bovine α-lactalbumin in the market. Due to the mixed quality of raw materials and different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/72
Inventor 任一平赖世云张京顺黄百芬蔡增轩
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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