Method for quantifying Apis mellifera honey MRJP2 through liquid chromatography-tandem mass spectrometry

Abstract

The invention provides a method for quantifying Apis mellifera honey MRJP2 through liquid chromatography-tandem mass spectrometry. Characteristic peptide fragments of Apis mellifera honey royal jellymajor protein 2 (MRJP2) are screened out, a quantitative method of liquid chromatography tandem mass spectrometry is developed, and the method comprises the steps of screening of the characteristic peptide fragments, making of a standard curve, performing sample pretreatment, performing liquid chromatography separation and tandem mass spectrometry detection, carrying out software quantification ofthe peptide fragments and the like. The method for quantifying the MRJP2 in the Apis mellifera honey, provided by the invention, has the advantages of higher accuracy, precision and sensitivity and strong stability, and is suitable for accurately quantifying the MRJP2 in the Apis mellifera honey. The method can be used for assisting authenticity evaluation of Apis mellifera honey. In addition, the quantitative method has important practical significance in protecting rights and interests of honey consumers and maintaining healthy development of the bee product industry.

Description

[0001] The invention relates to the field of food testing, in particular to a method for quantifying Italian honey MRJP2 by liquid chromatography tandem mass spectrometry. Background technique

[0002] Honey is a kind of sweet food that is formed after bees collect nectar or honeydew from nectar source plants and their secretions, and brew for a period of time. As a traditional natural food, honey has health care functions such as beautifying, beautifying and calming the nerves, and is deeply loved by the people. The production of honey has high labor intensity, long cycle, and the output is easily affected by factors such as nectar source and weather, which leads to high production cost of honey and limited total output, resulting in a shortage of honey on the market. In order to pursue huge profits, some unscrupulous companies manufacture adulterated honey by mixing sucrose, invert sugar, fructose, glucose, fructose syrup, etc. in the production, processing, and sales of hon...

Examples

Embodiment 1

[0056] Example 1 Method for Determining the Content of Italian Bee MRJP2 Using Liquid Chromatography Tandem Mass Spectrometry

[0057] 1. Sample source

[0058] A total of 20 samples of real Italian honey were purchased from the market or beekeepers.

[0059] 2. Experimental procedure

[0060] (1) Solution preparation

[0061] 5M Urea solution: Weigh 4.5 g of Urea, and dilute to 15 mL with ultrapure water.

[0062] 100 mM DTT: Weigh 308.5 mg DTT, 40 mM NH 4 HCO 3 Dilute the solution to 20 mL, dispense 1 mL into each tube, and store in a -20°C refrigerator until use.

[0063] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , dilute to 100 mL with ultrapure water, and store in a 4°C refrigerator until use.

[0064] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was fixed to 20 mL, and stored in a -20°C refrigerator until use.

[0065] 5% of B. Flow rate: 0.3 mL / min; injection volume: 5.0 µL.

[0066] The mass spectrometry condition...

Embodiment 2

[0074] Example 2 Authenticity detection of Italian honey samples by quantifying Italian bee MRJP2

[0075] 1. Sample source

[0076] Buy real samples of honey from the market

[0077] 2. Experimental procedure

[0078] (3) Pretreatment of honey samples

[0079] ① Accurately weigh 10 g of the honey to be tested into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new 2 mL centrifuge tube.

[0080] ② Pipette 200 μL of the protein solution and add 4 μL of 100 μg / mL internal standard peptide (IVNNDFNFNDVNFR*), and mix with 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution and react in dark at room temperature for 60 min.

[0081] ③Add 30 μL of trypsin solution to each sample, and digest at 37°C overnight. When the digestion r...

[0001] The invention relates to the field of food testing, in particular to a method for quantifying Italian honey MRJP2 by liquid chromatography tandem mass spectrometry. Background technique

[0002] Honey is a kind of sweet food that is formed after bees collect nectar or honeydew from nectar source plants and their secretions, and brew for a period of time. As a traditional natural food, honey has health care functions such as beautifying, beautifying and calming the nerves, and is deeply loved by the people. The production of honey has high labor intensity, long cycle, and the output is easily affected by factors such as nectar source and weather, which leads to high production cost of honey and limited total output, resulting in a shortage of honey on the market. In order to pursue huge profits, some unscrupulous companies manufacture adulterated honey by mixing sucrose, invert sugar, fructose, glucose, fructose syrup, etc. in the production, processing, and sales of hon...

Examples

Embodiment 1

[0056] Example 1 Method for Determining the Content of Italian Bee MRJP2 Using Liquid Chromatography Tandem Mass Spectrometry

[0057] 1. Sample source

[0058] A total of 20 samples of real Italian honey were purchased from the market or beekeepers.

[0059] 2. Experimental procedure

[0060] (1) Solution preparation

[0061] 5M Urea solution: Weigh 4.5 g of Urea, and dilute to 15 mL with ultrapure water.

[0062] 100 mM DTT: Weigh 308.5 mg DTT, 40 mM NH 4 HCO 3 Dilute the solution to 20 mL, dispense 1 mL into each tube, and store in a -20°C refrigerator until use.

[0063] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , dilute to 100 mL with ultrapure water, and store in a 4°C refrigerator until use.

[0064] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was fixed to 20 mL, and stored in a -20°C refrigerator until use.

[0065] 5% of B. Flow rate: 0.3 mL / min; injection volume: 5.0 µL.

[0066] The mass spectrometry condition...

Embodiment 2

[0074] Example 2 Authenticity detection of Italian honey samples by quantifying Italian bee MRJP2

[0075] 1. Sample source

[0076] Buy real samples of honey from the market

[0077] 2. Experimental procedure

[0078] (3) Pretreatment of honey samples

[0079] ① Accurately weigh 10 g of the honey to be tested into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new 2 mL centrifuge tube.

[0080] ② Pipette 200 μL of the protein solution and add 4 μL of 100 μg / mL internal standard peptide (IVNNDFNFNDVNFR*), and mix with 800 μL of 40 mM NH 4 HCO 3 mix. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution and react in dark at room temperature for 60 min.

[0081] ③Add 30 μL of trypsin solution to each sample, and digest at 37°C overnight. When the digestion r...

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