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A liquid chromatography-tandem mass spectrometry-based method for the detection of Apis mellifera mrjp1 and its application in the identification of Apis mellifera honey

A technology of tandem mass spectrometry and liquid chromatography, which is applied in the field of food testing, can solve the problems that the research on the authenticity of bee honey is still blank, and achieve the effect of maintaining the ecological diversity of bee genes, good accuracy and precision, and high sensitivity

Inactive Publication Date: 2021-01-12
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But research on the authenticity of bee honey is still blank

Method used

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  • A liquid chromatography-tandem mass spectrometry-based method for the detection of Apis mellifera mrjp1 and its application in the identification of Apis mellifera honey
  • A liquid chromatography-tandem mass spectrometry-based method for the detection of Apis mellifera mrjp1 and its application in the identification of Apis mellifera honey
  • A liquid chromatography-tandem mass spectrometry-based method for the detection of Apis mellifera mrjp1 and its application in the identification of Apis mellifera honey

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Discovery of specific peptides of Apis mellifera MRJP1

[0037] 1. According to the mass ratio of 1:1, weigh the small

[0038] Bee honey and PBS solution, vortex fully, centrifuge at 4°C for 20 min, and collect the supernatant;

[0039] 2. Take 50 μL supernatant and add 200 μL 40 mM NH 4 HCO 3 The solution was vortexed and mixed with it, and 25 μL of 100 mM DTT solution was added. After fully vortexed and mixed, the reaction was carried out at room temperature for 60 minutes, and then 125 μL of 100 mM IAA solution was added for 60 minutes of dark reaction at room temperature;

[0040] 3. Add 2 μg excess trypsin to the final solution in step 3, and digest overnight at 37°C. Add 1 μL of formic acid solution to terminate the digestion reaction. After the digestion product was mixed with 0.1% formic acid solution at a volume ratio of 2:3, it was desalted through a C18 column, and the desalted sample was vacuum-dried.

[0041] 4. Detection of peptides: The sa...

Embodiment 2

[0044] Example 2 Establishment of characteristic peptides of Apis mellifera MRJP1 and development of detection methods

[0045] All the specific peptides screened out in Example 1 were verified on the NCBI and Uniprot websites, and the specific peptides that only existed in the MRJP1 of the small bee were screened to obtain multiple specific peptides of the MRJP1 of the small bee. The sequences are as follows:

[0046] 1) IKEALPHVPILDR (SEQ ID No. 1)

[0047] 2) FFDYDFGNDER (SEQ ID No. 2)

[0048] 3) LTSNTFNYDPEYTK (SEQ ID No. 3)

[0049] 4) YNGVPSSLNVISNK (SEQ ID No. 4)

[0050] 5) NNYPSDIDQWHGK (SEQ ID No. 5)

[0051] 6) TVAQNNETLQMIVGMK (SEQ ID No. 6)

[0052] 7) QVQIPHDIAVNTTTGK (SEQ ID No. 7)

[0053] Although there are many specific peptides to choose from, considering factors such as the uniqueness, stability, and ion response of the peptides on the mass spectrometer, only one of the optimal characteristic peptides can be selected for quantitative analysis. Looki...

Embodiment 3

[0067] Example 3 The practical application of the method for detecting the characteristic peptides of Apis MRJP1

[0068]1. Buy honey made by Italian bees (acacia honey, linden honey and wattle honey), soil honey (honey made by Apis cerana), honey made by big bees (tree honey) and honey made by small bees from regular markets. Honey and other samples are tested on actual samples.

[0069] 2. Detection of samples of honey brewed by 8 different species of bees

[0070] (1) Weigh the same amount of different honeys into centrifuge tubes, add PBS solution at a ratio of 1:1, vortex fully, centrifuge at 4°C for 20 min, and collect the supernatant;

[0071] (2) Take 100 μL supernatant and add 400 μL 40 mM NH 4 HCO 3 The solution was vortexed and mixed with it, and 50 μL of 100 mM DTT solution was added. After fully vortexed and mixed, it was reacted at room temperature for 60 min, and then 250 μL of 100 mM IAA solution was added to react in the dark at room temperature for 60 min;...

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Abstract

The invention relates to the field of food detection. The invention specifically provides a method for detecting honeybee MRJP1 based on liquid chromatography-tandem mass spectrometry and an application thereof. The method specifically comprises steps of honeybee honey sample pretreatment, database searching and protein matching, specific peptide fragment screening, liquid chromatography separation, tandem mass spectrometry detection and the like. The method for detecting the MRJP1 of the honeybees in the honey has the advantages of high specificity, high sensitivity, high accuracy, high precision and the like, and is particularly suitable for identifying the authenticity of the honeybee honey. The method provided by the invention provides a method support for restraining supply and marketing of honeybee honey, and has important practical significance for protecting the endangered population honeybees and maintaining the genetic ecological diversity of the honeybees.

Description

technical field [0001] The invention relates to the field of food detection, in particular to a liquid chromatography tandem mass spectrometry-based method for detecting Apis MRJP1 and its application in identifying Apis honey. Background technique [0002] little bee ( Apis florea Fabricius ) is a social insect belonging to the genus Apis in the family Apis, which is mainly distributed in tropical and subtropical regions of the northern hemisphere, such as India, Pakistan, Thailand, Malaysia, and Indonesia. In China, small bees are mainly distributed in Yunnan, Guizhou, Guangxi and Hainan. Small bees generally inhabit areas below 1900 m above sea level, where the annual average temperature is 15-22°C, and they build open-air single combs in secondary shrubs or weeds. Small bee colonies are small and can hunt 1-2 kg of honey per year. Small bee honey is rich in nutrients, has a good taste and fragrance, and the shape of the honeycomb is unique. Out of the curiosity of eat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72G01N2030/045
Inventor 杨术鹏李熠傅怡蔡冬梅周金慧杨宇晖
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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