Method for detecting Apis dorsata MRJP1 through liquid chromatography-tandem mass spectrometry and application of method in identifying authenticity of Apis dorsata honey

Abstract

The invention relates to the field of food detection, in particular to a method for detecting Apis dorsata MRJP1 through liquid chromatography-tandem mass spectrometry and application of the method inidentifying the authenticity of Apis dorsata honey. According to the method, technical and method references can be provided for identification and authenticity evaluation of the Apis dorsata honey,and scientific and technological supports and method references are provided for protection of the endangered species Apis dorsata and prohibition of sale of the Apis dorsata honey. The method for detecting the MRJP1 in the Apis dorsata honey has the advantages of being strong in characteristic, high in sensitivity, good in accuracy and precision and the like, and is particularly suitable for authenticity identification of the Apis dorsata honey. The method provides a method support for a law enforcement institution to restrain the supply and marketing of Apis dorsata honey, and has importantpractical significance for protecting the endangered population Apis dorsatas and maintaining bee gene polymorphism.

Description

technical field

[0001] The invention relates to the field of food detection, in particular to a liquid chromatography tandem mass spectrometry method for detecting the MRJP1 of apis mellifera and its application in identifying the authenticity of honey of apis mellifera. Background technique

[0002] big bee ( Apis dorsata ) is a wild pollinating insect endemic to Asia, belonging to the genus Apis in the family Apis of the order Hymenoptera. The individuals of the big bees are larger than other bees in the genus Apis, so they are called big bees; and because their hives are arranged in rows and hang under the trunks of kapok trees, they are commonly called big row bees or big hanging bees. So far, the big bees have not been domesticated, and they are all wild bees with a more ferocious personality. It is mainly distributed in Southeast Asia and South Asia, and it is also distributed in Yunnan, Guangxi and Hainan in my country. The big bee is a very important pollinator in...

Examples

Embodiment 1

[0037] Example 1 Excavation of MRJP1 characteristic peptides in the honey of Apis mellifera

[0038] Solution preparation:

[0039] 1. Solution preparation

[0040] 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.

[0041] 100 mM DTT solution: weigh 0.308 g of DTT, add 40 mM NH 4 HCO 3 The solution was dissolved, mixed evenly and adjusted to 20mL, and stored in a -20°C refrigerator until use.

[0042] 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.

[0043] 2. Weigh the honey and PBS solution according to the ratio of 1:1, vortex fully, centrifuge at 4°C for 20 min, and collect the supernatant;

[0044] 3. 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...

Embodiment 2

[0055] Example 2 The establishment and detection method of the characteristic peptide segment of MRJP1 in bee honey

[0056] 1. All the characteristic peptides screened out in Example 1 are verified on the NCBI and Uniprot websites, and the characteristic peptides that only exist in the large bee MRJP1 are screened, and the mass spectrometry data generated in Example 1 is checked, and the response is selected The characteristic peptide with high concentration and no influence of other substances is the final characteristic peptide, ie ENAILSGEYDYTK.

[0057] 2. Synthesize the characteristic peptide segment ENAILSGEYDYTK and the stable isotope internal standard (IS) peptide segment ENAILSGEYDYTK*, K* means that all Cs in arginine are replaced by 13 C, all N replacements are 15 N, with a purity of over 98%, was stored at -20°C for future use.

[0058] 3. Drawing of Standard Curve

[0059] A series of characteristic peptide standards (1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL , 20 ...

Embodiment 3

[0070]Example 3 Practical Application of MRJP1 Characteristic Peptides in Big Bee Honey

[0071] 1. Purchase samples of honey made by Italian bees (acacia honey, linden honey and wattle honey), soil honey (honey made by Apis mellifera), big bees and small bees from the regular market for actual sample testing.

[0072] 2. Detection of 6 kinds of honey samples

[0073] (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;

[0074] (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 minutes, and then 250 μL of 100 mM IAA solution was added to react in the dark at room temperature for 60 minutes;

[0075] (3) Add 2 μg excess trypsin to the 6 final solutions obtained in (2), and digest ov...

technical field

[0001] The invention relates to the field of food detection, in particular to a liquid chromatography tandem mass spectrometry method for detecting the MRJP1 of apis mellifera and its application in identifying the authenticity of honey of apis mellifera. Background technique

[0002] big bee ( Apis dorsata ) is a wild pollinating insect endemic to Asia, belonging to the genus Apis in the family Apis of the order Hymenoptera. The individuals of the big bees are larger than other bees in the genus Apis, so they are called big bees; and because their hives are arranged in rows and hang under the trunks of kapok trees, they are commonly called big row bees or big hanging bees. So far, the big bees have not been domesticated, and they are all wild bees with a more ferocious personality. It is mainly distributed in Southeast Asia and South Asia, and it is also distributed in Yunnan, Guangxi and Hainan in my country. The big bee is a very important pollinator in...

Examples

Embodiment 1

[0037] Example 1 Excavation of MRJP1 characteristic peptides in the honey of Apis mellifera

[0038] Solution preparation:

[0039] 1. Solution preparation

[0040] 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.

[0041] 100 mM DTT solution: weigh 0.308 g of DTT, add 40 mM NH 4 HCO 3 The solution was dissolved, mixed evenly and adjusted to 20mL, and stored in a -20°C refrigerator until use.

[0042] 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.

[0043] 2. Weigh the honey and PBS solution according to the ratio of 1:1, vortex fully, centrifuge at 4°C for 20 min, and collect the supernatant;

[0044] 3. 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...

Embodiment 2

[0055] Example 2 The establishment and detection method of the characteristic peptide segment of MRJP1 in bee honey

[0056] 1. All the characteristic peptides screened out in Example 1 are verified on the NCBI and Uniprot websites, and the characteristic peptides that only exist in the large bee MRJP1 are screened, and the mass spectrometry data generated in Example 1 is checked, and the response is selected The characteristic peptide with high concentration and no influence of other substances is the final characteristic peptide, ie ENAILSGEYDYTK.

[0057] 2. Synthesize the characteristic peptide segment ENAILSGEYDYTK and the stable isotope internal standard (IS) peptide segment ENAILSGEYDYTK*, K* means that all Cs in arginine are replaced by 13 C, all N replacements are 15 N, with a purity of over 98%, was stored at -20°C for future use.

[0058] 3. Drawing of Standard Curve

[0059] A series of characteristic peptide standards (1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL , 20 ...

Embodiment 3

[0070]Example 3 Practical Application of MRJP1 Characteristic Peptides in Big Bee Honey

[0071] 1. Purchase samples of honey made by Italian bees (acacia honey, linden honey and wattle honey), soil honey (honey made by Apis mellifera), big bees and small bees from the regular market for actual sample testing.

[0072] 2. Detection of 6 kinds of honey samples

[0073] (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;

[0074] (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 minutes, and then 250 μL of 100 mM IAA solution was added to react in the dark at room temperature for 60 minutes;

[0075] (3) Add 2 μg excess trypsin to the 6 final solutions obtained in (2), and digest ov...

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