CN111796038B

CN111796038B - Liquid chromatography-tandem mass spectrometry method for detecting MRJP1 of honeybee and application thereof in identifying authenticity of honeybee honey

Info

Publication number: CN111796038B
Application number: CN202010937306.5A
Authority: CN
Inventors: 杨术鹏; 李熠; 傅怡; 蔡冬梅; 周金慧; 杨宇晖; 张金震
Original assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Apicultural Research of Chinese Academy of Agricultural Sciences
Priority date: 2020-09-09
Filing date: 2020-09-09
Publication date: 2021-01-15
Anticipated expiration: 2040-09-09
Also published as: CN111796038A

Abstract

The invention relates to the field of food detection, in particular to a liquid chromatography-tandem mass spectrometry method for detecting MRJP1 of honey bees and application thereof in identifying the authenticity of honey bees. The method can provide technical and method references for identification and authenticity evaluation of honey of Apis mellifera, and also provides scientific and technological support and method references for protecting Apis mellifera which is an endangered species and forbidding selling of Apis mellifera. The method for detecting MRJP1 in the honey of the honeybees provided by the invention has the advantages of strong characteristics, high sensitivity, good accuracy and precision and the like, and is particularly suitable for authenticity identification of the honey of the honeybees. The method provides method support for law enforcement agencies to restrain supply and sale of the honeybees of the big bees, and has important practical significance for protecting the endangered population of the big bees and maintaining the genetic polymorphism of the honeybees.

Description

Liquid chromatography-tandem mass spectrometry method for detecting MRJP1 of honeybee and application thereof in identifying authenticity of honeybee honey

Technical Field

The invention relates to the field of food detection, in particular to a liquid chromatography-tandem mass spectrometry method for detecting MRJP1 of honey bees and application thereof in identifying the authenticity of honey bees.

Background

Big bee (Apis dorsata) Is a wild pollinating insect peculiar to Asia, and belongs to Apidae of Hymenoptera. The big bee is called as big bee because the individual of the big bee is bigger than other bees of the genus Apis; the honeycomb is hung under the trunks of kapok trees and the like because the honeycomb is single-spleen and is arranged in rows, and is commonly called as big wasp or big hanging wasp. So far, big bees are not domesticated, are all wild bees and have fierce characters. It is mainly distributed in southeast Asia and south Asia, and also distributed in Yunnan, Guangxi and Hainan areas in China. The bee is a very important pollination insect in tropical region, and many fruit trees and crops depend on the pollination, such as licheePomelo and mango, etc., which have indispensable functions in ensuring the biological diversity of tropical zones and the crop yield. But the population quantity is increasingly reduced under the influence of global climate change, human activities, and habitat deterioration. The big bee honeycomb is large, the honey yield is high, the honey has excellent quality and delicious taste, and has rich nutritive value, so the big bee honeycomb is deeply loved by local people in Yunnan and the like, and the selling price of the big bee honeycomb is as high as hundreds of yuan per kilogram, so that a honey picker can hunt the honey of the big bee. In recent years, with the rise of network sales and the curiosity of people for eating wild taste, the sales of the honeybee honey is in short supply. As the big bees are all wild and are difficult to domesticate artificially, the emergence of the professional honey hunting people greatly damages the honeycombs of the big bees, so that the population quantity of the big bees is sharply reduced, and some places even reach endangered degree. The protection of the large bee population and the refusal of purchasing the large bee honey become social consensus.

Since the big bee honey has no obvious difference with the common honey in shape, the big bee honey is difficult to distinguish by the conventional method. In order to protect the endangered population of the big bees and maintain the diversity of bee genes, the species needs to be protected at the legislative level. In the law enforcement process, the authenticity identification of the bee honey sample for sale needs to be carried out, so that the basis is provided for subsequent law enforcement. In addition, driven by the interests, some trade companies can adopt ordinary honey to impersonate big honeybee honey and sell, conspire to get high profit, and this has not only deceived the consumer, has encouraged the custom that people consumed big honeybee honey moreover. At present, China does not set up an identification method and an authenticity evaluation index related to the honey of the big bees, which provides a good opportunity for the adulteration of the selling price of the honey of the big bees in the market. The honey of the big bees is the natural sweet food which is stored in the honeycomb after being fully brewed by collecting nectar or secretion of honey source plants by the bees and mixing the nectar or secretion with the secretion of the bees like common honey. Thus, the secretions from the bees in the honey can be used to indicate the species of bees that brew the honey. Many scientific researches show that the secretion mixed in the honey brewing process of bees mainly contains substances such as royal jelly major proteins (MRJPs) and various enzymes. Different varieties of bees in the genus Apis have evolved over the million years, and a part of amino acid sequences of homologous proteins MRJPs of the bees have certain changes, so that the honey brewing bee variety can be indicated based on the changes.

Disclosure of Invention

According to the invention, the MRJP1 content in the honey of the honey bees is relatively constant and the abundance is highest, so that the invention mainly focuses on MRJP1 research, and the developed characteristic peptide segment provides technical and method references for identification and authenticity evaluation of the honey bees, and also provides scientific and technological support and method references for protecting endangered species of the honey bees and forbidding sale of the honey bees.

Specifically, the first purpose of the invention is to provide a characteristic peptide fragment for detecting MRJP1 of a big bee, wherein the sequence of the characteristic peptide fragment is as follows: ENAILSGEYDYTK (SEQ ID No. 1).

The second purpose of the invention is to provide the application of the characteristic peptide segment in detecting the MRJP1 of the big bee.

Specifically, the invention firstly provides a method for identifying honey of a big bee, which comprises the following steps: detecting whether the honey sample contains the characteristic peptide segment; if the characteristic peptide segment is contained, determining that the honey sample contains honeybee honey; if the characteristic peptide segment is not contained, the honey sample is judged to contain no bee honey.

The invention further provides a method for detecting the MRJP1 of the big bee, which comprises the following steps: the characteristic peptide fragment with the sequence of ENAILSGEYDYTK was detected in the honey sample.

Preferably, the detection is performed by liquid chromatography tandem mass spectrometry, and the parent ion of the detection signal generated by the characteristic peptide fragment in the mass spectrum hasm/z 751.85410 ([M+2H]²⁺) The mass-to-charge ratio of; the daughter ions comprising a mass-to-charge ratio ofm/z875.37814，m/z962.41017, which is allowed to deviate within 5 ppm.

Preferably, the characteristic peptide fragment is quantitatively detected by adopting an isotope internal standard method, wherein the used internal standard peptide fragments are as follows: ENAILSGEYDYTK, K indicates all C substitutions in arginine¹³C, all N are replaced by¹⁵N; the internal standard peptideThe parent ion of the detection signal generated by the fragment in the mass spectrum hasm/z 757.86606 ([M+2H]²⁺) The mass-to-charge ratio of; the daughter ions comprising a mass-to-charge ratio ofm/z 886.39423 andm/z 973.42626, which is allowed to deviate within 5 ppm.

Only in the honey samplem/zThe value and the characteristic fragment ions simultaneously meet the characteristics, so that the content of the characteristic peptide ENAILSGEYDYTK in the honey sample can be determined reliably, and the quality of honey can be identified according to the content.

Preferably, the pretreatment of the honey sample comprises: extracting protein in honey to be detected, and carrying out enzymolysis on the protein by adopting trypsin.

Preferably, the detection is carried out by liquid chromatography tandem mass spectrometry by using UHPLC-Q active plus or triple quadrupole mass spectrometry.

Based on the precise mass number provided by the high-resolution mass spectrum, the method has higher characteristics and sensitivity. Based on the instruments and parameters adopted by the method, different analysis laboratories and detection mechanisms can carry out certain adjustment on the parameters according to the relevant knowledge of the liquid phase tandem high resolution mass spectrometry technology, and the adjustment belongs to the protection scope of the invention.

The invention also provides a kit for detecting the MRJP1 of the honey bee, and the standard substance comprises the characteristic peptide segment.

Preferably, the standard substance further comprises an internal standard peptide segment of the characteristic peptide segment, wherein the internal standard peptide segment is: ENAILSGEYDYTK, K indicates all C substitutions in arginine¹³C, all N are replaced by¹⁵N。

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the characteristic peptide segment of the invention provides technical and method references for the identification and authenticity evaluation of the honey of the honeybees, and also provides scientific and technological support and method references for protecting the endangered species of the honeybees and forbidding the sale of the honeybees. By means of the characteristic peptide segment, the detection method of MRJP1 in honey of the honeybees provided by the invention has the advantages of strong characteristics, high sensitivity, good accuracy and precision and the like. The method has certain practical significance for maintaining the health development of the honey consumption industry and the rights and interests of honey consumers.

Drawings

FIG. 1 is an ion flow diagram of characteristic peptide segment ENAILSGEYDYTK of MRJP1 extracted by UHPLC-Q active plus.

FIG. 2 is a mass spectrum of characteristic peptide ENAILSGEYDYTK of MRJP1 detected by UHPLC-Q active plus.

FIG. 3 is a secondary fragment mass spectrum of characteristic peptide ENAILSGEYDYTK of MRJP1 detected by UHPLC-Q active plus.

FIG. 4 shows peptide ENAILSGEYDYTK of isotope-labeled Internal Standard (IS) extracted from UHPLC-Q active plus¹³C₆, ¹⁵N₄) Ion flow diagram of (a).

FIG. 5 shows peptide ENAILSGEYDYTK of isotope-labeled Internal Standard (IS) detected by UHPLC-Q active plus¹³C₆, ¹⁵N₄) Mass spectrum of (2).

FIG. 6 shows peptide ENAILSGEYDYTK of isotope-labeled Internal Standard (IS) detected by UHPLC-Q active plus¹³C₆, ¹⁵N₄) Second order fragment mass spectrum of (1).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The instruments and reagents referred to in the following examples:

1. ultra-high performance liquid chromatography tandem quadrupole/high resolution electrostatic orbitrap mass spectrometer (UHPLC-Q active Plus), Thermo Fisher Scientific, usa;

2. a table low temperature Centrifuge (Microfuge 22R Centrifuge), BeckMAN Coul TER corporation, usa;

3. a full wavelength microplate reader (Multiskan GO), Thermo Fisher Scientific, USA;

4. electronic analytical balance ((PL203), mettleteloledo, germany;

5. a pH meter (DELTA 320), METTLER TOLEDO, Germany;

6. evaporative concentrators (Speed-Vacsvstem, RVC2-18), MarinChrist, Germany;

7. ultra pure water machines (Milli-Q Gradient), Millipore Inc. of USA;

8. ultra-low temperature refrigerator (MDF-U3286S), SANYO, Japan;

urea (Urea) was purchased from Solambio; thiourea, CHAPS, Tris base, Dithiothreitol (DL-Dithiothreitol, DTT) from Amresco; iodoacetamide (IAA) from Merk (Kenilworth, NJ, USA); acetone, Ti (SO)₄)₂Trifluoroacetic acid (TFA) was purchased from j.t.baker; the Bradford method protein quantification kit was purchased from plerian corporation; bovine Serum Albumin (BSA) was purchased from Roche (Basel, Switzerland); ammonium bicarbonate (NH)₄HCO₃) Purchased from Sigma Aldrich; mass-spectral Trypsin (Trypsin) was obtained from Promega (Madison, Wis., USA) and Formic Acid (FA) from MREDA Technology (Carlsbad, Calif., USA); acetonitrile was purchased from Fisher corporation; Zip-Tip desalting elution columns were purchased from Millipore; the remaining chemicals were purchased from Beijing Chemicals, Inc.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1 discovery of MRJP1 characteristic peptide fragment in honey of Apis mellifera

Solution preparation:

1. solution preparation

40 mM NH₄HCO₃Solution: 0.316 g of NH are weighed out₄HCO₃The volume is 100 mL by ultrapure water, and the mixture is stored in a refrigerator at 4 ℃ for later use.

100 mM DTT solution: 0.308 g of DTT was weighed out and added 40 mM NH₄HCO₃Dissolving the solution, mixing uniformly and fixing the volume to 20 mL, and storing the solution in a refrigerator at the temperature of minus 20 ℃ for later use.

100 mM IAA solution: 0.39 g of IAA was weighed out and 40 mM NH was used₄HCO₃The solution is dissolved to 20 mL and stored in a refrigerator at-20 ℃ for later use.

2. Weighing honeybee honey and PBS solution at a ratio of 1:1, fully vortexing, centrifuging at 4 deg.C for 20 min, and collecting supernatant;

3. 100 μ L of the supernatant was added 400 μ L of 40 mM NH₄HCO₃Mixing the solution with the solution, adding 50 μ L of 100 mM DTT solution, mixing, reacting at room temperature for 60 min, adding 250 μ L of 100 mM IAA solution, and reacting at room temperature for 60 min;

4. to the final solution of

step

3, 2. mu.g excess trypsin was added and the cleavage was carried out overnight at 37 ℃. When the reaction was complete, 1. mu.L of FA solution was added to inactivate trypsin. Desalting the enzyme digestion product through a C18 column, and drying the desalted sample in vacuum.

5. Detection of peptide fragments: and (4) dissolving the sample obtained in the step (4) by using a 0.1% FA solution, and sequencing and identifying the peptide fragment by using a Full MS-ddMS2 under a positive ion mode by using a UHPLC-Q active plus.

And collecting and storing data generated by mass spectrum in Xcalibur software, and importing raw data collected by mass spectrum into PEAKS 8.0 for qualitative analysis. The search parameter settings are as follows: the mass error of The parent ion (The precursor mass tolerances) is 15 ppm, The mass error of The child ion (The fragment mass tolerances) is 0.05 Da, and The enzymes are: carrying out Trypsin enzyme digestion, wherein the maximum number of missed cutting sites is 2; the variable modification is oxidation (M, +15.99) and the fixed modification is carbamidomethyl (C, + 57.02). All search results adopt an algorithm of forward-reverse library fusion to control the False positive rate (FDR) of the protein and peptide fragment, wherein the FDR is less than 1%.

6. Screening of peptide fragments: after the qualitative analysis of PEAKS 8.0, aiming at the data analysis and treatment of the peptide segment of the matched big bee MRJP1 (the full-length sequence is shown in SEQ ID No. 5), the peptide segments which have higher response, no modification, no enzyme cutting site and the length of 8-20 and exist in different big bee honeys are selected.

7. Through the treatment and analysis of the steps, a plurality of characteristic peptide fragments of the big bee MRJP1 are obtained, and the sequences are respectively as follows:

1）ENAILSGEYDYTK（SEQ ID No.1）

2）NYPSDVDEWHGK（SEQ ID No.2）

3）RENAILSGEYDYTK（SEQ ID No.3）

4）NYPSDVDEWHGKIFVSMLR（SEQ ID No.4）

although there are many characteristic peptide fragments to choose from, only the optimal one of them can be selected for quantitative analysis in consideration of the uniqueness, stability, ion response in mass spectrum, etc. of the peptide fragments.

Example 2 method for establishing and detecting MRJP1 characteristic peptide fragment in honey of Apis mellifera

1. All the peptide fragments with characteristics screened in example 1 are verified on NCBI and Uniprot websites, the characteristic peptide fragments only existing in MRJP1 of the big bees are screened, mass spectrum data generated in example 1 are checked, and the characteristic peptide fragment with high response and no influence of other substances is selected as a final characteristic peptide fragment, namely ENAILSGEYDYTK.

2. Synthesis of characteristic peptide ENAILSGEYDYTK and Stable isotope Internal Standard (IS) peptide ENAILSGEYDYTK, K indicates substitution of all C's in arginine to¹³C, all N are replaced by¹⁵N, the purity is over 98 percent, and the product is stored at the temperature of minus 20 ℃ for standby.

3. Drawing of standard curve

A series of signature peptide fragment standards (1 ng/mL, 3 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL) were prepared in an initial mobile phase (97: 3, v/v; water/acetonitrile, 0.1% formic acid) and then an IS peptide fragment was added to each concentration of standards as configured to a concentration of 10 ng/mL each. And drawing a standard curve through the ratio of the peak areas of the characteristic peptide fragment and the IS peptide fragment and the concentration of the characteristic peptide fragment corresponding to the ratio.

4. Pretreatment of honey samples

(1) Weighing honeybee honey and PBS solution at a ratio of 1:1, fully vortexing, centrifuging at 4 deg.C for 20 min, and collecting supernatant;

(2) 100 μ L of the supernatant was added 400 μ L of 40 mM NH₄HCO₃Mixing the solution with vortex, adding 50 μ L100 mM DTT solution, mixing, reacting at room temperature for 60 min, adding 250 μ L100 mM IAA solution, and reacting at room temperature for 60 min；

(3) To the final solution of (2), 2. mu.g of excess trypsin was added and the digestion was carried out overnight at 37 ℃. At the completion of the reaction, 1 μ L of FA was added to inactivate trypsin, and an IS peptide fragment was added to each sample to ensure that its concentration remained at 10 ng/mL upon subsequent reconstitution. Desalting the enzyme digestion product, drying the desalted sample in vacuum, dissolving the obtained sample by using 0.1% FA solution, and performing liquid chromatography tandem mass spectrometry by adopting 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometry.

5. Data processing of honey samples

Substituting the peak area ratio of the characteristic peptide fragment/stable isotope internal standard peptide fragment into a formula to obtain the concentration of the characteristic peptide fragment, and obtaining the content of the characteristic peptide fragment ENAILSGEYDYTK according to the formula 1:

x = (phi cV)/m formula 1

Wherein X is (ng/g) the content of the characteristic peptide segment ENAILSGEYDYTK in the honey sample, phi is the ratio of zymoprotein volume to total sample volume, c (ng/mL) is the concentration of the characteristic peptide in the trypsin digest, V (mL) is the volume of the trypsin digest, and m (g) is the mass of the honey sample. So as to achieve the purpose of quantifying the characteristic peptide ENAILSGEYDYTK in the honey sample.

Through the detection of the honey sample of the big bee, the ion flow graph, the mass spectrogram and the secondary fragment mass spectrogram of the characteristic peptide segment are respectively shown as figure 1-figure 3, the ion flow graph, the mass spectrogram and the secondary fragment mass spectrogram of the Internal Standard (IS) peptide segment are respectively shown as figure 4-figure 6, and the accurate mass number of ENAILSGEYDYTK which should contain the characteristic peptide segment in the spectrogram of the honey sample ISm/z 751.85410 ([M+2H]²⁺) (ii) a The accurate mass number of the characteristic peptide segment ENAILSGEYDYTK of the stable isotope internal standard peptide segment in the sample map ism/z 757.86606 ([M+2H]²⁺) The allowable deviation should be within 5 ppm.

The MS/MS spectrum (sub-ion spectrum) should contain characteristic fragment ions of the honey characteristic peptide ENAILSGEYDYTK of Apis melliferam/z 875.37814，m/z962.41017, corresponding to characteristic peptide ENAILSGEYDYTK of stable isotope internal standard peptide fragment, the fragment should be contained in the sub-ion spectrum (MS/MS)Sheet ionm/z 886.39423，m/z973.42626 and the error in its exact mass number should be less than 5 ppm. Only in the honey samplem/zThe value and the characteristic fragment ions simultaneously meet the characteristics, so that the content of the characteristic peptide ENAILSGEYDYTK in the honey sample can be determined reliably, and the quality of honey can be identified according to the content.

Example 3 practical application of MRJP1 characteristic peptide fragment in honey of Apis mellifera

1. The actual sample detection is carried out by purchasing honey (acacia honey, linden honey and vitex honey) brewed by Italian bees, honey (honey brewed by Chinese bees), big bees and small bees from normal markets.

2.6 Honey sample detection

(1) Weighing equal amount of different honey in centrifuge tubes, adding PBS solution at a ratio of 1:1, centrifuging at 4 deg.C for 20 min after sufficient vortex, and collecting supernatant;

(2) 100 μ L of the supernatant was added with 400 μ L of 40 mM NH₄HCO₃Mixing the solution with the solution, adding 50 μ L of 100 mM DTT solution, mixing, reacting at room temperature for 60 min, adding 250 μ L of 100 mM IAA solution, and reacting at room temperature for 60 min;

(3) to 6 parts of the final solution obtained in (2), 2. mu.g of excess trypsin was added, and the cleavage was carried out overnight at 37 ℃. At the completion of the reaction, 1 μ L of FA was added separately to inactivate trypsin, and an IS peptide fragment was added to each sample to ensure that the concentration remained at 10 ng/mL upon subsequent reconstitution. Desalting the enzyme digestion product, drying the desalted sample in vacuum, dissolving the obtained sample by using 0.1% FA solution, and performing liquid chromatography tandem mass spectrometry by adopting 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometry.

Through detection, as shown in table 1, the characteristic peptide fragment information is detected only in the honey sample of the big bee, and the characteristic peptide fragment information is not detected in other honey samples.

Watch (A)

Detecting 6 different honey samples

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> bee institute of Chinese academy of agricultural sciences

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Claims

1. A method for detecting MRJP1 of a honeybee based on liquid phase tandem mass spectrometry is characterized by comprising the following steps:

extracting protein in honey to be detected, carrying out enzymolysis on the protein by adopting trypsin to obtain a sample to be detected, and carrying out liquid chromatography tandem mass spectrometry detection;

judging the information of the characteristic peptide segment in the sample to be detected by contrasting the ion flow diagram, the mass spectrogram and the secondary fragment mass spectrogram;

wherein the characteristic peptide segment is ENAILSGEYDYTK.

2. The method of claim 1, wherein the precursor ions of the detection signal generated by the signature peptide fragment in the mass spectrum havem/z751.85410 mass-to-charge ratio; the daughter ions comprising a mass-to-charge ratio ofm/z 875.37814，m/z962.41017, which is allowed to deviate within 5 ppm.

3. The method according to claim 2, characterized in that the characteristic peptide fragment is quantitatively detected by an isotope internal standard method, wherein the internal standard peptide fragment is used as follows: ENAILSGEYDYTK, K indicates all C substitutions in arginine¹³C, all N are replaced by¹⁵N; the internal standard peptide fragment has the characteristics of the parent ion of a detection signal generated in mass spectrumm/z 757.86606 mass-to-charge ratio; the daughter ions comprising a mass-to-charge ratio ofm/z 886.39423 andm/z 973.42626, which is allowed to deviate within 5 ppm.

4. The method of claim 3, wherein the quantitative determination comprises:

preparing a characteristic peptide fragment standard substance with the internal standard peptide fragment, detecting the standard substance through liquid chromatography tandem mass spectrometry, and drawing a standard curve;

extracting protein in honey to be detected, and carrying out enzymolysis on the protein by adopting trypsin; adding the internal standard peptide segment into a sample to be detected, and performing liquid chromatography tandem mass spectrometry detection;

and judging the information of the characteristic peptide segment in the sample to be detected by referring to the ion flow diagram, the mass spectrogram and the secondary fragment mass spectrogram, and calculating to obtain the content of the characteristic peptide segment according to the standard curve and the detection result.

5. The method according to claim 2 or 3, characterized in that the detection by liquid chromatography tandem mass spectrometry is performed using UHPLC-Q active plus or triple quadrupole mass spectrometry.

6. Use of the method of any one of claims 1 to 5 for identifying the authenticity of honey from a honeybee.

7. The use of claim 6, wherein if the honey sample contains the characteristic peptide segment with the sequence of ENAILSGEYDYTK, the honey sample is judged to contain honeybee honey; if the honey sample does not contain the characteristic peptide segment, the honey sample is judged to contain no bee honey.

8. A kit for detecting MRJP1 of a big bee, which is characterized by comprising DTT, IAA, trypsin and standard substances;

wherein the standard substance comprises a characteristic peptide segment with the sequence of ENAILSGEYDYTK.

9. The kit according to claim 8, wherein the standard substance further comprises an internal standard peptide fragment of the characteristic peptide fragment, wherein the internal standard peptide fragment is: ENAILSGEYDYTK, K indicates all C substitutions in arginine¹³C, all N are replaced by¹⁵N。