CN111579705B

CN111579705B - Jujube flower honey authenticity evaluation method and application

Info

Publication number: CN111579705B
Application number: CN202010512066.4A
Authority: CN
Inventors: 杨术鹏; 李熠; 周金慧; 傅怡; 张金震; 金钥; 杨宇辉; 黄京平; 袁媛; 赵文
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: 2020-06-08
Filing date: 2020-06-08
Publication date: 2023-06-09
Anticipated expiration: 2040-06-08
Also published as: CN111579705A

Abstract

The invention discloses a novel jujube flower honey authenticity evaluation method. In particular to a method for detecting primary amine oxidase in honey by utilizing liquid chromatography-tandem mass spectrometry, which is used for evaluating the authenticity of jujube flower honey. Comprising the following steps: screening characteristic peptide fragments after primary amine oxidase in jujube flower honey is hydrolyzed, pre-treating a sample, separating by liquid chromatography, detecting by tandem mass spectrometry and the like. The jujube flower honey authenticity evaluation method provided by the invention has higher accuracy, precision and sensitivity and strong stability, and is used for jujube flower honey authenticity evaluation by accurately quantifying primary amine oxidase in honey. The method has important practical significance for protecting the rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry.

Description

Jujube flower honey authenticity evaluation method and application

Technical Field

The invention relates to the field of food detection, and particularly discloses a novel jujube flower honey authenticity evaluation method, which mainly adopts liquid chromatography-tandem mass spectrometry to quantitatively detect primary amine oxidase in jujube flower honey and is used for jujube flower honey authenticity evaluation.

Background

Honey is a natural sweet food and is popular with people. Sugar and water are main substances in honey, and in addition, the honey also contains substances beneficial to human beings, such as rich amino acids, phenolic acids, flavone, trace elements and the like. The honey is various and can be classified into single nectar such as acacia, basswood, jujube flower, wattle, litchi, longan, rape and the like according to the different honey source plants. The jujube nectar has sweet taste and fragrance and is very popular with women and the elderly, and has the effects of moistening intestines, enriching blood, regulating immunity, aiding sleep and the like. The jujube nectar is a unique honey variety in summer in northern areas of China and mainly concentrated in Shandong, henan, hebei, shanxi and other areas. Because the bees are easy to generate a certain poisoning reaction when collecting the jujube nectar, the colony potential is quickly reduced, so that the yield of the jujube nectar is relatively low. Although the selling price of the jujube nectar is significantly higher than that of other types of single nectar, the harvested jujube nectar can not meet the market demand. In order to chase high profit, bad enterprises often manufacture and sell adulterated jujube nectar, mainly other types of honey are doped in the jujube nectar honey, and even more, a large amount of cheap syrup is doped, so that the jujube nectar sales market is disturbed. How to effectively evaluate the authenticity of the jujube flower honey and protect the rights of beeworkers and consumers becomes one of the problems to be solved urgently in the current bee products.

The honey contains a small amount of proteins, mainly from royal jelly main proteins secreted by honey, animal-derived proteins such as enzymes, and plant-derived proteins secreted by honey plants into nectar. Because of the specificity of the plant-derived protein source in the honey, the honey can be used as a specific index for evaluating the honey type. The present research team found that there was a large amount of primary amine oxidase (primary amine oxidase, prAO) in date flower honey, which had a molecular weight of about 18kDa. PrAO in the jujube flower nectar is quite rich, and migrates and remains in the honey through the collecting behavior of the bees. PrAO is only present in date flower honey, is not found in other types of honey, has specificity, and is a special component of date flower honey. In addition, the PrAO content in the jujube flower honey of different producing areas is relatively stable, so that the existence and content of PrAO in the honey can be used as key indexes for evaluating the authenticity and purity of the jujube flower honey.

Disclosure of Invention

The invention provides a method for detecting primary amine oxidase in honey, a characteristic peptide fragment applied to the method and application, and the characteristic peptide fragment is used for carrying out high-precision quantitative detection on the primary amine oxidase in the honey, so that the problem that the primary amine oxidase in the honey is difficult to identify after adulteration in the prior art is solved.

The method can be used for quality control and authenticity identification of jujube flower honey in production and business.

In a first aspect, the invention provides a method for identifying plant-derived protein-primary amine oxidase and screening characteristic peptide fragments in jujube flower honey, comprising the following steps:

extracting total protein in the jujube flower honey sample, separating and recovering PrAO by an SDS-PAGE electrophoresis method, carrying out in-gel enzymolysis on PrAO and desalting, and carrying out liquid chromatography tandem mass spectrometry detection after redissolution; searching a library by using PEAKS Studio software aiming at a mass spectrum result, and inquiring and comparing to obtain PrAO specific protein;

b, screening PrAO characteristic peptide fragments and sequences thereof from mass spectrum detection results, and artificially synthesizing the characteristic peptide fragments and corresponding stable isotope internal standard peptide fragments;

further, the present invention provides a characteristic peptide fragment useful for liquid chromatography tandem mass spectrometry for determining PrAO protein in jujube flower honey, said characteristic peptide fragment selected from any one of the following:

i)AGPVISLASIYDR；

ii)AGPVISLASIYDREQGK；

iii)IIPGSLSSISLLSEDDYPQIR。

further, characteristic peptide fragment i) may be used as a quantitative peptide fragment, and characteristic peptide fragments ii) and iii) may be used as qualitative peptide fragments.

In a second aspect, the invention provides a method for determining whether honey is jujube flower honey or not based on a liquid phase tandem high resolution mass spectrometry technology and quantifying a characteristic peptide segment of PrAO of a jujube flower honey sample, which comprises the following specific steps:

a: synthesizing and storing peptide fragment markers: the characteristic peptide AGPVISLASIYDR and the stable isotope labeled Internal Standard (IS) peptide AGPVISLASIYDR are synthesized by Shanghai blaze biotechnology limited company with purity over 98%; storing at-20deg.C for use.

B: drawing a standard curve: a series of standard peptides (5 ng/mL,10ng/mL,25ng/mL,50ng/mL,100ng/mL,200ng/mL,400 ng/mL) of the characteristic peptides were prepared in an initial mobile phase (97:3 v/v, water/ACN, 0.1% formic acid), and IS peptides were added to the prepared standard at each concentration to a concentration of 500ng/mL. Calibration curves were drawn by plotting the analyte/IS peptide peak area ratio versus analyte concentration.

C: pretreatment of honey sample detection: diluting a honey sample by using a PBS buffer solution, extracting proteins in the honey by using an ultrafiltration method, extracting proteins by using trypsin hydrolysis, desalting an enzymolysis product, adding a stable isotope internal standard peptide segment to enable the concentration to be the same as that in the step C, and performing liquid chromatography tandem mass spectrometry detection by adopting the same method; .

Further, in step B, C, a liquid chromatography tandem mass spectrometry detection is performed using UHPLC-Q actual plus (or triple quadrupole mass spectrometry):

it should be understood that the technical scheme of expanding or shrinking the dosage of the above-mentioned used reagent or raw material in equal proportion is substantially equivalent to the above-mentioned content, and all the technical schemes are within the protection scope of the present invention.

Specifically, the UHPLC-Q exact Plus is used for detecting the accurate m/z value of the PrAO characteristic peptide AGPVISLASIYDR of the jujube flower honey: 681.37481 ([ M+2H)] ²⁺ ) The method comprises the steps of carrying out a first treatment on the surface of the The profile of the sample should contain the exact m/z values of the added stable isotope internal standard peptide AGPVISLASIYDR x: 686.37849 ([ M+2H)] ²⁺ ) The allowable deviation should be within 5ppm.

The MS/MS spectrum (sub-ion spectrum) should contain the characteristic fragment ion m/z 924.48, m/z 1037.56 of the characteristic peptide fragment AGPVISLASIYDR of date flower honey PrAO, correspondingly, the sub-ion spectrum (MS/MS) of the stable isotope internal standard peptide fragment AGPVISLASIYDR should contain the fragment ion m/z 934.48, m/z 1047.57, and the error of the precise mass number should be less than 5ppm. The honey sample only meets the characteristics in terms of the accurate m/z value and the characteristic fragment ions, the PrAO content in the honey sample can be determined to be reliable, and the honey quality can be identified according to the content.

The method adopts a UHPLC-Q exact plus instrument to quantify PrAO in the jujube flower honey, and has higher specificity and sensitivity based on the accurate mass number provided by high-resolution mass spectrum. Based on the instrument and parameters adopted by the method, different analysis laboratories and detection mechanisms can adjust the parameters to a certain extent according to the related knowledge of the liquid phase tandem high resolution mass spectrometry technology, and the adjustment belongs to the protection scope of the invention.

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

the research shows that the content of primary amine oxidase in the jujube flower honey is relatively constant, and the invention establishes a set of method for identifying primary amine oxidase specific proteins in the jujube flower honey according to the content, which is used for assisting in identifying honey adulteration. The detection method for the primary amine oxidase protein content of the jujube flower honey has the advantages of being strong in specificity, high in sensitivity, good in accuracy and precision and the like, and is suitable for accurate quantification of the primary amine oxidase of the jujube flower honey. The method has great significance for maintaining healthy development of the honey consumption industry and rights and interests of honey consumers.

Drawings

FIG. 1 is an ion flow of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 extracted by UHPLC-Q actual plus provided in example 1 of the present invention;

FIG. 2 is a mass spectrum of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 detected by UHPLC-Q actual plus provided in example 1 of the present invention;

FIG. 3 is a mass spectrum of secondary fragments of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 detected by UHPLC-Q actual plus provided in example 1 of the present invention;

FIG. 4 shows ion flow of a stable isotope internal standard peptide AGPVISLASIYDR extracted by UHPLC-Q real plus provided in example 2 of the present invention;

FIG. 5 is a mass spectrum of a stable isotope internal standard peptide fragment AGPVISLASIYDR detected by UHPLC-Q actual plus provided in example 2 of the present invention;

FIG. 6 is a mass spectrum of a secondary fragment of the stable isotope internal standard peptide AGPVISLASIYDR detected by UHPLC-Q actual plus provided in example 2 of the present invention;

FIG. 7 is a graph showing the responsivity of each peptide fragment selected according to example 3 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The following examples relate to the apparatus and reagents:

1. mass spectrometer (Q-actual), company Thermo Fisher Scientific usa;

2. a tabletop cryogenic Centrifuge (Microfuge 22R Centrifuge), beckMAN coulter company, usa;

3. full wavelength microplate reader (Multiskan GO), company Thermo Fisher Scientific in the united states;

4. an electronic analytical balance ((PL 203), mettlertolo company, germany;

pH meter (DELTA 320), METTER TOLEDO, germany;

6. evaporative concentration apparatus (Speed-Vacsvstem, RVC 2-18), marinChrist, germany;

7. ultra-pure water machine (Milli-QGracurrent), millipore, inc., USA;

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

9.1290 Infinicity liquid chromatography-6495 triple quadrupole mass spectrometry, agilent Technologies, usa;

urea (Urea) was purchased from Solarbio company; thiourea, CHAPS, tris base, dithiothreitol (DL-Dithiothreitol, DTT) available from Amresco; iodoacetamide (IAA) is available from Merk (Kenilworth, NJ, USA); acetone, ti (SO) ₄ ) ₂ Trifluoroacetic acid (TFA) from j.t.baker company; the Bradford protein quantification kit was purchased from priley company; bovine serum albumin (Bovine serum albumin, BSA) was purchased from Roche (Basel, switzerland); ammonium bicarbonate (NH 4HCO 3) was purchased from Sigma Aldrich company; mass spectrum grade Trypsin (Trypsin) is available from Promega (Madison, wis., USA) and Formic Acid (FA) is available from MREDA Technology (Carlsbad, calif., USA); acetonitrile(can) available from Fisher corporation; zip-Tip desalting elution column was purchased from Millipore; the remaining chemicals were purchased from beijing chemicals.

Example 1

The protein in the jujube flower honey is analyzed in the embodiment, and the specific steps are as follows:

(1) Dissolving jujube flower honey in deionized water (or PBS) at a ratio of 1:1, centrifuging for 10min, collecting supernatant, transferring to a 10kD molecular flux ultrafiltration tube, centrifuging at 5000 Xg, concentrating protein solution to minimum volume, collecting the rest liquid in the tube to obtain protein extract, and determining total protein concentration in the extract by using Bradford method;

(2) SDS-PAGE analysis of proteins: performing SDS-PAGE analysis on 40 μg of protein, separating primary amine oxidase from other proteins in 12% concentrated gel and 10% separation gel, and staining gel with Coomassie blue;

(3) Enzymatic hydrolysis of PrAO: cutting a strip corresponding to PrAO into cubes with proper size, adding decolorizing liquid until gel strips are not passed, and rotating for decolorizing to colorless; discarding the decolorized solution, uniformly reacting and dehydrating by using acetonitrile, removing the acetonitrile, evaporating, concentrating and drying; trypsin (40 mM NH) was added to the dried gel block ₄ HCO ₃ Solution dissolution) immersing the adhesive tape, and enzyme cutting overnight at 37 ℃; adding 70% acetonitrile water (0.1% formic acid) by volume ratio into an immersed adhesive tape, standing for 10-20min to extract protein, collecting extract, repeating extraction once, combining the two extracts, desalting the extract, and vacuum drying to obtain PrAO peptide fragment

(4) Determination of primary amine oxidase peptide fragment: dissolving PrAO peptide fragments in mobile phase A (0.1% formic acid water), using UHPLC-Q active plus to forward the peptide fragments in Full MS-ddMS ² Sequencing and identification.

Data generated by mass spectrometry are collected and stored in Xcalibur software, and raw data acquired by mass spectrometry are imported into PEAKS 8.0 for qualitative analysis. The search parameters were set as follows: the parent ion mass error (The precursor mass tolerances) was 15ppm, the child ion mass error (The fragment mass tolerances) was 0.05Da, and the enzyme was: the Trypsin is digested, and the number of maximum missed cleavage sites is 2; variable modification is oxidation (M, +15.99), fixed modification is carbamidomethyl (C, + 57.02). All search results control false positive rate of protein and peptide fragments by adopting a forward and reverse library fusion algorithm (False discovery rate FDR), and FDR is less than 1%. The UHPLC-Q actual plus is used for identifying that the PrAO peptide fragment is the date flower honey PrAO, and the nucleotide sequence of the PrAO peptide fragment is shown as SEQ ID NO. 1.

(5) Determination of the characteristic peptide fragment: three candidate peptide fragments were obtained during UHPLC-Q actual plus analysis, and the charged state and corresponding molecular weights were well in agreement with their theoretical values. The AGPVISLASIYDRR, AGPVISLASIYDREQGK and IIPGSLSSISLLSEDDYPQIR peptide fragment sequences were finally determined. Verification of three candidate peptide specificity by UniProt search peptide AGPVISLASIYDR was ultimately selected as the quantitative peptide, AGPVISLASIYDREQGK and IIPGSLSSISLLSEDDYPQIR as the qualitative peptide.

FIG. 1 is an ion flow of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 extracted by UHPLC-Q actual plus.

FIG. 2 is a mass spectrum of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 detected by UHPLC-Q actual plus.

FIG. 3 is a mass spectrum of secondary fragments of three peptide fragments of primary amine oxidase SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 detected by UHPLC-Q actual plus.

Example 2

The embodiment provides a method for detecting primary amine oxidase in honey, which comprises the following specific steps of

1. Synthetic signature peptide AGPVISLASIYDR and stable isotope Internal Standard (IS) peptide AGPVISLASIYDR, R represents all C substitutions in arginine as ¹³ C, all N are replaced by ¹⁵ N, the purity exceeds 98%, store at-20 ℃ for standby.

Fig. 4-6 show ion flow, mass spectrum and secondary fragment mass spectrum of stable isotope internal standard peptide AGPVISLASIYDR in sequence.

2. Drawing of a Standard Curve

A series of characteristic peptide standards (5 ng/mL,10ng/mL,25ng/mL,50ng/mL,100ng/mL,200ng/mL,400 ng/mL) were prepared in an initial mobile phase (97:3 v/v, water/ACN, 0.1% formic acid) and then added to the formulated standards at 500ng/mL. And drawing a standard curve through the ratio of peak areas of the characteristic peptide fragments and the stable isotope internal standard peptide fragments and the concentration of the characteristic peptide fragments corresponding to the ratio.

3. Pretreatment of honey samples

(1) Dissolving appropriate amount of Mel to be tested in deionized water (or PBS) at a mass ratio of 1:1, completely dissolving at room temperature, and centrifuging at 12000g at 4deg.C for 10min. The supernatant was collected and transferred to a 10kD molecular flux ultrafiltration tube, centrifuged at 5000 x g, and the protein extract was obtained by collecting the remaining liquid after the minimum volume of concentrated protein solution, and the total protein concentration in the protein extract was determined using Bradford method. The protein solution was diluted with 5M urea to a final concentration of total protein of 2mg/mL as the sample to be tested.

(2) To 50. Mu.L of the sample to be tested, 8. Mu.L of 100. Mu.g/mL of the stable isotope internal standard peptide fragment was added, and 200. Mu.L of 40mM NH was taken ₄ HCO ₃ To this mixture was added 25.8. Mu.L of 30mM DTT solution, and the mixture was reacted at room temperature for 60 minutes, and then 129. Mu.L of 100mM IAA solution was added and the mixture was dark reacted at room temperature for 60 minutes.

(3) A proper amount of trypsin solution (the mass ratio of trypsin to substrate protein is 1:50) is added into a honey protein sample and is subjected to enzyme digestion at 37 ℃ overnight. At the completion of the reaction, 1. Mu.L of FA was added to inactivate trypsin. Desalting the enzyme-digested 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 using 1290 Infinicity liquid chromatography-6495 triple quadrupole mass spectrometry.

4. Data processing of honey samples

Obtaining the concentration of the characteristic peptide fragment according to the peak area ratio of the characteristic peptide fragment/stable isotope internal standard peptide fragment brought into a formula, and obtaining the content of the jujube flower honey primary amine oxidase according to the formula 1:

X＝(ФcVM ₁ )/(M ₂ m) 1

Wherein X is (ng/g) the content of primary amine oxidase in the honey sample, phi is the ratio of the volume of enzymolysis protein to the total sample volume, c (ng/mL) is the concentration of characteristic peptide in trypsin digestion product, and V (mL) is trypsin digestion productVolume, M ₁ 、M ₂ Is the molar mass of primary amine oxidase and the characteristic peptide fragment, and m (g) is the mass of the honey sample. So as to achieve the aim of quantifying the primary amine oxidase in the honey sample.

Through detection of a part of the honey sample, as shown in fig. 1-6, the map of the honey sample should contain the accurate m/z value of the jujube flower honey primary amine oxidase characteristic peptide AGPVISLASIYDR: 681.37481 ([ M+2H)] ²⁺ ) The method comprises the steps of carrying out a first treatment on the surface of the The profile of the sample should contain precise m/z values for the stable isotope internal standard peptide AGPVISLASIYDR: 686.37849 ([ M+2H)] ²⁺ ) The allowable deviation should be within 5ppm.

The MS/MS spectrum (sub-ion spectrum) should contain the characteristic fragment ion m/z 924.48, m/z 1037.56 of the primary amine oxidase characteristic peptide fragment AGPVISLASIYDR of the jujube flower honey, the corresponding sub-ion spectrum (MS/MS) of the stable isotope internal standard peptide fragment AGPVISLASIYDR should contain the fragment ion m/z 1047.57085, m/z 934.48679, and the error of the precise mass number should be less than 5ppm. The honey sample only meets the characteristics in terms of the accurate m/z value and the characteristic fragment ions, the required primary amine oxidase content in the honey sample can be confirmed to be reliable, and the honey quality can be identified according to the content.

Example 3

This example provides a primary amine oxidase specific peptide screening, comprising the following steps:

(3) Enzymatic hydrolysis of PrAO: cutting a strip corresponding to PrAO into cubes with proper size, adding decolorizing liquid until gel strips are not passed, and rotating for decolorizing to colorless; discarding decolorizing solution, and using BUniformly reacting and dehydrating nitrile, removing acetonitrile, evaporating, concentrating and drying; trypsin (40 mM NH) was added to the dried gel block ₄ HCO ₃ Solution dissolution) immersing the adhesive tape, and enzyme cutting overnight at 37 ℃; adding 70% acetonitrile water (0.1% formic acid) by volume ratio into an immersed adhesive tape, standing for 10-20min to extract protein, collecting extract, repeating extraction once, combining the two extracts, desalting the extract, and vacuum drying to obtain PrAO peptide fragment

Data generated by mass spectrometry are collected and stored in Xcalibur software, and raw data acquired by mass spectrometry are imported into PEAKS 8.0 for qualitative analysis. The search parameters were set as follows: the parent ion mass error (The precursor mass tolerances) was 15ppm, the child ion mass error (The fragment mass tolerances) was 0.05Da, and the enzyme was: the Trypsin is digested, and the number of maximum missed cleavage sites is 2; variable modification is oxidation (M, +15.99), fixed modification is carbamidomethyl (C, + 57.02). All search results control false positive rate of protein and peptide fragments by adopting a forward and reverse library fusion algorithm (False discovery rate FDR), and FDR is less than 1%. Identifying the PrAO peptide fragment as jujube flower honey PrAO through UHPLC-Q actual plus.

(5) And (3) screening PrAO peptide fragment data matched by PEAKS software after the PrAO peptide fragment data are derived, removing modified and overlong and too short peptide fragments, specifically screening the preliminarily screened peptide fragments into NCBI and UNIPAT websites, and calculating theoretical m/z of the specific peptide fragments which only exist in PrAO, namely WANWEFHLGFD AR, IGVGLTGILGVK, AGPVISLASIYDREQGK, AGPVISLASIYDR, GSI SELFVPYMDETEEWYYK and IIPGSLSSISLLSEDDYPQIR, so that response values of the peptide fragments are observed in a sample raw file as shown in figure 7. Because two peptides WANWEFHLGFDAR, IGVGLTGILGVK were not detected in all samples, three peptides AGPVISLASIYDREQGK, AGPVISLASIYDR and IIPGSLSSISLLS EDDYPQIR were selected based on the response level, with the highest AGPVISLASIYDR being the qualitative peptide and the remaining two peptides being the qualitative peptide.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

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20

Claims

1. The application of the primary amine oxidase PrAO quantitative method in the aspect of evaluating the authenticity of the jujube honey is characterized in that: the primary amine oxidase PrAO quantification method comprises the following steps:

(1) Preparing a standard solution containing stable isotope internal standard peptide fragments with fixed concentrations and characteristic peptide fragments with different concentrations, and detecting the standard solution by a liquid chromatography tandem mass spectrometry method; drawing a standard curve according to the concentration of the standard solution and the peak area ratio of the characteristic peptide segment/the stable isotope internal standard peptide segment;

(2) Diluting a honey sample to be detected by using a PBS buffer solution, extracting protein in the honey sample to be detected by using an ultrafiltration method, hydrolyzing the extracted protein by using trypsin, desalting an enzymolysis product, adding the stable isotope internal standard peptide segment to enable the concentration to be the same as that in the step (1), and detecting by using the same liquid chromatography tandem mass spectrometry detection method in the step (1);

(3) According to the detection result, comparing the standard curve, and calculating the concentration of the characteristic peptide in the honey sample to be detected, thereby realizing quantitative detection of PrAO in the jujube nectar;

the characteristic peptide segment comprises:

i）AGPVISLASIYDR；

ii）AGPVISLASIYDREQGK；

iii）IIPGSLSSISLLSEDDYPQIR；

characteristic peptide fragment i) as a quantitative peptide fragment, characteristic peptide fragments ii) and iii) as qualitative peptide fragments;

if the concentration of primary amine oxidase in the honey is detected to be 0.095 mg/g-0.12 mg/g, judging that the honey is the date nectar.