CN111157635B

CN111157635B - Method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry

Info

Publication number: CN111157635B
Application number: CN201911368845.5A
Authority: CN
Inventors: 杨术鹏; 李熠; 丛晓蕾; 周金慧; 张金震; 金钥; 黄京平; 杨宇辉; 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: 2019-12-26
Filing date: 2019-12-26
Publication date: 2021-01-15
Anticipated expiration: 2039-12-26
Also published as: CN111157635A

Abstract

The invention provides a method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry, which comprises the following steps: screening characteristic peptide fragments, making a standard curve, pretreating a sample, separating liquid chromatography and tandem mass spectrometry, quantifying the peptide fragments by software and the like; the characteristic peptide fragments comprise characteristic peptide fragments 1: MLNDSVFAFSR and signature peptide stretch 2: IYTHDIPETYNIVR are provided. The method for quantifying the alpha-glucosidase in the Chinese honey has high accuracy, precision and sensitivity and strong stability, is suitable for accurately quantifying the alpha-glucosidase in the Chinese honey, and can be used for assisting in evaluating the authenticity of the Chinese honey. The quantitative method has important practical significance for protecting the rights and interests of honey consumers and maintaining the healthy development of the honey product industry.

Description

Method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to the field of food detection, in particular to a method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry, which is suitable for identifying and evaluating authenticity of the honey.

Background

Chinese honey, also known as native honey, is brewed from Chinese bees by collecting honey-derived plants. The Chinese honeybees are smaller than the Italian honeybees, the back is black, the movement is more agile, the two populations have no relation of marriage, the two populations cannot be bred in a hybridization way, the yield of the Chinese honeybees is far lower than that of the Italian honeybees, and therefore the market price of the Chinese honeybees is higher and is about 5-10 times that of the Italian honeybees. In order to chase up high profit, bad vendors often adopt Italian honey to serve as medium honey, or mix Italian honey with medium honey to adulterate, even mix cheap fructose syrup.

Alpha-glucosidase, one of the major proteins in honey, is widely present in various kinds of honey, but has different amino acid sequences in different kinds of honey such as honey and honey. The content of the Chinese bee alpha-glucosidase in different honey species and different honey sources is slightly different, but is relatively constant, and if exogenous substances such as syrup and the like are added, the content of the Chinese bee alpha-glucosidase is greatly reduced, so that the content of the Chinese bee alpha-glucosidase can be used for assisting in identifying the adulteration phenomenon of honey.

Due to the high selectivity and high sensitivity of the liquid chromatography-mass spectrometry technology compared with other technologies, the method is more beneficial to the accurate characterization, identification and quantification of the protein in the complex biological matrix. The qualitative requirements of peptide level detection can be met by the sequence of the target protein obtained by trypsin digestion, and then quantification is performed. The key point of this method is the selection of one or more specific signature peptides for the target protein and suitable internal standard peptides. The characteristic peptide fragment of the Chinese bee alpha-glucosidase is determined by using mass spectrum and software, and a quantitative method of the characteristic peptide fragment is established, so that the method has important significance for assisting identification of adulteration of honey.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry.

The method can be used for quality control and authenticity identification of Chinese honey in production and commerce.

In the first aspect, the invention screens out a characteristic peptide fragment group of alpha-glucosidase in honey, which can be used in quantitative detection of liquid chromatography-tandem mass spectrometry:

characteristic peptide fragment 1: MLNDSVFAFSR, for quantification;

characteristic peptide fragment 2: IYTHDIPETYNIVR, for qualitative purposes.

The method successfully screens the characteristic peptide fragment 1 of the honey alpha-glucosidase in the Chinese honey: MLNDSVFAFSR, characteristic peptide fragment 2: IYTHDIPETYNIVR, wherein the characteristic peptide segment 1: MLNDSVFAFSR can be used as quantitative peptide fragment, characteristic peptide fragment 2: IYTHDIPETYNIVR can be used as qualitative peptide fragment, and the specificity of both is verified by Uniprot database; the invention simultaneously designs stable isotope Internal Standard (IS) peptide segments corresponding to the characteristic peptide segment 1 so as to accurately and sensitively quantify the alpha-glucosidase in honey.

Preferably, the stable isotope internal standard peptide fragment is: MLNDSVFAFSR, wherein R represents the substitution of carbon in arginine to¹³C, nitrogen is replaced by¹⁵N。

Further, the parent ion of the detection signal generated by the characteristic peptide segment 1 in the mass spectrum has 643.81353 mass-to-charge ratio; contains daughter ions with mass-to-charge ratios of 1042.49523, 803.42539.

The stable isotope internal standard peptide fragment generates a detection signal in a mass spectrum, wherein the parent ion is m/z:648.81766, and the parent ion comprises daughter ions with mass-to-charge ratios of 1052.50350 and 637.33319.

In a second aspect, the invention provides a method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry, which comprises the following steps: detecting characteristic peptide fragments of alpha-glucosidase in honey to be detected by a liquid chromatography tandem mass spectrometry, wherein the characteristic peptide fragments comprise peptide fragments:

characteristic peptide fragment 1: MLNDSVFAFSR the flow of the air in the air conditioner,

characteristic peptide fragment 2: IYTHDIPETYNIVR are provided.

Further, before detection, the honey to be detected is treated by a detection kit, wherein the detection kit comprises the following components:

(1) extracting buffer solution 0.01M PBS, pH 7.4;

(2) trypsin 20 ng/. mu.L in a solvent of 40mM ammonium bicarbonate;

(3) standard substance of

characteristic peptide sections

1 and 2.

Specifically, the method comprises the following specific steps:

A. preparing characteristic peptide fragment standard solutions with different concentrations and containing stable isotope internal standard peptide fragments with fixed concentrations, performing liquid chromatography tandem mass spectrometry detection, and drawing a standard curve;

B. extracting protein in the honey to be detected, carrying out enzyme digestion by using trypsin, and desalting an enzymolysis product to be used as a sample to be detected;

C. adding the stable isotope internal standard peptide segment into the sample to be detected to enable the concentration of the stable isotope internal standard peptide segment to be the same as that in the step A, and performing liquid chromatography tandem mass spectrometry detection by adopting the same method in the step A;

D. and C, obtaining the concentration of the characteristic peptide segment in the sample to be detected by contrasting the standard curve obtained in the step A, thereby realizing the quantitative detection of the alpha-glucosidase.

Further, in the steps A and C, liquid chromatography tandem mass spectrometry detection is carried out by using UHPLC-Q active plus.

a1, liquid chromatography conditions were as follows:

a chromatographic column: is a C18 column;

mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile containing 0.1% formic acid;

the gradient elution conditions were: 0-0.5min, 5% of B; 0.5-1.0min, 5-15% of B; 1.0-6.5min, 15-40% of B; 6.5-7.0min, 40-95% of B; 7.0-8.5min, 95% of B; 8.5-8.6min, 95-5% B, 8.6-10.0min, 5% B; flow rate: 0.30 mL/min; sample introduction amount: 5.0 mu L;

a2, mass spectrometry conditions were as follows:

ion source parameters: a flow rate 38 of the sheath gas; flow rate of the auxiliary gas 15; the flow rate of cone blocking gas is 0; the electrospray voltage was 3.2 kV; the temperature of the ion conduit was 275 ℃; s-lensRF level is set to 60; the ion source temperature is 380 ℃; the collection mode is Full MS-ddMS in positive ion mode²。

In the above method, the standard curve obtained in step C is Y-0.01238X-0.3023, R²0.9988, wherein Y is the peak area ratio of the characteristic peptide fragment/stable isotope internal standard peptide fragment, and X is the concentration of the characteristic peptide fragment; the detection range is 5-1000ng/mL, and the lowest detection limit is 2 ng/mL.

The method comprises the steps of detecting a honey sample by adopting UHPLC-Q active Plus (ultra high performance liquid chromatography-quadrupole tandem high resolution electrostatic orbit trap), calculating the content of alpha-glucosidase by comparing the peak area ratio of alpha-glucosidase characteristic peptide segment/IS peptide segment in a map, and further judging whether honey IS adulterated, wherein the peak area of the parent ion of the alpha-glucosidase characteristic peptide segment on the liquid IS mainly based on the peak area of the stable isotope internal standard peptide segment on the liquid.

It should be understood that the technical solutions of the above-mentioned reagents or raw materials with proportionally enlarged or reduced dosage are substantially equivalent to the above-mentioned contents, and all fall within the protection scope of the present invention.

The method adopts a UHPLC-Q active plus instrument to quantify the alpha-glucosidase in the honey, and has higher specificity and sensitivity based on the accurate mass number provided by the high-resolution mass spectrum. 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.

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

in view of the fact that the content of alpha-glucosidase in the medium honey is relatively constant, the invention establishes a set of honey authenticity evaluation method for assisting in identification of honey adulteration. The method for detecting the content of the alpha-glucosidase protein in the Chinese honey has the advantages of strong specificity, high sensitivity, good accuracy and precision and the like, and is suitable for accurate quantification of the alpha-glucosidase of the Chinese honey. The method is significant for maintaining health development of honey consumption industry and interests of honey consumers.

Drawings

FIG. 1 is an ion flow diagram of a characteristic peptide fragment of alpha-glucosidase in honey extracted by UHPLC-Q active plus provided in embodiment 1 of the present invention;

FIG. 2 is a mass spectrum of a characteristic peptide fragment of alpha-glucosidase in honey detected by UHPLC-Q active plus provided in example 1 of the present invention;

FIG. 3 is a second-order fragment mass spectrum of a characteristic peptide fragment of alpha-glucosidase in honey detected by UHPLC-Q active plus provided in example 1 of the present invention;

FIG. 4 is the response (relative abundance) of alternative signature peptides in example 1 of the present invention in a reference sample;

fig. 5 is a schematic diagram of the results of liquid chromatography tandem mass spectrometry detection of pure Chinese honey and adulterated Chinese honey provided in embodiment 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The instruments and reagents referred to in the following examples:

1. mass spectrometer (Q-exact), 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;

pH meter (DELTA 320), METTLER TOLEDO, Germany;

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

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

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

9.1290Infinity liquid chromatography-6495 triple four-bar mass spectrometry, Agilent technologies, USA;

10. dithiothreitol (DL-Dithiothreitol, DTT), Solarbio, china;

11. iodoacetamide (IAA), Merk corporation, usa;

bradford method protein quantification kit, Solambio, China;

13. ammonium bicarbonate (NH)₄HCO₃) Sigma, USA.

Example 1 method for determining the content of alpha-glucosidase in honey using liquid chromatography tandem mass spectrometry

1. Sample source

The actual Chinese honey samples are purchased from the market or a bee farmer for 20 parts.

2. Experimental procedure

2.1 preparation of the solution

5M Urea solution: weighing 4.5g of Urea, and metering the volume of ultrapure water to 15 mL;

100mM DTT: weighing DTT 308.5mg, 40mM NH₄HCO₃The solution is metered to 20mL, and each tube is subpackaged with l mL, and the solution is stored in a refrigerator at the temperature of-20 ℃ for standby.

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

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

Activating solution: mu.L of ACN, 3. mu.L of TFA, and ultrapure water were added to a volume of 1mL, and the mixture was stored at 4 ℃.

The equilibrium solution was 1. mu.L of TFA, diluted to 1mL with ultrapure water and stored at 4 ℃.

Eluent: 800. mu.L of ACN and 1. mu.L of TFA were diluted to 1mL with ultrapure water and stored at 4 ℃.

2.2 the invention carries out liquid chromatography tandem mass spectrometry detection on the medium honey, and two characteristic peptide fragments 1 of the alpha-glucosidase are obtained by screening the result: MLNDSVFAFSR the flow of the air in the air conditioner,

characteristic peptide fragment 2: IYTHDIPETYNIVR are provided.

The invention selects the characteristic peptide segment 1 as the characteristic peptide segment of quantitative analysis by comparison and correspondingly designs a stable isotope internal standard peptide segment MLNDSVFAFSR, wherein R represents that carbon in arginine is replaced by carbon¹³C, nitrogen is replaced by¹⁵N。

Characteristic peptide MLNDSVFAFSR and stable isotope Internal Standard (IS) peptide MLNDSVFAFSR were synthesized with purity over 98% and stored at-20 ℃ until use.

2.3 drawing of standard curve: a series of signature peptide fragment standards (2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL and 1000ng/mL) were prepared in the initial mobile phase (97: 3, v/v, water/ACN with 0.1% formic acid) and 200ng of IS peptide fragment was added to each concentration of standard configured to a concentration of 100 μ g/mL.

1290Infinity liquid chromatography-6495 triple quadrupole mass spectrometry liquid phase conditions were as follows:

a chromatographic column: kinetex C18(50 × 2.1mm)2.6 μm, column temperature room temperature: at 20 ℃.

Mobile phase composition: mobile phase a was 0.1% formic acid water and mobile phase B was 0.1% formic acid acetonitrile. The gradient elution conditions were: 0-0.8min, 10% of B; 0.8-1.3min, 10-20% of B; 1.3-4.5min, 20-30% of B; 4.5-5.4min, 30-95% of B; 5.4-6.0min, 95% of B; 6.0-6.1min, 95-5% of B, 6.1-7.0min and 5% of B.

Flow rate: 0.30 mL/min; sample introduction amount: 5.0. mu.L.

The mass spectrum conditions were as follows:

ion source parameters: the temperature of the sheath gas is 350 ℃; the flow rate of the sheath gas is 12L/min; the temperature of the auxiliary gas is 290 ℃; the auxiliary airflow rate is 11L/min; the capillary voltage positive mode is 3.5 kV; the iFunnel parameters are in positive mode, high voltage is 200V, low voltage is 100V, and the collection mode is MRM mode in positive ion mode. And drawing a standard curve by drawing the peak area ratio of the characteristic peptide fragment/IS peptide fragment and the concentration of the characteristic peptide fragment.

2.4 pretreatment of Honey samples

(1) Accurately weighing 10g to 50mL of uniform honey to be detected, adding 10mL of deionized water, and vortexing until the honey is fully dissolved. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min. Collect the supernatant in a new 2mL centrifuge tube.

(2) 200. mu.L of the protein solution was removed and 4. mu.L of 100. mu.g/mL stable isotope internal standard peptide fragment was added and mixed with 800. mu.L of 40mM NH₄HCO₃And (4) mixing. To the above mixed solution, 100. mu.L of 30mM DTT solution was added and the reaction was carried out at room temperature for 60min, and then 500. mu.L of 100mM IAA solution was added and the reaction was carried out at room temperature for 60min in a dark state.

(3) 30. mu.L of trypsin solution was added to each sample and cleaved overnight at 37 ℃. When the cleavage reaction was complete, 1 μ L TFA was added to inactivate trypsin. And then desalting the enzyme digestion product, and performing mass spectrometry on the desalted sample.

(4) Mass spectrometric analysis of honey samples

Honey samples were tested using 1290Infinity liquid chromatography-6495 triple quadrupole mass spectrometry.

(5) Data processing of honey samples

The content of alpha-glucosidase in honey is obtained according to formula 1:

X＝(ФcVM₁)/(M₂m) a compound of the formula 1,

in formula 1, X (ng/g) is the content of alpha-glucosidase in the honey sample, phi is the ratio of zymoprotein volume to total sample volume, c (ng/mL) is the concentration of characteristic peptide in the trypsin digest, V (mL) is the volume of the trypsin digest, M1 and M2 are the molar masses of alpha-glucosidase and characteristic peptide fragments, and M (g) is the mass of the honey sample. So as to achieve the purpose of quantifying the alpha-glucosidase in the honey sample.

As shown in figures 1-3, the detected honey sample spectrum should contain Chinese bee alpha-glucosidase characteristic peptide MLNDSVFAFSRPrecise m/z value of (2): 643.81353([ M + 2H)]²⁺) (ii) a The sample should contain added stable isotope Internal Standard (IS) peptide segment MLNDSVFAFSR (R-¹³C₆,¹⁵N₄) Precise m/z value of (2): 648.81766([ M + 2H)]²⁺) The allowable deviation should be within 5 ppm.

The sub-ion spectrum should contain the characteristic fragment ions m/z1042.49523 and m/z 803.42539 of the characteristic peptide segment MLNDSVFAFSR, and correspondingly, the sub-ion spectrum (MS/MS) of the stable isotope internal standard peptide segment MLNDSVFAFSR contains the fragment ions m/z 1052.50350 and 637.33319, and the error of the accurate mass number of the fragment ions should be less than 5 ppm. The honey sample only meets the above characteristics in the aspects of accurate m/z value and characteristic fragment ions, so that the content of the apis cerana alpha-glucosidase in the honey sample can be determined to be credible, and the quality of honey can be identified according to the content.

The peptide fragments of the alpha-glucosidase of the real Chinese honey sample collected by a bee farmer are subjected to matching detection, the results are compiled into a graph result shown in figure 4, and it can be seen from figure 4 that in Chinese honey, the response of the characteristic peptide fragments MLNDSVFAFSR and IYTHDIPETYNIVR of the alpha-glucosidase screened by the invention is highest, so that the content of the alpha-glucosidase in different reference samples is relatively stable, and the alpha-glucosidase in the Chinese honey sample can be used as the peptide fragment suitable for quantifying the content of the alpha-glucosidase in the Chinese honey sample.

Example 2 quantitative determination of adulteration of honey

1. Sample source

The actual Chinese honey sample and syrup are purchased from the market or a beekeeper.

2. Experimental procedure

(1) Solution preparation

The same as in example 1.

(2) Mixing honey: mixing the syrup with Mel at different ratio.

The syrup is added into Mel at ratio of 5%, 10%, 50%.

(3) Pretreatment of honey samples

Accurately weighing 10g to 50mL of uniform honey to be detected, adding 10mL of deionized water into a centrifuge tube, and vortexing until the honey is fully dissolved. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min. Collect the supernatant in a new 2mL centrifuge tube.

② removing 200. mu.L protein solution and adding 4. mu.L 100. mu.g/mL stable isotope internal standard peptide segment MLNDSVFAFSR, and mixing with 800. mu.L 40mM NH₄HCO₃And (4) mixing. To the above mixed solution, 100. mu.L of 30mM DTT solution was added and the reaction was carried out at room temperature for 60min, and then 500. mu.L of 100mM IAA solution was added and the reaction was carried out at room temperature for 60min in a dark state.

③ 30. mu.L of trypsin solution was added to each sample and cleaved overnight at 37 ℃. When the cleavage reaction was complete, 1 μ L TFA was added to inactivate trypsin. And then desalting the enzyme digestion product, and performing mass spectrometry on the desalted sample.

(4) Mass spectrometric analysis of honey samples

(5) Data processing of honey samples

And extracting the mass-to-charge ratio of the characteristic peptide segment MLNDSVFAFSR from mass spectrum data, and taking the peak area of m/z 643.81353 as a comparison basis. The result is shown in figure 5, and the result shows that the invention can detect the adulteration of the medium honey which is mixed into the syrup by more than 5 percent.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

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Claims

1. A method for quantitatively detecting alpha-glucosidase in honey by liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps: detecting characteristic peptide fragments of alpha-glucosidase in honey to be detected by a liquid chromatography tandem mass spectrometry, wherein the characteristic peptide fragments comprise peptide fragments:

characteristic peptide fragment 2: IYTHDIPETYNIVR, respectively;

treating the honey to be detected with a detection kit before detection, wherein the detection kit comprises the following components:

(1) extracting buffer solution 0.01M PBS, pH 7.4;

(2) trypsin 20 ng/. mu.L in a solvent of 40mM ammonium bicarbonate;

(3) standard substance of characteristic peptide sections 1 and 2.

2. The method according to claim 1, characterized in that it comprises in particular the steps of:

3. The method of claim 2, wherein steps a and C are performed using triple quadrupole or Q active plus lc.

4. The method of claim 2, wherein MLNDSVFAFSR is used as a quantitative characteristic peptide fragment, the parent ion of the detection signal generated in the mass spectrum has a mass-to-charge ratio of 643.81353, and contains daughter ions with mass-to-charge ratios of 1042.49523 and 803.42539, and the allowable deviation is within 10 ppm.

5. The method of claim 2, wherein the stable isotope internal standard peptide fragment is: MLNDSVFAFSR, wherein R represents the substitution of carbon in arginine to¹³C, nitrogen is replaced by¹⁵N；

The stable isotope internal standard peptide fragment generates a detection signal in a mass spectrum, the parent ion is m/z:648.81766, and the parent ion comprises daughter ions with mass-to-charge ratios of 1052.50350 and 637.33319.

6. The method of claim 2, wherein the standard curve obtained in step a is Y-0.01238X-0.3023, R²0.9988, wherein Y is the peak area ratio of the characteristic peptide fragment/stable isotope internal standard peptide fragment, and X is the concentration of the characteristic peptide fragment; the detection range is 5-1000ng/mL, and the lowest detection limit is 2 ng/mL.