CN110850002A - Method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry - Google Patents
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Abstract
The invention provides a method for quantifying honey MRJP2 in liquid chromatography-tandem mass spectrometry. The invention screens out the characteristic peptide segment of royal jelly main protein 2 (MRJP 2) in medium honey and develops a quantitative method of liquid chromatography tandem mass spectrometry, wherein the method comprises the following steps: screening characteristic peptide fragments, making a standard curve, pretreating a sample, separating by liquid chromatography and tandem mass spectrometry, quantifying the peptide fragments by software and the like. The method for quantifying the MRJP2 of the medium honey has high accuracy, precision and sensitivity and strong stability, and is suitable for accurately quantifying the MRJP2 of the medium honey. Can be used for assisting in evaluating the authenticity of honey. In addition, 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
Technical Field
The invention relates to the field of food detection, in particular to a method for quantifying honey MRJP2 in liquid chromatography-tandem mass spectrometry.
Background
The Chinese honey, also called as native honey, is brewed by native honey in China, namely Chinese honey, which collects honey source plants. Currently, the amount of the bee stocks in China is about 300 million, and the bee stocks are mainly distributed in mountainous areas in provinces and cities in south of China. The Italian bees mainly adopt a transition breeding mode of chasing flowers and gradually breeding honey to produce single flower honey; the Chinese bees are mainly raised on fixed places to produce the hundred-flower honey. The yield of medium honey is far lower than that of Italian honey, so that the market price of the medium honey is higher, which is about 5-10 times that of Italian honey. In order to chase after high profit, bad enterprises often adopt honey to pretend to be medium honey, or blend honey into medium honey to adulterate, even dope cheap fructose syrup, thus seriously disturbing the normal production and sale order of honey. Therefore, there is a need to develop a scientific and effective identification technology for honey.
Royal jelly major protein 2 (MRJP 2) is one of the major proteins in honey. The amino acid sequences of Italian bees and Chinese bees MRJP2 have certain difference, and corresponding Chinese bee honey authenticity evaluation methods can be developed according to the difference. The content of the Chinese bee MRJP2 in different honey species and different honey sources is slightly different, but the content is relatively constant, and if exogenous substances such as syrup and the like are added, the content is greatly reduced, so that the content of the Chinese bee MRJP2 can be used as an auxiliary identification of 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 requirement of peptide level detection can be met by the sequence of the target protein obtained by trypsin enzyme digestion, and then quantification is carried out. 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 mass spectrum and software are used for determining the MRJP2 characteristic peptide segment of the Chinese bee, and a quantitative method is established, so that the method has important significance for assisting the identification of adulteration of honey.
Disclosure of Invention
The invention aims to provide a method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry.
It is another object of the present invention to use the method for quality control and authenticity verification of honey in manufacturing and commerce.
In order to achieve the object, the invention provides a method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry, which comprises detecting a characteristic peptide fragment of MRJP2 in honey by mass spectrometry, wherein the characteristic peptide fragment comprises the following characteristic peptide fragments:
characteristic peptide fragment 1: IVNNDFNFNDVNFR, respectively;
characteristic peptide fragment 2: MTSNTFDYDPR are provided.
The method also comprises the step of pretreating the honey sample, wherein reagents used in pretreatment comprise:
(1) extracting a buffer solution: 0.01M PBS, pH7.4;
(2) 20 ng/mu L of trypsin solution and 40mM ammonium bicarbonate solution as a solvent;
(3) standard substance of characteristic peptide sections 1 and 2.
The method for determining the content of MRJP2 protein in honey by using liquid chromatography-tandem mass spectrometry comprises the following steps:
A. preparing characteristic peptide fragment standard solutions with different concentrations and containing stable isotope internal standard peptide fragments with fixed concentrations, performing mass spectrometry detection, and drawing a standard curve;
B. pretreatment of a honey sample to be detected: extracting protein in honey, carrying out enzyme digestion on sample protein by using trypsin liquid, and desalting an enzymolysis product;
C. b, adding stable isotope internal standard peptide sections into the sample to be detected to enable the concentration of the stable isotope internal standard peptide sections to be the same as that in the step A, and performing liquid chromatography tandem mass spectrometry detection by adopting the same method;
D. and (3) obtaining the concentration of the characteristic peptide fragment in the sample to be detected by contrasting with the standard curve, thereby realizing the quantitative detection of MRJP 2.
Preferably, the liquid chromatography conditions are as follows:
a chromatographic column: 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: 0-0.5 min, 5% of B; 0.5-1.0 min, 5-15% of B; 1.0-6.5 min, 15-40% of B; 6.5-7.0 min, 40-95% of B; 7.0-8.5 min, 95% of B; 8.5-8.6 min, 95-5% B, 8.6-10.0 min, 5% B; flow rate: 0.3 mL/min; sample introduction amount: 5.0 muL.
Preferably, the mass spectrometry conditions are as follows:
ion source parameters: the flow rate of sheath gas (sheath gas flow rate) was 38L/min; flow rate of auxiliary gas (aux gasflow rate) 15L/min; electrospray voltage (spray voltage) 3.2 KV; temperature of the ion conduit (capitalrytemperature) 275 ℃; s-lens RF level is set to 60; ion source temperature (Heater temperature) 380 ℃;
the collection mode is Full MS-ddMS in positive ion mode2：
Wherein, the specific parameters of Full MS are set as follows: resolution (Resolution): 70000; AGC Target: 3e 6; maximum IT: 100 ms; scan range: 200-; spectrum data: a Centroid; inclusion: on;
wherein dd-MS2The specific parameter settings are as follows: resolution (Resolution): 17500, AGC Target: 1e 5; maximum IT: 50 ms; loop count: 2; isolation window: 2.0 Da; NCE: 15, 25, 35; spectrumdata: a Centroid; and in dd settings, Minimum AGC: 8.0e 3; apex trigger: 2-6 s; excludeisotope: on; dynamic exclus: 4.0 s; if idle: pick other.
The quantitative characteristic peptide segment IVNNDFNFNDVNFR of the Chinese bee is successfully screened out, and the specificity of the quantitative characteristic peptide segment IVNNDFNFNDVNFR is verified by a Uniprot database; and selecting a stable Internal Standard (IS) peptide fragment for accurate and sensitive quantification of MRJP2 in honey.
Preferably, the stable isotope internal standard peptide fragment is: IVNNDFNFNDVNFR, 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 fragment in the mass spectrum has 864.41044 mass-to-charge ratio; contains daughter ions with mass-to-charge ratio of 535.29883 and m/z 1172.54797.
The stable isotope internal standard peptide fragment generates a detection signal in a mass spectrum, wherein the parent ion is m/z, the parent ion is m/z: 869.41458, and the stable isotope internal standard peptide fragment also comprises daughter ions with mass-to-charge ratios of 1035.48755 and 1182.55713.
In the method, the standard curve obtained in the step C is Y =0.001118X-0.01915, R2=0.9988, 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 linear detection range of MRJP2 protein is 10-1000 ng/mL, and the lowest detection limit is 5 ng/mL.
The method comprises the steps of detecting a honey sample by adopting UHPLC-Q active Plus (ultra high performance liquid chromatography-four-stage rod series high-resolution electrostatic orbit trap), calculating the content of MRJP2 by comparing the peak area ratio of MRJP2 characteristic peptide segment/IS peptide segment in a spectrum, and further judging whether honey IS adulterated, wherein the peak area of the parent ion of the characteristic peptide segment of MRJP2 on the liquid IS mainly based on the peak area of the stable isotope labeled Internal Standard (IS) 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.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
in view of the relatively constant content of MRJP2 in Chinese bee, the invention establishes a set of honey authenticity evaluation method for assisting the identification of honey adulteration. The method for detecting the content of the protein of the Chinese bee MRJP2 has the advantages of strong specificity, high sensitivity, good accuracy and precision and the like, and is suitable for accurate quantification of the Chinese bee MRJP 2. The method has great significance for maintaining the healthy development of the honey consumption industry and protecting the rights and interests of honey consumers.
Drawings
FIG. 1 is an ion flow diagram of two candidate peptide fragments of Apis cerana MRJP2 extracted by UHPLC-Q active plus in example 1 of the present invention.
FIG. 2 is the mass spectra of two candidate peptide fragments of Apis cerana MRJP2 detected by UHPLC-Q active plus in example 1 of the present invention.
FIG. 3 is the second-order fragment mass spectrum of two candidate peptide fragments of Apis cerana MRJP2 detected by UHPLC-Q active plus in example 1 of the present invention.
FIG. 4 shows peptide IVNNDFNFNDVNFR (R-13C6,15N4) Ion flow diagram of (a).
FIG. 5 shows peptide IVNNDFNFNDVNFR (R-13C6,15N4) Mass spectrum of (2).
FIG. 6 shows peptide IVNNDFNFNDVNFR (R-13C6,15N4) Second order fragment mass spectrum of (1).
FIG. 7 is the response (relative abundance) of alternative signature peptides in example 1 of the present invention in a reference sample.
FIG. 8 shows the quantitative results of the MRJP2 content in different honey samples according to example 1 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
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;
5. a 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;
9.1290 Infinity liquid chromatography-6495 triple quadrupole 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 content of Mesema MRJP2 by 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
(1) Solution preparation
5M Urea solution: 4.5 g of Urea is weighed out and the volume of the ultrapure water is adjusted to 15 mL.
100 mM DTT: weighing DTT 308.5 mg, 40mM NH4HCO3The solution is metered to 20 mL, and each tube is subpackaged with l mL, and the solution is stored in a refrigerator at the temperature of-20 ℃ for standby.
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 IAA solution: 0.39 g of IAA was weighed out and 40mM NH was used4HCO3The solution is dissolved to 20 mL 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 1 mL, and the mixture was stored at 4 ℃.
The balance solution was 1. mu.L of TFA, and the volume was adjusted to 1 mL with ultrapure water, and the mixture was stored at 4 ℃.
Eluent: 800. mu.L of ACN and 1. mu.L of TFA were diluted to 1 mL with ultrapure water and stored at 4 ℃.
(2) Drawing a standard curve: a series of signature peptide fragment standards (2, 5, 10, 20, 40, 80, 100, 200, 400, 800 and 1000 ng/mL) were prepared in the initial mobile phase (97: 3, v/v, water/ACN with 0.1% formic acid) and 200 ng of IS peptide fragment was added to each concentration of standard that was formulated.
1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometry liquid chromatography conditions were as follows:
a chromatographic column: kinetex C18 (50 mm. times.2.1 mm) 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: 0-0.8 min, 10% of B; 0.8-1.3 min, 10-20% of B; 1.3-4.5 min, 20-30% of B; 4.5-5.4 min, 30-95% of B; 5.4-6.0min, 95% of B; 6.0-6.1 min, 95-5% of B, 6.1-7.0 min and 5% of B.
Flow rate: 0.3 mL/min; sample introduction amount: 5.0 muL.
The mass spectrometry 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. A calibration curve was prepared by plotting the characteristic peptide/IS peptide peak area ratio versus analyte concentration.
(3) Pretreatment of honey samples
① the honey to be tested is weighed accurately into 10 g to 50 mL centrifuge tube, 10 mL deionized water is added, vortex is carried out until the honey is fully dissolved, centrifugation is carried out for 10 min at 12000 rpm and 4 ℃, and the supernatant is collected into a new 2 mL centrifuge tube.
② mu.L of the protein solution was removed and 4. mu.L of 100. mu.g/mL internal standard peptide fragment (IVNNDFNFNDVNFR) was added and mixed with 800. mu.L of 40mM NH4HCO3And (4) mixing. To the above mixed solution, 100. mu.L of 30 mM DTT solution was added and the reaction was carried out at room temperature for 60 min, and then 500. mu.L of 100 mM IAA solution was added and the reaction was carried out at room temperature for 60 min in a dark state.
③ mu.L of trypsin solution was added to each sample and the digestion was carried out overnight at 37 ℃ when the digestion was complete, 1. mu.L of FA was added to inactivate the trypsin.
(4) Mass spectrometric analysis of honey samples
Honey samples were tested using 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometry.
The chromatographic conditions were as follows:
a chromatographic column: kinetex C18 (50 x 2.1 mm) 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: 0-0.8 min, 10% of B; 0.8-1.3 min, 10-20% of B; 1.3-4.5 min, 20-30% of B; 4.5-5.4 min, 30-95% of B; 5.4-6.0min, 95% of B; 6.0-6.1 min, 95-5% of B, 6.1-7.0 min and 5% of B. Flow rate: 0.3 mL/min; sample introduction amount: 5.0 muL.
The mass spectrometry 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.
(5) Data processing of honey samples
Substituting the peak area ratio of the characteristic peptide fragment/IS peptide fragment into a formula to obtain the concentration of the characteristic peptide fragment, and obtaining the content of the Mewasp MRJP2 protein according to the formula (2).
X=(ФcVM1)/(M2m) formula (2)
Wherein X is (ng/g) the content of bee MRJP2 in the honey sample, Φ is the ratio of the volume of zymoprotein to the total sample volume, c is the concentration of the characteristic peptide fragment in the tryptic digest, V (mL) is the volume of the tryptic digest, M is1、M2Is the molar mass of MRJP2 and the characteristic peptide stretch, and m (g) is the mass of the honey sample. So as to achieve the aim of quantifying MRJP2 in the honey sample.
Detecting the accurate m/z value of the characteristic peptide segment IVNNDFNFNDVNFR of the Chinese bee MRJP2 in a map of a honey sample: 864.41044 ([ M + 2H)]2+) (ii) a The sample map should contain added stable isotope labeled Internal Standard (IS) peptide segment IVNNDFNFNDVNFR (R-13C6,15N4) Precise m/z value of (2): 869.41458 ([ M + 2H)]2+) The fragment ion spectrum should contain the characteristic fragment ions m/z 535.29883, 1172.54797 of characteristic peptide segment IVNNDFNFNDVNFR, and correspondingly, the fragment ions 1035.48755, 1182.55713 should be contained in the sub-ion spectrum (MS/MS) of stable isotope labeled Internal Standard (IS) peptide segment IVNNDFNFNDVNFR. Only in the honey samplem/zThe value and the characteristic fragment ions simultaneously meet the characteristics, so that the calculated content of the MRJP2 protein in the honey sample can be determined to be credible, and the quality of the honey can be identified according to the content.
The peptide segments of MRJP2 of real medium honey samples collected by bee farmers are subjected to matching detection, the results are compiled into a graph, the graph results are shown in figure 7, and it can be seen from figure 7 that in medium honey, the MRJP2 characteristic peptide segments IVNNDFNFNDVNFR and MTSNTFDYDPR screened by the invention have the highest response, so that the content of the medium honey samples is relatively stable, the medium honey samples can be shown to show that the content of the medium honey samples is relatively stable, and the medium honey samples can be used as peptide segments in quantitative medium honey samples, which are suitable for quantifying the content of MRJP2 in the medium honey samples.
According to the quantitative result of the MRJP2 content in the real medium honey sample collected from the bee farmer, the result is shown in figure 8, and it can be seen from figure 8 that the MRJP2 content in the medium honey is relatively stable, and the result repeatability is good.
Example 2 detection of authenticity of a quantitative Apis cerana MRJP2 Honey sample in a Michelia
1. Sample source
Purchasing real honey sample from market
2. Experimental procedure
(3) Pretreatment of honey samples
① the honey to be tested is weighed accurately into 10 g to 50 mL centrifuge tube, 10 mL deionized water is added, vortex is carried out until the honey is fully dissolved, centrifugation is carried out for 10 min at 12000 rpm and 4 ℃, and the supernatant is collected into a new 2 mL centrifuge tube.
② mu.L of the protein solution was removed and 4. mu.L of 100. mu.g/mL internal standard peptide fragment (IVNNDFNFNDVNFR) was added and mixed with 800. mu.L of 40mM NH4HCO3And (4) mixing. To the above mixed solution, 100. mu.L of 30 mM DTT solution was added and the reaction was carried out at room temperature for 60 min, and then 500. mu.L of 100 mM IAA solution was added and the reaction was carried out at room temperature for 60 min in a dark state.
③ mu.L of trypsin solution was added to each sample and the digestion was carried out overnight at 37 ℃ when the digestion was complete, 1. mu.L of FA was added to inactivate the trypsin.
(4) Mass spectrometric analysis of honey samples
Honey samples were tested using 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/IS peptide fragment into a formula to obtain the concentration of the characteristic peptide fragment, and obtaining the content of the Mewasp MRJP2 protein according to the formula (2).
The peptide fragment of MRJP2 of the Chinese honey sample purchased from the market is detected, the result is shown in Table 1, and as can be seen from the table 1, the concentration of the MRJP2 characteristic peptide fragment IVNNDFNFNDVNFR screened according to the invention in the mass spectrum is obviously seen, and the content of MRJP2 in the Chinese honey is shown to be abnormal or 0 in the samples which are not the Chinese honey.
The ion flow diagram of two candidate peptide fragments of Chinese bee MRJP2 extracted by UHPLC-Q active plus is shown in figure 1.
The mass spectra of two candidate peptide fragments of Apis cerana MRJP2 detected by UHPLC-Q active plus are shown in FIG. 2.
The secondary fragment mass spectra of two candidate peptide fragments of Apis cerana MRJP2 detected by UHPLC-Q active plus are shown in FIG. 3.
Isotopically labeled Internal Standard (IS) peptide segment IVNNDFNFNDVNFR (R-13C6,15N4) See fig. 4.
Isotopically labeled Internal Standard (IS) peptide segment IVNNDFNFNDVNFR (R-13C6,15N4) The mass spectrum of (A) is shown in FIG. 5.
Isotopically labeled Internal Standard (IS) peptide segment IVNNDFNFNDVNFR (R-13C6,15N4) The secondary fragment mass spectrum of FIG. 6.
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.
Table 1 concentration of characteristic peptide fragment IVNNDFNFNDVNFR in different honey samples
Sequence listing
<110> bee institute of Chinese academy of agricultural sciences
<120> method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry
<130>KHP191115396.6
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Ile Leu Lys Tyr Asp Gly Val Pro Ser Thr Leu Asn Met Ile Ser Asn
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Ala Ile Asp Lys Phe Asp Arg Leu Trp Val Leu Asp Ser Gly Leu Ile
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Asn Arg Thr Glu Pro Ile Cys Ala Pro Lys Leu His Val Phe Asp Leu
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Lys Asn Thr Lys His Leu Lys Gln Ile Glu Ile Pro His Asp Ile Ala
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Val Asn Ala Thr Thr Gly Lys Gly Gly Leu Val Ser Leu Val Val Gln
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Ala Met Asp Pro Met Asn Thr Leu Val Tyr Ile Ala Asp His Lys Gly
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Asp Ala Leu Ile Val Tyr Gln Asn Ser Asp Asp Ser Phe His Arg Met
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Asn Gly Glu Ser Phe Thr Leu Lys Asn Gly Ile Cys Gly Met Ala Leu
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Leu Tyr Tyr Val Asn Thr Glu Pro Phe Met Lys Ser Gln Phe Gly Asp
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Asn Asn Asn Val Gln Tyr Glu Gly Ser Gln Asp Thr Leu Asn Thr Gln
290 295 300
Ser Leu Ala Lys Ala Val Ser Lys Asp Gly Val Leu Phe Val Gly Leu
305 310 315 320
Val Gly Asn Ser Ala Leu Gly Cys Leu Asn Glu His Gln Pro Leu Gln
325 330 335
Arg Glu Asn Leu Glu Leu Val Ala Gln Asn Glu Lys Thr Leu Gln Met
340 345 350
Ile Ala Gly Met Lys Ile Lys Glu Glu Leu Pro His Phe Val Gly Ser
355 360 365
Asn Lys Pro Val Lys Asp Glu Tyr Met Leu Val Leu Ser Asn Lys Met
370 375 380
Gln Lys Ile Val Asn Asn Asp Phe Asn Phe Asn Asp Val Asn Phe Arg
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Ile Leu Gly Ala Asn Val Lys Glu Leu Met Arg Asn Thr His Cys Ala
405 410 415
Asn Phe Asn Asn Lys Asn Asn Gln Lys Asn Asn Asn Gln Lys Asn Asn
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Asn Gln Asn Asn Asn Asn Gln Lys Asn Asn Asn Gln Lys Asn Asn Asn
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Gln Lys Asn Asn Asn Gln Lys Asn Asn Asn Gln Lys Asn Asn Asn Gln
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Asn Thr Asn Asn
465
Claims (8)
1. The method for quantifying honey MRJP2 in liquid chromatography tandem mass spectrometry is characterized in that the method provides a characteristic peptide fragment of honey MRJP2 for accurate quantification of mass spectrometry, and the characteristic peptide fragment comprises the following characteristic peptide fragments:
characteristic peptide fragment 1: IVNNDFNFNDVNFR, respectively;
characteristic peptide fragment 2: MTSNTFDYDPR are provided.
2. The method according to claim 1, further comprising the step of pre-treating the honey sample with reagents comprising:
(1) extracting a buffer solution: 0.01M PBS, pH7.4;
(2) 20 ng/mu L of trypsin solution and 40mM ammonium bicarbonate solution as a solvent; and/or
(3) Standard substance of characteristic peptide sections 1 and 2.
3. Method according to claim 1 or 2, characterized in that it comprises the following steps:
A. preparing characteristic peptide fragment standard solutions with different concentrations and containing stable isotope internal standard peptide fragments with fixed concentrations, performing mass spectrometry detection, and drawing a standard curve;
B. pretreatment of a honey sample to be detected: extracting protein in honey, carrying out enzyme digestion on sample protein by using trypsin liquid, and desalting an enzymolysis product;
C. b, adding stable isotope internal standard peptide sections into the sample to be detected to enable the concentration of the stable isotope internal standard peptide sections to be the same as that in the step A, and performing liquid chromatography tandem mass spectrometry detection by adopting the same method;
D. and (3) obtaining the concentration of the characteristic peptide fragment in the sample to be detected by contrasting with the standard curve, thereby realizing the quantitative detection of MRJP 2.
4. The method of claim 3, wherein the step A and the step C are performed by liquid chromatography tandem mass spectrometry using a triple quadrupole or Q Exactiveplus LC-MS.
5. The method of claim 3, wherein the parent ion of the detection signal generated by the quantitative signature peptide segment IVNNDFNFNDVNFR in the mass spectrum has a mass-to-charge ratio of 864.41044; contains 535.29883 mass/charge ratio, m/z1172.54797 daughter ions, the allowable deviation should be within 10 ppm.
6. The method of claim 3, wherein the stable isotope internal standard peptide fragment is: IVNNDFNFNDVNFR, 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: 869.41458, and the parent ion comprises daughter ions with mass-to-charge ratios of 1035.48755 and 1182.55713.
7. The method of claim 3, wherein step A results in a standard curve of Y =0.001118X-0.01915, R2=0.9988, 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.
8. The method of claim 7 wherein the MRJP2 has a linear detection range of 10-1000 ng/mL and a minimum detection limit of 5 ng/mL.
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