Method for identifying organophosphate and application thereof

Abstract

The invention discloses application of a peptide fragment with an amino acid sequence shown as SEQ ID NO: 1 in identification of organophosphorus esters, and provides a method for identifying organophosphorus esters, and the method comprises the following steps: pretreating a sample to be detected, performing high performance liquid chromatography, mixing with the peptide fragment with the amino acid sequence shown as SEQ ID NO: 1, and performing mass spectrometry. The method comprises the following steps: obtaining a to-be-detected sample mass spectrum result, setting a control group and obtaining a to-be-detected sample control mass spectrum result, performing analysis based on the mass spectrum result, selecting a characteristic peak which is remarkably up-regulated and meets m / z = 560.2918 in the to-be-detected sample mass spectrum result as a primary screening characteristic peak, and combining the primary screening characteristic peak with a database for identification so as to identify the organophosphorus ester. By utilizing the method disclosed by the invention, the organophosphate in the to-be-detected sample can be accurately identified, and the interference of a large number of unknown substances in the to-be-detected sample is avoided.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically, to a method for identifying organophosphates and its applications. Background Art

[0002] Organophosphate diesters (di-OPEs) have been widely detected in environmental media such as water bodies, sediments, the atmosphere and biological samples. A major source of di-OPEs is the direct use of industrial products or impurities in the industrial production of tri-OPEs.

[0003] Diphenyl phosphate, dibutyl phosphate, bis(2-ethylhexyl) phosphate, etc. are all widely used flame retardants or plasticizers, with an annual output as high as 17,050 tons. In addition, with the gradual phasing out of toxic halogenated flame retardants such as polybrominated diphenyl ethers, the demand for organophosphate triesters (tri-OPEs) in global consumption and production has increased sharply, with a production volume exceeding 2.5 million tons. The extensive use of tri-OPEs has led to their widespread presence in environmental media, and the products formed by their biological or abiotic transformation are another important source of di-OPEs in the environment. The ubiquitous di-OPEs' negative impacts on organisms cannot be ignored.

[0004] Toxicological studies have shown that di-OPEs can have toxic effects on aquatic organisms, including cytotoxicity, developmental toxicity, teratogenicity, reproductive toxicity, endocrine disruption effects, etc. Epidemiological studies have also revealed the potential adverse reactions of di-OPEs to humans. For example, the concentration level of di-OPEs in pregnant women's urine is related to abnormal growth and development of infants and symptoms such as neurodevelopmental damage. More worryingly, compared with the parent compound tri-OPEs, some di-OPEs can produce similar or even higher toxic effects. Therefore, identifying organophosphate diesters and their transformation products in the environment is of great significance.

[0005] Based on the screening technology of high performance liquid chromatography-high resolution mass spectrometry, it has become possible to simultaneously detect thousands of compounds in a single sample run. By establishing a compound library, the targeted screening technology can reveal the occurrence of di-OPEs of high concern in various environmental media. However, the types and quantities of di-OPEs covered by the compound library are relatively few and the coverage is limited. The non-targeted screening technology greatly improves the structural identification efficiency of unknown di-OPEs and their transformation products by mining molecular feature information such as retention time, quasi-molecular ion peak, characteristic fragment ions and isotope peak distribution. However, the large number of co-existing interfering substances in the environment makes the structural analysis of compounds still a time-consuming and difficult task.

[0006] Therefore, there are still certain challenges in identifying unknown organophosphoric acid diesters and their transformation products in the current environment. At the same time, a large number of interfering molecules coexisting in complex matrices greatly increase the difficulty and time consumption of data processing. In view of the problems of wide distribution, diverse types, and different structures of organophosphoric acid diesters existing in the environment, developing a rapid, sensitive, economical, and high-throughput method for identifying and screening organophosphoric acid diesters and their transformation products is conducive to improving the screening efficiency of high-risk substances. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for identifying organophosphates and its application.

[0008] The first purpose of the present invention is to provide the application of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in identifying organophosphates.

[0009] The second purpose of the present invention is to provide a method for identifying organophosphates.

[0010] The third purpose of the present invention is to provide the application of the above method in identifying organophosphates.

[0011] In order to achieve the above purpose, the present invention is realized through the following solutions: The present invention claims the application of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in identifying organophosphates.

[0012] Peptide segment (SEQ ID NO: 1): Ac-SGAGKT-NH 2 ; Among them, the N-terminal of the peptide segment with the amino acid sequence shown in SEQ ID NO: 1 has an acetylation modification, and the C-terminal has an amino modification.

[0013] Preferably, the organophosphate is an organophosphoric acid monoester and / or an organophosphoric acid diester.

[0014] More preferably, the organic phosphoric acid monoester is 2-ethylhexyl phosphate (EHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP); the organic phosphoric acid diester is dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), phenylethylene hydrogen phosphate (EPP), and bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP).

[0015] The present invention also claims protection for a method for identifying organic phosphates, comprising the following steps: S1. Pretreatment of the sample to be tested: The sample to be tested is successively extracted and eluted, the eluate is collected and dried, and then dissolved in a control solution to obtain an extract of the sample to be tested; the control solution is methanol or acetonitrile; S2. Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with a peptide solution having an amino acid sequence as shown in SEQ ID NO: 1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain a mass spectrometry result of the sample to be tested; Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with the control solution shown in step S1 through a three-way joint and then injected into a mass spectrometer for mass spectrometry analysis to obtain a control mass spectrometry result of the sample to be tested; S3. Select the characteristic peaks that are significantly up-regulated in the mass spectrometry result of the sample to be tested obtained in step S2 compared with the control mass spectrometry result of the sample to be tested, and select the characteristic peak with △m / z = 560.2918 as the preliminary screening characteristic peak; S4. Identify the structure of the preliminary screening characteristic peak obtained in step S3, and further identify the organic phosphate in the sample to be tested.

[0016] Preferably, in step S1, the extraction is performed on the sample to be tested using an HLB solid-phase extraction column.

[0017] More preferably, the HLB solid-phase extraction column is an HLB solid-phase extraction column rinsed with a control solution and balanced with deionized water; the control solution is methanol or acetonitrile.

[0018] Preferably, in step S1, the elution is performed using a control solution; the control solution is methanol or acetonitrile.

[0019] Preferably, the chromatographic conditions of the high-performance liquid chromatography are as follows: the flow rate is 200 - 400 μL / min; the column temperature is 25 - 40 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set to 10%, then it is increased to 50% within 10 min, then it is increased to 90% at 12 min and maintained for 3 min, then it is decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile.

[0020] Further preferably, the chromatographic column is a Poroshell 120 EC-C18 chromatographic column; the specifications of the chromatographic column are 3.0 × 150 mm, 1.9 μm.

[0021] Preferably, the peptide solution with the amino acid sequence shown in SEQ ID NO: 1 in step S2 is mixed with the substance output from the high-performance liquid chromatograph at a flow rate of 20 μL / min through a three-way joint.

[0022] More preferably, the concentration of the peptide solution is 50 μmol / L.

[0023] Preferably, the conditions for mass spectrometry in step S2 are as follows: Detection is performed in the negative ion mode, the sheath gas temperature is 100 - 400 °C, the sheath gas flow rate is 5 - 11 L / min, the nozzle voltage is 1000 - 4000 V, the nebulizing gas pressure is 5 - 50 psi, the drying gas temperature is 100 - 400 °C, the drying gas flow rate is 5 - 11 L / min, the capillary voltage is 2000 - 4000 V, the cone voltage is 65 V, the octopole voltage is 750 V, and the fragmentation voltage is 100 - 200 V.

[0024] More preferably, the conditions for mass spectrometry are as follows: Detection is performed in the negative ion mode, the sheath gas temperature is 400 °C, the sheath gas flow rate is 11 L / min, the nozzle voltage is 1800 V, the nebulizing gas pressure is 10 psi, the drying gas temperature is 250 °C, the drying gas flow rate is 11 L / min, the capillary voltage is 4000 V, the cone voltage is 65 V, the octopole voltage is 750 V, and the fragmentation voltage is 125 V.

[0025] Further preferably, the sheath gas is nitrogen.

[0026] Preferably, in step S2, the mass spectrometry analysis is performed using a quadrupole time-of-flight mass spectrometer equipped with an ESI source.

[0027] Preferably, before selecting the significantly up-regulated characteristic peaks in step S3, the missing values in the mass spectrometry results are filled using MetaboAnalyst.

[0028] More preferably, the filling of the missing values is specifically: replacing the missing values of the samples with 1 / 5 of the minimum positive value of the variable corresponding to the missing value.

[0029] Preferably, the significantly up-regulated characteristic peaks in step S3 are the characteristic peaks with up-regulation, Fold change > 5.0 and p-value < 0.05.

[0030] Preferably, in step S4, the structure identification is specifically carried out by searching the secondary spectrum database, predicting using sirius software and verifying with standards; the standards are analytical pure grade reagents of dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), and 2-ethylhexyl phosphate (EHP).

[0031] The present invention also claims the application of any of the above methods in identifying organophosphates.

[0032] Preferably, the organophosphate is an organophosphoric acid monoester and / or an organophosphoric acid diester.

[0033] More preferably, the organic phosphoric acid monoester is 2-ethylhexyl phosphate (EHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP); the organic phosphoric acid diester is dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), phenylethylene hydrogen phosphate (EPP), and bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP).

[0034] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the application of a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 in identifying organic phosphates, and provides a method for identifying organic phosphates. After pre-treating a sample to be tested, it is mixed with a peptide segment with the amino acid sequence shown in SEQ ID NO: 1 after high performance liquid chromatography and then subjected to mass spectrometry to obtain the mass spectrometry result of the sample to be tested. At the same time, a control group is set up to obtain the control mass spectrometry result of the sample to be tested. Based on the mass spectrometry result, analysis is carried out and characteristic peaks that are significantly up-regulated in the mass spectrometry result of the sample to be tested and satisfy △m / z = 560.2918 are selected as the preliminary screening characteristic peaks, and the preliminary screening characteristic peaks are combined with a database for identification, thereby identifying organic phosphates. Using the method shown in the present invention, the organic phosphates in the sample to be tested can be accurately identified, avoiding the interference of a large number of unknown substances in the sample to be tested. Description of the Drawings

[0035] Figure 1 It is a schematic flow chart of a method for identifying organic phosphates in Example 1; Figure 2 It is a collision-induced dissociation curve diagram of the labeled group in Example 2; Figure 3 It is a principal component analysis result diagram in Example 2; Figure 4The volcano plot drawn for step S3 when the marker group in Example 2 is processed according to the equation shown in Example 1; Figure 5 The distribution diagram of the m / z signal versus retention time during the mass spectrometry process of the marker group in Example 2; Figure 6 The volcano plot drawn for step S3 when processed according to the method shown in Example 1 in Example 3; Figure 7 The change diagram of the number of characteristic peaks screened in Example 3. Detailed implementation manners

[0036] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] Example 1 A method for identifying organophosphates The schematic flow diagram of a method for identifying organophosphates is as Figure 1 shown.

[0038] 1. Peptide synthesis Based on the solid-phase peptide synthesis method, a peptide segment (6P) with the amino acid sequence shown in SEQ ID NO: 1 is synthesized; 6P (SEQ ID NO: 1): Ac-SGAGKT-NH 2 .

[0039] 2. Method for identifying organophosphates Combined with 6P with the amino acid sequence shown in SEQ ID NO: 1 to identify organophosphates, and the binding-guided analysis technology is used to identify organophosphates in the sample to be tested, which specifically includes the following steps: S1. Place the HLB solid-phase extraction column on the solid-phase extraction device. After flushing it 2 times with 5 mL of methanol to remove the impurities in the HLB solid-phase extraction column itself, and then equilibrating it 2 times with 5 mL of deionized water, the equilibrated HLB solid-phase extraction column is obtained; After extracting 500 mL of the sample to be tested with the equilibrated HLB solid-phase extraction column, then eluting the HLB solid-phase extraction column 3 times with 5 mL of methanol, collecting and combining the eluate, drying the eluate under nitrogen, redissolving it in 500 μL of methanol, and then filtering it with a 0.22 μm organic nylon filter head, and collecting the filtrate to obtain the extract of the sample to be tested.

[0040] S2. Dissolve 6P with the amino acid sequence shown in SEQ ID NO: 1 in methanol to prepare a 25 mM 6P stock solution, and then dilute the 6P stock solution with methanol to a 50 μM 6P solution.

[0041] Experimental group: Inject the extract of the sample to be tested obtained in step S1 into a high-performance liquid chromatograph (Agilent, 1290) with an injection volume of 3 μL for high-performance liquid chromatography. The substance output by the high-performance liquid chromatograph is mixed with a 50 μM 6P solution through a three-way joint and then injected into a quadrupole time-of-flight mass spectrometer (Agilent, 6545) equipped with an ESI source for mass spectrometry analysis to obtain the mass spectrometry result (spectrum) of the sample to be tested; The conditions for high-performance liquid chromatography are as follows: The chromatographic column is a Poroshell 120 EC-C18 chromatographic column (3.0×150 mm, 1.9 μm); the flow rate is 200 μL / min; the column temperature is 35 °C; the mobile phase gradient is: the initial gradient of mobile phase B is set to 10%, then increased to 50% within 10 min, then increased to 90% at 12 min and maintained for 3 min, then decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A is water and mobile phase B is acetonitrile; The 6P solution is mixed with the substance output by the high-performance liquid chromatograph at a flow rate of 20 μL / min through a micro-injection pump; The conditions for mass spectrometry analysis are as follows: An electrospray ionization source is used, and gas-phase complexes are detected in the negative ion mode. The sheath gas temperature is 400 °C, the sheath gas flow rate is 11 L / min, the nozzle voltage is 1800 V, the nebulizing gas pressure is 10 psi, the drying gas temperature is 250 °C, the drying gas flow rate is 11 L / min, the capillary voltage is 4000 V, the cone voltage is 65 V, the octopole voltage is 750 V, the fragmentation voltage is 125 V, and the sheath gas is nitrogen.

[0042] The only difference between the control group and the experimental group is that the 50 μM 6P solution is replaced with methanol, and the rest of the treatments are exactly the same, to obtain the mass spectrometry result (spectrum) of the control sample to be tested; S3. Use the ProteoWizard MSconvert software to convert the mass spectrometry result of the sample to be tested obtained in step S2 into a mass spectrometry result in mzML format, and then import the mass spectrometry result in mzML format into the Mzmine 2.53 software. Based on the default parameters, perform mass feature detection, ADAP chromatographic reconstruction, isotope feature detection, peak filtering, and peak alignment to obtain a CSV file of the mass spectrometry result of the sample to be tested recording retention time, mass-to-charge ratio, and abundance; Perform the same treatment on the mass spectrometry result of the control sample to be tested obtained in step S2 to obtain a CSV file of the mass spectrometry result of the control sample to be tested recording retention time, mass-to-charge ratio, and abundance.

[0043] Import the mass spectrometry result CSV file of the sample to be tested and the control mass spectrometry result CSV file of the sample to be tested into the MetaboAnalyst data processing platform (https: / / www.metaboanalyst.ca), and fill in the missing values using 1 / 5 of the minimum positive value of the variable corresponding to the missing values in the two mass spectrometry result CSV files. Then, perform statistical analysis and draw a volcano plot based on the preset parameters of the MetaboAnalyst data processing platform. Select the characteristic peaks that are significantly up-regulated in the mass spectrometry result CSV file of the sample to be tested compared to the control mass spectrometry result CSV file of the sample to be tested (Fold change > 5 and p-value < 0.05) from the volcano plot, and select the characteristic peak with △m / z = 560.2918 as the candidate characteristic peak; S4. Based on the candidate characteristic peaks obtained in step S3, set the mass-to-charge ratio deviation △ppm = 20, retrieve through the secondary spectrum database (https: / / mona.fiehnlab.ucdavis.edu / downloads), predict using sirius software, and verify with standards to identify the information of the candidate characteristic peaks, and then identify the organophosphates in the sample to be tested; The standards are analytical reagent grades of dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), bis-(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), and 2-ethylhexyl phosphate (EHP).

[0044] Example 2 Verification of a method for identifying organophosphates I. Experimental method Take 300 mL of river water from the Pearl River in Guangzhou, add 8 organophosphate compounds (diphenyl phosphate, dibutyl phosphate, diethyl phosphate, dimethyl phosphate, bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate, monophenyl phosphate, and monomethyl phosphate) to a final concentration of 2 ng / L for each organophosphate compound to obtain spiked river water. After filtering the spiked river water through a medium-speed qualitative filter paper (30 - 50 μm), sonicate for 10 min to obtain pretreated spiked river water.

[0045] Place the HLB solid-phase extraction column on the solid-phase extraction device. After rinsing twice with 5 mL of methanol to remove the impurities in the HLB solid-phase extraction column itself, balance it twice with 5 mL of deionized water to obtain a balanced HLB solid-phase extraction column; After extracting 300 mL of pretreated spiked river water using a balanced HLB solid-phase extraction column, the HLB solid-phase extraction column was then eluted three times with 5 mL of methanol. The eluates were collected and combined, dried under nitrogen, dissolved in 300 μL of methanol, filtered through a 0.22 μm organic nylon filter head, and the filtrate was collected to obtain the extract of the river water to be tested. As shown in Example 1, a 50 μM 6P solution was prepared.

[0046] Labeling group (6P): The extract of the river water to be tested was injected into a high-performance liquid chromatograph (Agilent, 1290) with an injection volume of 3 μL for high-performance liquid chromatography. The substances output by the high-performance liquid chromatograph were mixed with a 50 μM 6P solution through a three-way joint and then injected into a quadrupole time-of-flight mass spectrometer (Agilent, 6545) equipped with an ESI source for mass spectrometry analysis to obtain the mass spectrometry results (spectra) of the extract of the river water to be tested. At the same time, collision-induced dissociation (CID) scanning was performed during the mass spectrometry process to obtain the collision-induced dissociation curve of the labeling group, and the collision-induced dissociation curve was fitted with a Boltzmann function to obtain the centroid collision energy (E cm，1 / 2 ) when the dissociation ratio of 6P to each organophosphate was 50%.

[0047] The conditions for high-performance liquid chromatography were as follows: The chromatographic column was a Poroshell 120 EC-C18 chromatographic column (3.0×150 mm, 1.9 μm); the flow rate was 200 μL / min; the column temperature was 35 °C; the mobile phase gradient was: the initial gradient of mobile phase B was set at 10%, then increased to 50% within 10 min, then increased to 90% at 12 min and maintained for 3 min, then decreased to 10% at 15.1 min and maintained for 5 min, where mobile phase A was water and mobile phase B was acetonitrile; The 6P solution was mixed with the substances output by the high-performance liquid chromatograph at a flow rate of 20 μL / min through a micro-injection pump; The conditions for mass spectrometry analysis were as follows: An electrospray ionization source was used, and gas-phase complexes were detected in the negative ion mode. The sheath gas temperature was 400 °C, the sheath gas flow rate was 11 L / min, the nozzle voltage was 1800 V, the nebulizing gas pressure was 10 psi, the drying gas temperature was 250 °C, the drying gas flow rate was 11 L / min, the capillary voltage was 4000 V, the cone voltage was 65 V, the octapole voltage was 750 V, the fragmentation voltage was 125 V, and the sheath gas was nitrogen.

[0048] The only difference between Control Group 1 (Control) and the labeling group was that the 50 μM 6P solution was replaced with methanol, and the other treatments were exactly the same, obtaining the control mass spectrometry results (spectra) of the extract of the river water to be tested.

[0049] Next, according to the methods shown in steps S3 and S4 in Example 1, identify the organophosphates in the extracted river water to be tested.

[0050] At the same time, obtain the mixture (at the three-way joint) after mixing the substances output by the high-performance liquid chromatograph during the treatment of the labeled group with the 6P solution, and the mixture 1 (at the three-way joint) after mixing the substances output by the high-performance liquid chromatograph during the treatment of the control group with methanol, and perform principal component analysis.

[0051] II. Experimental Results The collision-induced dissociation curve diagram of the labeled group is as Figure 2 shown, and the centroid collision energy when the dissociation ratio of peptide 6P to organophosphate is 50% is shown in Table 1.

[0052] Table 1 Centroid collision energy when the dissociation ratio of peptide 6P to organophosphate is 50%

[0053] The results show that in the anion mode, peptide 6P with the amino acid sequence shown in SEQ ID NO: 1 can form complexes with 8 different organophosphate compounds and generate stable composite signals, and the 8 organophosphate compounds added to the sample can all be accurately identified.

[0054] The principal component analysis result diagram is as Figure 3 shown, and the results show that the labeled group coincides with the control group on the PCA2 axis and cannot be distinguished; while the labeled group is significantly separated from the control group on the PCA1 axis.

[0055] Moreover, methanol is a common component in the labeled group and the control group, while peptide 6P in the labeled group is a single variable; therefore, the PCA1 axis is used to explain the contribution of the introduction of 6P to the change in signal abundance, and the PCA2 axis is used to explain the contribution of the solvent effect of the introduction of methanol to the change in signal abundance; in addition, since the contribution of the PCA1 axis to data analysis is 70.6% and the contribution of the PCA2 axis is 11.5%, it shows that the contribution of peptide 6P to the change in signal abundance dominates, and combined with peptide 6P with the amino acid sequence shown in SEQ ID NO: 1, more accurate identification of organophosphates can be achieved.

[0056] When the labeled group is processed according to steps S3 and S4 in Example 1, the volcano plot drawn in step S3 is as Figure 4 shown, and the distribution diagram of the m / z signal in the mass spectrometry process of the labeled group with respect to the retention time is as Figure 5 shown.

[0057] The results show that based on Figure 4As shown in the volcano plot, after introducing the peptide 6P with the amino acid sequence as shown in SEQ ID NO: 1 into the marker group, 981 significantly up-regulated characteristic peak signals were generated, and the models of the 8 spiked organophosphorus ester compounds also appeared in the significantly up-regulated characteristic peak signals; at the same time, in the distribution map of m / z versus retention time, the peptide 6P with the amino acid sequence as shown in SEQ ID NO: 1 showed good anti-interference performance, specifically manifested as that within 0.2 min of the change in retention time and in the presence of interference signals, the peptide 6P could non-covalently label organophosphorus esters (phosphodiesters and monoester compounds) and did not form gas-phase composite ions with other interfering components.

[0058] Based on the above results, the peptide 6P with the amino acid sequence as shown in SEQ ID NO: 1 can accurately bind to the organophosphorus esters in the sample to form a complex and is not affected by other interfering components in the sample.

[0059] Example 3 Application of a method for identifying organophosphorus esters I. Experimental method Taking 500 mL of underground wastewater from Yancheng Industrial Park, Jiangsu as the sample to be tested, identifying organophosphorus esters according to the method shown in Example 1, and recording the number of characteristic peak signals collected during the identification of organophosphorus esters; During the identification of organophosphorus esters, the standards used were standards of dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), 2-ethylhexyl phosphate (EHP), bis-(1-chloro-2-propyl) phosphate (BCPP) and 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)).

[0060] II. Experimental results When identifying organophosphorus esters in the underground wastewater according to the method shown in Example 1, the volcano plot drawn in step S3 is as Figure 6 shown, and the change diagram of the number of characteristic peaks screened is as Figure 7 shown.

[0061] 9346 characteristic peak signals were collected in the mass spectrometry results of the sample to be tested obtained in step S2; based on the Figure 6 shown volcano plot, 816 characteristic peaks significantly up-regulated compared to the mass spectrometry results CSV file of the control of the sample to be tested were collected from the mass spectrometry results CSV file of the sample to be tested, and 215 characteristic peaks were determined as candidate characteristic peaks from these 816 significantly up-regulated characteristic peaks (simultaneously meeting the conditions of significant up-regulation and △m / z = 560.2918).

[0062] Based on 215 candidate characteristic peaks, 14 organophosphates were identified from the underground wastewater, including 9 diphosphates and 5 monophosphates. The 14 organophosphates are shown in Table 2 and are respectively: dibutyl phosphate (DBP), diphenyl phosphate (DPP), bis(2-ethylhexyl) phosphate (DEHP), 2-ethylhexyl phosphate (EHP), bis(1-chloro-2-propyl) phosphate (BCPP), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(18:1(9Z)0:0)), 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine PE(16:1(9Z) / 0:0), 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine Lyso(PE(0:0 / 16:0)), ethenylphenyl hydrogen phosphate (EPP), bis(5-hydroxy-4-oxohex-6-en-1-yl) hydrogen phosphate (BHOHP), 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate (PTP), 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate (CFOBP), 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate (APDP), and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazin-7-yl)pentyl dihydrogen phosphate (FMPDP).

[0063] Table 2 14 Organophosphates

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Application of a peptide having an amino acid sequence as shown in SEQ ID NO: 1 in identifying organophosphates.

2. The use according to claim 1, characterized in that: The organic phosphate ester is an organic phosphate monoester and / or an organic phosphate diester.

3. The use according to claim 2, characterized in that: The organic phosphate monoester is 2-ethylhexyl phosphate, 2-[1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-2-methylpyridin-1-ium-3-yl]ethyl dihydrogen phosphate, 4-chloro-3-fluoro-4-oxobutyl dihydrogen phosphate, 3-(allyloxy)-2-phenoxyphenyl dihydrogen phosphate and 2-(fluoromethyl)-5-(4-methylimidazo[2,1-f][1,2,4]triazine-7-yl)pentyl dihydrogen phosphate; The organic phosphoric acid diesters are dibutyl phosphate, diphenyl phosphate, di(2-ethylhexyl) phosphate, bis-(1-chloro-2-propyl) phosphate, 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine, 1-(9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine, 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine, phenylvinyl hydrogen phosphate and bis(5-hydroxy-4-oxohept-6-en-1-yl) hydrogen phosphate.

4. A method for identifying an organophosphate, characterized in that: The following steps are involved: S1. Pretreatment of the sample to be tested: extracting and eluting the sample to be tested in sequence, collecting the eluate and drying it, and then dissolving it in a control solution to obtain an extract of the sample to be tested; the control solution is methanol or acetonitrile; S2. The sample extract obtained in step S1 is injected into a high performance liquid chromatograph for high performance liquid chromatography, and the substance output by the high performance liquid chromatograph is mixed with a peptide solution with an amino acid sequence such as SEQ ID NO: 1 through a three-way connector and then injected into a mass spectrometer for mass spectrometry analysis to obtain a mass spectrometry result of the sample to be tested; The sample extract obtained in step S1 is injected into a high performance liquid chromatograph for high performance liquid chromatography, and the substance output by the high performance liquid chromatograph is mixed with the reference solution shown in step S1 through a three-way connector and then injected into a mass spectrometer for mass spectrometry analysis to obtain a reference mass spectrometry result of the sample to be tested; S3. Select the characteristic peaks in the mass spectrometry results of the test sample obtained in step S2 that are significantly upregulated compared to the mass spectrometry results of the control sample, and select the characteristic peak with △m / z=560.2918 as the primary screening characteristic peak; S4. Perform structural identification on the primary screening characteristic peaks obtained in step S3, thereby identifying the organophosphate in the sample to be tested.

5. The method according to claim 4, characterized in that The chromatographic conditions of the high performance liquid chromatography described in step S2 are: a flow rate of 200-400 μL / min; a column temperature of 25-40° C.; a mobile phase gradient: the initial gradient of mobile phase B is set to 10%, then increased to 50% within 10 minutes, then increased to 90% at 12 minutes and maintained for 3 minutes, then decreased to 10% at 15.1 minutes and maintained for 5 minutes, wherein mobile phase A is water and mobile phase B is acetonitrile.

6. The method according to claim 4, characterized in that In step S2, the peptide solution having an amino acid sequence as shown in SEQ ID NO: 1 is mixed with the material output from the high performance liquid chromatograph through a three-way connector at a flow rate of 5 to 50 μL / min.

7. The method according to claim 6, characterized in that The concentration of the peptide solution is 1-100 μmol / L.

8. The method according to claim 4, characterized in that The conditions for mass spectrometry analysis in step S2 are: Negative ion mode detection was adopted, with sheath gas temperature of 100-400°C, sheath gas flow rate of 5-11 L / min, nozzle voltage of 1000-4000 V, nebulizing gas pressure of 5-50 psi, drying gas temperature of 100-400°C, drying gas flow rate of 5-11 L / min, capillary voltage of 2000-4000 V, cone voltage of 65 V, octapole voltage of 750 V, and fragmentation voltage of 100-200 V.

9. The method according to claim 4, characterized in that Step S3: Before selecting the significantly upregulated characteristic peaks, MetaboAnalyst is used to fill the missing values ​​of the mass spectrometry results.

10. Use of the method according to any one of claims 4 to 9 in identifying organophosphates.

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