CN115925808A - Characteristic polypeptide of asparagus sea, application thereof and method for identifying asparagus sea - Google Patents
Characteristic polypeptide of asparagus sea, application thereof and method for identifying asparagus sea Download PDFInfo
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Abstract
The invention relates to a characteristic polypeptide of radix cynanchi bungei and application thereof and a method for identifying radix cynanchi bungei, belonging to the technical field of biotechnology detection. The invention provides two characteristic polypeptides unique to asparagus sea, and the amino acid sequences of the characteristic polypeptides are shown in SEQ ID NO.1 and SEQ ID NO. 2. The characteristic polypeptide provided by the invention has excellent specificity and stability aiming at the asparagus sea, has strong specificity, can be used for identifying the asparagus sea and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biotechnology detection, and particularly relates to two characteristic polypeptides of asparagus sea, application of the characteristic polypeptides and a method for identifying the asparagus sea.
Background
The sea dragon is an important animal medicinal material in China, has the effects of warming kidney, tonifying yang, dissipating stagnation and relieving swelling, and has long history and numerous primitive sources. The pharmaceutical base source of Syngnathus is defined in the Chinese pharmacopoeia as dried bodies of Syngnathus caudatum hardwickii (Gray), syngnathus biaceutus (Bloch) or Syngnathus acus cusa cusLinnaeus, which are Syngnathus animals of the Syngnaceae family.
The sea dragon as a tonifying medicinal material has an increasing market demand year by year, and because the sea dragon cannot effectively realize artificial breeding at present, the wild sea dragon resources are increasingly reduced, and the sea dragon is mostly confused or is secondary to good by other varieties. At present, the sale proportion of the Syngnathus from different sources, which is not included in the Chinese pharmacopoeia, is increased year by year, which causes the market circulation of the Syngnathus medicinal materials to be disordered. The multi-asparagus sea dragon is a kindred variety, has similar names, is the most common confused product of the asparagus sea dragon, and is usually sold as a genuine asparagus sea dragon in the market. The commercial sea dragon medicinal materials are classified according to the body size of the sea dragon, and the market price of the sea dragon is even higher than that of the original sea dragon and other sea dragon commodities due to the large body size of the sea dragon.
The species identification and quality control methods of the current Syngnathus mainly comprise a sex identification method, an HPLC fingerprint spectrum method and a molecular biology method. The character identification method generally requires that the sea dragon individual is complete, and the person needing to be identified has rich experience, and the method has certain subjectivity; the specificity of an HPLC fingerprint spectrum method is low; the molecular biology method has high specificity, but is limited by the intact preservation degree of genetic materials in samples, and for the samples of the Syngnathus medicated liquor, the processed products, the extracts, the worm-eaten mildew and a plurality of individual mixed samples, the successful experiment can not be carried out due to the difficulty in extracting DNA. In recent years, more and more methods and standards have been used to identify or quantify natural drugs using characteristic peptide fragments. The protein contained in each species has difference of amino acid at individual site, and the difference site can be intercepted by using proper protease to be used as polypeptide segment with identification meaning, i.e. characteristic peptide segment. The detection of the characteristic peptide fragment generally uses a mass spectrum means, protein activity does not need to be considered, the preparation method is simple, and the property of the peptide fragment is relatively stable. The existing method for identifying the Syngnathus has more limiting factors, can not be applied to derivative products such as Syngnathus powder, syngnathus medicated liquor, processed products, extracts and the like, and the characteristic peptide segment in the Syngnathus protein has potential to be used as an index component for identifying the species of the Syngnathus. At present, each database does not contain information of related protein sequences of the asparagus sea, and differential peptide segments cannot be obtained through protein sequence comparison, so related records of characteristic polypeptides of the asparagus sea are not available.
Disclosure of Invention
The invention aims to provide two characteristic polypeptides of asparagus sea, application thereof and a method for identifying the asparagus sea. The characteristic polypeptide provided by the invention has excellent specificity and stability aiming at the asparagus sea dragon, has strong specificity, can be used for identifying the species of the sea dragon medicinal material, particularly the asparagus sea dragon, and has good application prospect.
The invention provides two characteristic polypeptides of asparagus polycephalus, and the amino acid sequences of the characteristic polypeptides are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention also provides application of the characteristic polypeptide in the technical scheme in identifying the asparagus officinalis.
Further, the application of SEQ ID NO.1 and SEQ ID NO.2 in identifying the asparagus officinalis separately or together.
Preferably, the identifying is performed using mass spectrometry, and detecting the ion pair comprises: the quantitative ion with the mass-to-charge ratio of m/z676.3 → 418.2 and the qualitative ion with the mass-to-charge ratio of m/z676.3 → 603.3 in SEQ ID NO. 1; the mass-to-charge ratio of the quantitative ion in SEQ ID NO.2 is m/z521.7 → 631.3, and the mass-to-charge ratio of the qualitative ion in m/z521.7 → 517.2.
The invention also provides an identification method of the asparagus polycephalus, which adopts the characteristic polypeptide shown in SEQ ID NO.1 and/or SEQ ID NO.2 as a reference substance.
The invention also provides a method for identifying the asparagus polyclone based on the characteristic polypeptide in the technical scheme, which comprises the following steps:
(1) Preparation of a test solution: mixing the pretreated pipefish extract or the pretreated pipefish medicinal liquor extract with trypsin respectively to obtain a mixed solution, mixing the mixed solution with an ammonium bicarbonate water solution, performing enzymolysis, filtering, and taking a filtrate to obtain a test sample solution;
(2) Preparation of control solutions: taking the characteristic peptide of the asparagus polycephalus as a reference substance, and adding water to dissolve the characteristic peptide to obtain a reference substance solution;
(3) Detection and analysis: detecting and analyzing by using a triple quadrupole mass spectrometry;
the steps (1) or (2) are not limited in chronological order.
Preferably, in the step (1), the preparation method of the pretreated pipefish extract comprises the following steps: pulverizing Syngnathus, mixing with water, decocting for three times (4 h,3h and 2 h), mixing decoctions, and diluting with ammonium bicarbonate water solution to obtain pretreated Syngnathus extract;
the preparation process of the pretreated Syngnathus medicinal liquor extract comprises the following steps: taking out Syngnathus in Syngnathus medicated liquor, drying at low temperature, decocting with water for three times (4 h,3h and 2h respectively), mixing decoctions, filtering, and collecting filtrate to obtain pretreated Syngnathus medicated liquor extract;
preferably, the trypsin is added in the form of an aqueous trypsin solution, and the mass concentration of the trypsin in the aqueous trypsin solution is 1mg/ml.
Preferably, the mass percentage content of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.
Preferably, the enzymolysis condition is enzymolysis at 37 ℃ for 2h.
Preferably, the triple quadrupole mass spectrometry is performed by taking 0.1% formic acid acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and performing gradient elution: 0 to 3min,5% by weight A → 8% by weight A; the flow rate was 0.5ml per minute.
Preferably, the triple quadrupole mass spectrometry adopts a mass spectrometry detector and an electrospray positive ion mode to carry out multi-reaction monitoring, wherein the SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m/z676.3 → 418.2, the qualitative ion with the mass-to-charge ratio of m/z676.3 → 603.3 is taken as a detection ion pair, the SEQ ID NO.2 selects a quantitative ion with the mass-to-charge ratio of m/z521.7 → 631.3, and the qualitative ion with the mass-to-charge ratio of m/z521.7 → 517.2 is taken as a detection ion pair.
The invention provides two polypeptide with the characteristics of asparagus. The characteristic polypeptide provided by the invention is used as an index component for identifying the asparagus sea, can be distinguished from sea of other species, and lays a foundation for developing an identification method of the asparagus sea and related products. Specifically, the characteristic polypeptide of the invention has the following beneficial effects:
(1) According to the method, the difference polypeptide segments in the protein sequence are intercepted, the mass spectrum is adopted for detection, the operation is simple and rapid, the detection result is stable, the interference of the shape and the processing process of a sea dragon sample is avoided, and the blank of the method for identifying the species of the asparagus sea dragon and detecting the asparagus sea dragon in the sea dragon product is filled;
(2) The characteristic polypeptide and the detection method provided by the invention provide reference for searching characteristic peptide fragments in closely related species lacking in a database;
(3) The characteristic polypeptide provided by the invention has excellent specificity and stability aiming at the asparagus fern, has strong specificity, can be used for identifying the asparagus fern medicinal material, and has good application prospect.
Drawings
FIGS. 1-9 are maps of the characteristic peptide SEQ ID NO.1 of the asparagus polycephalus provided by the invention;
wherein: FIG. 1: characteristic peptide control of SEQ ID NO. 1; FIG. 2 is a schematic diagram: a asparagus polycephalus sample; FIG. 3: a simulated Syngnathus sample; FIG. 4: a Shu's dragon sample; FIG. 5: a cunninghamia sample; FIG. 6: baojia gun kiss sea dragon sample; FIG. 7: a long thick oscorea sample; FIG. 8: a Geoscorea polystachya sample; FIG. 9: a sample of Geshi sea bracelet.
FIGS. 10-18 are maps of the characteristic peptide SEQ ID NO.2 of Cynanchum polycephalum provided by the present invention;
wherein: FIG. 10: characteristic peptide control of SEQ ID NO. 2; FIG. 11: a asparagus polycephalus sample; FIG. 12: a simulated Syngnathus sample; FIG. 13: a Schulelen sample; FIG. 14: a cunninghamia sample; FIG. 15 is a schematic view of: baojia gun kiss sea dragon sample; FIG. 16: a long thick oscillatoria sample; FIG. 17: a oscilla sample; FIG. 18: a sample of Geshi sea bracelet.
Detailed Description
The invention provides two characteristic polypeptides of asparagus polycephalus, wherein the amino acid sequence of the characteristic polypeptides is shown as SEQ ID NO.1: VGPAPGGAGPGPGPGGPVGKDGAR, shown in SEQ ID NO.2: GA- (HYP) -GLGGPTGSR. The invention provides a peptide segment with characteristic recognition significance for asparagus sea dragon. The method comprises the steps of carrying out trypsin enzymolysis on water extracts of different kinds of syngnathus such as cunninghamia paniculata, syngnathus, sulindachys syngnathus and the like, and analyzing by using a nano-liter liquid chromatography-high resolution mass spectrometer to obtain a synaptosomal spectrogram of a syngnathus protein polypeptide segment; carrying out chemometric analysis on the mass spectrum data, and then carrying out specificity verification on the experimental result by using a triple quadrupole mass spectrum; finally, 2 characteristic ion pairs are provided, corresponding to 2 characteristic peptide fragments, and the specificity verification result is good.
The invention also provides application of the characteristic polypeptide in the technical scheme in identifying the asparagus polycephalus.
In the present invention, the identifying is performed using mass spectrometry, and detecting the ion pair includes: the mass-to-charge ratio of the quantitative ion of SEQ ID NO.1 is m/z676.3 → 418.2, and the mass-to-charge ratio of the qualitative ion of m/z676.3 → 603.3; the mass-to-charge ratio of the quantitative ion of SEQ ID NO.2 is m/z521.7 → 631.3, and the mass-to-charge ratio of the qualitative ion of m/z521.7 → 517.2.
The invention also provides a method for identifying the asparagus polyclone based on the characteristic polypeptide in the technical scheme, which comprises the following steps:
(1) Mixing the pretreated pipefish extract or the pretreated pipefish medicinal liquor extract with trypsin to obtain a mixed solution, mixing the mixed solution with an ammonium bicarbonate water solution, performing enzymolysis, filtering, and taking a filtrate to obtain a test solution;
(2) Adding an ammonium bicarbonate aqueous solution to dissolve the characteristic peptide of the asparagus sea dragon serving as a reference substance to obtain a reference substance solution;
(3) Detecting and analyzing by using a triple quadrupole mass spectrometry;
the step (1) or the step (2) is not limited in chronological order.
According to the method, the difference polypeptide segments in the protein sequence are intercepted, the mass spectrum is adopted for detection, the operation is simple and rapid, the detection result is stable, the interference of the shape and the processing process of a Syngnathus sample is avoided, the blank of the method for identifying the species of the Cynanchum paniculatum and detecting the Cynanchum paniculatum in the Syngnathus product is filled, and reference is provided for searching the characteristic peptide segments in the kindred species lacking in a database.
The method comprises the steps of mixing a pretreated pipefish extracting solution or a pretreated pipefish medicinal liquor extracting solution with trypsin to obtain a mixed solution, mixing the mixed solution with an ammonium bicarbonate water solution, carrying out enzymolysis, filtering, and taking a filtrate to obtain a test solution. In the present invention, the preparation method of the pretreated Syngnathus extract preferably comprises the following steps: pulverizing Syngnathus, mixing with water, decocting for three times (4 h,3h and 2 h), mixing decoctions, and diluting with ammonium bicarbonate water solution to obtain pretreated Syngnathus extract. The invention preferably takes 100g of crushed sea dragon, and the total amount of the decoction is preferably 500mL. In the present invention, the dilution is preferably 10-fold. Specifically, the method preferably takes 5mL of the decoction, places the decoction in a 50mL measuring flask, uses an ammonium bicarbonate aqueous solution to dilute the decoction to a scale, and shakes the solution uniformly to obtain the pretreated Syngnathus extract. In the present invention, the preparation process of the pretreated Syngnathus medicated liquor extract preferably comprises the following steps: taking out Syngnathus in Syngnathus medicated liquor, drying at low temperature, decocting with water for three times (4 hr, 3 hr and 2 hr respectively), mixing decoctions, filtering, and collecting filtrate to obtain pretreated Syngnathus medicated liquor extract. After obtaining the pretreated pipefish extract and the pretreated pipefish medicinal liquor extract, the invention preferably measures 1-5 mL pipefish extract or pipefish medicinal liquor extract, preferably adds 100 μ L trypsin solution, shakes evenly, adds ammonium bicarbonate aqueous solution to 50mL, shakes evenly, carries out enzymolysis after sealing. In the present invention, the enzymolysis condition is preferably enzymolysis at 37 ℃ for 2h. After enzymolysis, the method is preferably cooled, more preferably cooled to normal temperature, and the temperature range of the normal temperature is preferably 10-30 ℃. After cooling, the invention filters and takes the filtrate as the test solution.
In the invention, the trypsin is added in the form of an aqueous trypsin solution, and the mass concentration of the trypsin in the aqueous trypsin solution is 1mg/ml. In the invention, the mass percentage of ammonium bicarbonate in the ammonium bicarbonate aqueous solution is 1%.
The invention takes the characteristic peptide of the asparagus as a reference substance, and water is added for dissolution to obtain a reference substance solution. The source of the characteristic peptide of the asparagus is not specially limited, and the characteristic peptide can be obtained by adopting an artificial synthesis method, for example, the 2 characteristic peptides of the asparagus are all obtained by entrusting Nanjing Source peptide biotechnology and technology limited company to synthesize according to a specified amino acid sequence.
The invention utilizes triple quadrupole mass spectrometry to carry out detection and analysis. In the present invention, the triple quadrupole mass spectrometry is preferably performed by gradient elution with 0.1% formic acid acetonitrile as mobile phase a and 0.1% formic acid solution as mobile phase B: 0 to 3min,5% by weight A → 8% by weight A; the flow rate was 0.5ml per minute. In the invention, the triple quadrupole mass spectrometry preferably adopts a mass spectrometry detector, an electrospray positive ion mode and multiple reaction monitoring, wherein the SEQ ID NO.1 selects a quantitative ion with a mass-to-charge ratio of m/z676.3 → 418.2 and a qualitative ion with a mass-to-charge ratio of m/z676.3 → 603.3; SEQ ID NO.2 selects the quantitative ion with mass/charge ratio m/z521.7 → 631.3, the qualitative ion with m/z521.7 → 517.2.
The two characteristic polypeptides of asparagus according to the present invention, the use thereof and the method for identifying asparagus according to the present invention will be described in further detail with reference to the following embodiments, which include but are not limited to the following embodiments.
Triple quadrupole mass spectrometry conditions
Gradient elution was carried out using 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B (0-3min, 5%. A → 8%. A); the flow rate was 0.5ml per minute. Performing Multiple Reaction Monitoring (MRM) by adopting a mass spectrum detector and an electrospray positive ion mode (ESI +), wherein the mass charge ratio of quantitative ions with m/z676.3 → 418.2 and the mass charge ratio of qualitative ions with m/z676.3 → 603.3 are selected from SEQ ID NO. 1; SEQ ID NO.2 selects the quantitative ion with mass/charge ratio m/z521.7 → 631.3, the qualitative ion with m/z521.7 → 517.2.
Example 1: characteristic peptide segment searching method for asparagus fern
(1) Preparation of test solution
Sea dragon water extract: taking 100g of a test sample of Syngnathus, crushing, putting into a triangular flask, adding water, decocting for three times, namely, 4h,3h and 2h, respectively, combining the decoctions, totally 500mL, slightly boiling, concentrating to obtain a viscous liquid, transferring into a silica gel bowl, and drying in an electric heating constant-temperature air drying oven at 60 ℃ until the solid is obtained to obtain a Syngnathus aqueous extract;
test solution: weighing 0.1g of Syngnathus aqueous extract sample, adding 50ml of 1% ammonium bicarbonate aqueous solution, performing ultrasonic treatment for 30min to dissolve, filtering with microporous membrane, collecting 100 μ L of filtrate, adding 10 μ L of 1mg/ml trypsin aqueous solution, performing enzymolysis at 37 deg.C for 2h, taking out, and cooling to room temperature.
(2) Selection of characteristic ions and sequence estimation
After the test solution is subjected to nanoliter liquid chromatography-high resolution mass spectrometry, mass spectrometry data is introduced into PEAKS 8.5 software, de novo sequencing and sequence prediction of all peptide fragments of a sample are carried out, and the obtained result is analyzed. Selecting peptide fragments which can be detected only in the asparagus sea dragon and are hardly detected in other species sea dragons as parent ions from the analysis results, analyzing secondary maps one by one, and selecting daughter ions with better response. Further analysis yielded mass spectral ion pair information m/z676.3 (double charge) → 418.2, 603.3 and m/z521.7 (double charge) → 631.3, 517.2, representing the variability of asparagus polysporus. Through further analysis, the sequence of the cunninghamia paniculata characteristic peptide fragment corresponding to the ion is presumed, and the sequence shown in SEQ ID NO.1: VGPAPGGAAGPAGGPGGPVGKDGAR, SEQ ID No.2: GA- (HYP) -GLGGPTGSR, wherein HYP is hydroxyproline.
And (3) entrusting and synthesizing a multi-asparagus sea dragon characteristic peptide reference substance according to the deduced amino acid sequence, simultaneously detecting the reference substance and the multi-asparagus sea dragon test solution, and ensuring that the retention time of the reference substance and the multi-asparagus sea dragon test solution are consistent with the second-level mass spectrum information so as to confirm the correctness of the sequence.
Example 2: research on specificity of characteristic peptide of asparagus
Detecting the aqueous extracts of all species of the sea dragon by adopting triple quadrupole mass spectrometry, wherein the result shows that the three batches of the sea dragon have characteristic peptide chromatographic peaks of the sea dragon, and no corresponding chromatographic peak appears in other Panasonic cunninghamia, pseudo-Heilonghamia, shu Panasonic cunninghamia, hedgeon piscichoria, changchinosaurs and Ku's sea bracelet. See fig. 1-9 and fig. 10-18. The characteristic peptide is specific to the asparagus fern and can be used as an index component for identifying the asparagus fern.
Example 3: application of characteristic peptide of asparagus polycephalus in syngnathus extract and syngnathus medicinal liquor
(1) Sample preparation
Preparing a test solution of a Syngnathus extract: taking 100g of a test sample Syngnathus, crushing, putting into a triangular flask, adding water, decocting for three times, wherein the decoction is respectively 4h,3h and 2h, and combining the decoctions to obtain 500mL; taking 5ml of the pipefish extract, placing the pipefish extract in a 50ml measuring flask, adding 1% ammonium bicarbonate water solution to dilute to a scale, shaking up, measuring 1ml to 5ml of the measuring flask, adding 100 mu l of 1mg/ml trypsin water solution, shaking up, adding 1% ammonium bicarbonate water solution to a constant volume to a scale, shaking up, sealing, carrying out enzymolysis at a constant temperature of 37 ℃ for 2 hours, cooling down, filtering, and taking a subsequent filtrate as a sample solution.
Preparing a test solution of the Syngnathus medicinal liquor: taking 10g of Syngnathus in the Syngnathus medicated liquor, drying at low temperature, decocting with water for three times, respectively for 4h,3h and 2h, and mixing the decoctions to obtain 50mL; filtration was carried out, and the filtrate was taken in 1ml to 5ml measuring bottles and prepared as above from "100. Mu.l of 1mg/ml trypsin aqueous solution was added".
Reference solution of characteristic peptide fragment of asparagus fern: respectively weighing 10mg of each of the reference substances SEQ ID NO.1 and SEQ ID NO.2 of the characteristic peptide of the asparagus, placing the reference substances in a 50ml measuring flask, adding water to dissolve the reference substances, and fixing the volume to a scale to obtain the asparagus polypeptide.
(2) The measurement was performed under triple quadrupole mass spectrometry conditions.
(3) Sample assay
And (3) measuring 10 collected samples of the extraction solution of the asparagus polycephalus powder and 8 collected samples of the medicinal liquor of the asparagus polycephalus, and detecting characteristic peptides of the asparagus polycephalus respectively according to results, wherein the two characteristic peptides can be used for identifying the components of the asparagus polycephalus in the syngnathus respectively.
Example 4: ion information with discrimination potential
5 pieces of ion information with identification potential are screened from 368 candidate ions, triple quadrupole mass spectrometry is performed, identification of the cynanchum paniculatum component can not be realized by any ions, specificity of part of ions to cynanchum paniculatum is insufficient, and only the ions with the mass-nucleus ratios of 676.3 and 521.7 are good in specificity, and specific results are shown in table 1.
TABLE 1 results of specificity verification
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (10)
1. A characteristic polypeptide of asparagus officinalis, characterized in that the amino acid sequence of the characteristic polypeptide is shown in SEQ ID No.1 and/or SEQ ID No. 2.
2. Use of a polypeptide according to the characteristics of claim 1 for identifying asparagus officinalis l.
3. Use according to claim 2, wherein said identification is carried out using mass spectrometry, and wherein the detection of ion pairs by SEQ ID No.1 comprises: quantitative ions with mass-to-charge ratio of m/z676.3 → 418.2, and qualitative ions with mass-to-charge ratio of m/z676.3 → 603.3; the detection ion pair of SEQ ID NO.2 comprises: the mass-to-charge ratio of the quantitative ions is m/z521.7 → 631.3, and the mass-to-charge ratio of the qualitative ions is m/z521.7 → 517.2.
4. A method of identifying asparagus polycephalus, characterized in that the characteristic polypeptide of claim 1 is used as a reference.
5. The authentication method according to claim 4, comprising the steps of:
(1) Preparation of a test solution: mixing the pretreated pipefish extract or the pretreated pipefish medicinal liquor extract with trypsin respectively to obtain a mixed solution, mixing the mixed solution with an ammonium bicarbonate water solution, performing enzymolysis, filtering, and taking a filtrate to obtain a test sample solution;
(2) Preparation of control solutions: respectively dissolving the multi-asparagus sea dragon characteristic peptides of claim 1 in water to obtain reference substance solutions;
(3) Detection and analysis: detecting and analyzing by using a triple quadrupole mass spectrometry;
the step (1) or the step (2) is not limited in chronological order.
6. The method according to claim 5, wherein in the step (1), the preparation method of the pretreated Syngnathus extract comprises the following steps: pulverizing Syngnathus, mixing with water, decocting for three times (4 h,3h and 2 h), mixing decoctions, and diluting with ammonium bicarbonate water solution to obtain pretreated Syngnathus extract;
the preparation process of the pretreated pipefish medicinal liquor extracting solution comprises the following steps: taking out Syngnathus in Syngnathus medicated liquor, drying at low temperature, decocting with water for three times (4 hr, 3 hr and 2 hr respectively), mixing decoctions, filtering, and collecting filtrate to obtain pretreated Syngnathus medicated liquor extract.
7. The method according to claim 5, wherein the trypsin is added in the form of an aqueous trypsin solution, wherein the mass concentration of the trypsin in the aqueous trypsin solution is 1mg/ml.
8. The method of claim 5, wherein the aqueous ammonium bicarbonate solution comprises 1% ammonium bicarbonate by weight; the enzymolysis condition is enzymolysis for 2 hours at 37 ℃.
9. The method of claim 5, wherein the triple quadrupole mass spectrometry is performed with a gradient elution with 0.1% formic acid acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B: 0 to 3min,5% by weight A → 8% by weight A; the flow rate was 0.5ml per minute.
10. The method of claim 5, wherein the triple quadrupole mass spectrometry is monitored by multiple reactions using a mass spectrometer in electrospray positive ion mode, and wherein SEQ ID No.1 selects a quantitative ion with mass to charge ratio m/z676.3 → 418.2, a qualitative ion with mass to charge ratio m/z676.3 → 603.3 as the detecting ion pair, and SEQ ID No.2 selects a quantitative ion with mass to charge ratio m/z521.7 → 631.3, and a qualitative ion with mass to charge ratio m/z521.7 → 517.2 as the detecting ion pair.
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