CN112679580B - Sipunculus nudus oligopeptide capable of promoting bone development and preparation method and application thereof - Google Patents
Sipunculus nudus oligopeptide capable of promoting bone development and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses Sipunculus nudus oligopeptide capable of promoting bone development and a preparation method and application thereof, belonging to the technical field of biology. The sipunculus nudus is subjected to enzymolysis through pancreatin, after an enzymolysis product is subjected to centrifugal separation, a filtrate with the concentration of less than 2.5K is separated through a 2.5K ultrafiltration membrane, and the enzymolysis short peptide is obtained through freeze drying; and (3) carrying out reductive alkylation pretreatment and trypsin enzymolysis on the enzymolysis short peptide, and separating by using a liquid chromatography-mass spectrometry combined technology to obtain the sipunculus nudus oligopeptide capable of promoting bone development. The Sipunculus nudus oligopeptide composition for promoting bone development is simple in preparation method, is derived from Sipunculus nudus, is a natural and harmless biological polypeptide, can remarkably promote bone development and bone formation, and is expected to be applied to preparation of medicines for promoting bone development or treating osteoporosis.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to sipunculus nudus oligopeptide capable of promoting bone development and a preparation method and application thereof.
Background
Osteoporosis is a common skeletal disease characterized by decreased bone density and mass, resulting in decreased bone strength and increased risk of fracture. Epidemiological data show that about 8 million people in the united states suffer from osteoporosis, 2 million people have reduced bone mass, and 150 million patients suffer from osteoporosis causing fractures every year. The incidence rate of osteoporosis of white women after menopause is 17 percent, and the incidence rate of osteoporosis of black women is 6 percent. Approximately 50% of women and 25% of men over the age of 50 have the potential risk of osteoporotic fractures. China is a country with the largest absolute number of old people, and the 'white paper book for preventing and treating osteoporosis' shows that about 6944 thousands of people (1534 thousands of men and 5410 thousands of women) suffer from osteoporosis and 2.1 hundred million people suffer from low bone mass in people over 50 years old nationwide according to the national Chinese population sampling survey result in 2006, and the risk of osteoporosis is estimated to increase to 2.8 hundred million by 2020 in China.
Osteoporosis is a result of imbalance between bone resorption and bone formation in a human body, and both inhibition of bone resorption and promotion of bone formation can treat osteoporosis and promote bone formation by osteoblasts, and a drug for treating osteoporosis is called a bone formation promoting drug. The current drugs for promoting bone formation mainly include hormones, fluorides, statins, and the like. However, existing drugs often require chemical synthesis, are complex to produce, and are prone to side effects. With the continuous development of biotechnology, the biological peptide drug is paid attention to many researchers as an emerging research field, but nowadays, a truly feasible bone growth promoting biological peptide drug is still lacking. Therefore, a natural bone growth promoting polypeptide is urgently needed to be found, and a foundation is laid for further developing a natural bone growth promoting medicament which is convenient to produce and has no side effect.
Disclosure of Invention
The invention aims to provide sipunculus nudus oligopeptide capable of promoting bone development and a preparation method and application thereof, so as to solve the problems in the prior art, provide a natural biological peptide capable of promoting bone growth, and lay a foundation for further developing medicines for promoting bone formation and treating osteoporosis.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides Sipunculus nudus oligopeptide capable of promoting bone development, wherein the Sipunculus nudus oligopeptide is polypeptide with an amino acid sequence shown as SEQ ID NO.1 or polypeptide with an amino acid sequence shown as SEQ ID NO. 2.
The invention also provides a Sipunculus nudus oligopeptide composition for promoting bone development, which consists of the polypeptide shown in SEQ ID NO.1 and the polypeptide shown in SEQ ID NO.2 in amino acid sequence.
The invention also provides a preparation method of the Sipunculus nudus oligopeptide capable of promoting bone development or the Sipunculus nudus oligopeptide composition capable of promoting bone development, which comprises the following steps:
1) Preparing enzymolysis short peptide: carrying out enzymolysis on fresh Sipunculus nudus by using pancreatin, centrifuging an enzymolysis product, separating filtrate with a 2.5K ultrafiltration membrane to obtain filtrate with the pressure of below 2.5K, and freeze-drying to obtain enzymolysis short peptide;
2) Separation of Sipunculus nudus oligopeptide promoting bone development: and (3) carrying out reductive alkylation pretreatment and trypsin enzymolysis on the enzymolysis short peptide, and separating by using a liquid chromatography-mass spectrometry combined technology to obtain the sipunculus nudus oligopeptide capable of promoting bone development.
Preferably, the specific steps of the pancreatin enzymolysis of the sipunculus nudus in the step 1) are as follows: removing viscera of fresh Sipunculus nudus, mincing completely, adjusting pH to 7.5, adding pancreatin with a weight fraction of 0.5% of Sipunculus nudus, homogenizing completely, stirring continuously in 45 deg.C constant temperature water bath, performing enzymolysis for 5 hr, and inactivating enzyme in boiling water bath for 5min.
Preferably, the centrifugal separation parameter in the step 1) is 8000r/min and 20min.
Preferably, the specific steps of the reductive alkylation pretreatment in the step 2) are as follows: by ddH 2 O dissolving the short peptide, taking 10 mu g of short peptide and adding ddH 2 O to 100 mu L, adding Dithiothreitol (DTT) solution to make the final concentration to be 10mmol/L, and reducing in a water bath at 56 ℃ for 1h; adding Iodoacetamide (IAA) solution to a final concentration of 50mmol/L, reacting in dark for 40min, desalting with C18 desalting column, and volatilizing solvent in vacuum centrifugal concentrator at 45 deg.C.
Preferably, the specific steps of trypsin enzymolysis in step 2) are as follows: by ddH 2 Dissolving the short peptide by O, taking a 10 mu g protein sample, and adding 50mmol/L NH 4 HCO 3 The solution was brought to 100. Mu.L, DTT solution was added to give a final concentration of 10mmol/LReducing in 56 ℃ water bath for 1h; adding IAA solution to make the final concentration 50mmol/L, and reacting for 40min in dark; adding trypsin according to the mass ratio of the trypsin to the substrate of 1; desalting the peptide fragment after enzyme digestion by using a C18 desalting column, and volatilizing the solvent in a vacuum centrifugal concentrator at 45 ℃.
Preferably, the liquid chromatography conditions in step 2) are: analytical column C18, 150 μm i.d.. Times.150mm, 1.9 μm,
mobile phase A0.1% formic acid, 2% ACN; and (3) mobile phase B:0.1% formic acid, 80% acn; flow rate: 600nL/min; gradient elution conditions: 0-8min,6% by volume B;8-24min,9% by weight B;24-60,14% by weight B;60-75min,30% by weight B;75-78min,40% by weight B;78min,95% B.
Preferably, the mass spectrum conditions in step 2) are as follows:
1) Primary mass spectrum parameters: resolution setting: 70,000@ m/z; MS1 AGC:3e 6 (ii) a Maximum IT:40ms; ion scanning range: 300-1600m/z;
2) Secondary mass spectrum parameters: and (3) setting the resolution ratio: 17,500; MS2 AGC:1e 5 ；Maximum IT：60ms；Top N：20；NCE/stepped NCE：27。
The invention also provides application of the Sipunculus nudus oligopeptide capable of promoting bone development or the Sipunculus nudus oligopeptide composition capable of promoting bone development in preparation of a medicine for promoting bone development.
The invention also provides application of the Sipunculus nudus oligopeptide capable of promoting bone development or the Sipunculus nudus oligopeptide composition capable of promoting bone development in preparation of a medicine for treating osteoporosis.
The invention discloses the following technical effects:
the sipunculus nudus oligopeptide composition capable of promoting bone development provided by the invention can obviously promote bone development and is expected to be applied to preparation of medicines for treating osteoporosis and promoting bone development.
The Sipunculus nudus oligopeptide composition for promoting bone development provided by the invention is derived from Sipunculus nudus, belongs to a natural biological peptide medicine, has no side effect, has no adverse effect on human bodies, and is beneficial to clinical popularization and application.
The Sipunculus nudus oligopeptide composition provided by the invention is simple and convenient in preparation method, low in production cost and suitable for forming an industrial chain.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a chromatogram of total ion current in Sipunculus nudus short peptide analyzed by LC-MS/MS;
FIG. 2 is an amino acid analysis diagram of Sipunculus nudus extract polypeptide GSP 1;
FIG. 3 is an amino acid analysis diagram of Sipunculus nudus extract polypeptide GSP 2;
FIG. 4 is a graph of results of staining with calcein fluorescent dye of zebrafish treated with GSP 1; in the figure, the A picture is a blank control group; the B diagram is a GSP1 group with the concentration of 1 mug/ml, the C diagram is a GSP1 group with the concentration of 5 mug/ml, and the D diagram is a GSP1 group with the concentration of 50 mug/ml; ABCD is 10 times of the scope field under confocal field;
FIG. 5 is a graph of statistical analysis of calcein fluorescent dye from Zebra fish treated with GSP 1;
FIG. 6 is a graph of results of staining with calcein fluorescent dye of zebrafish treated with GSP 2; in the figure, the A picture is a blank control group; the B diagram is a GSP2 group with the concentration of 1 mug/ml, the C diagram is a GSP2 group with the concentration of 5 mug/ml, and the D diagram is a GSP2 group with the concentration of 50 mug/ml; ABCD is 10 times of the scope field under confocal field;
fig. 7 is a graph of calcein fluorescent dye statistical analysis of zebrafish treated with GSP2.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The materials and reagents used in the present invention are commercially available unless otherwise specified.
Example 1
1. Materials and methods
1.1 materials and reagents
Sipunculus nudus (purchased from Tofeng wholesale market, zhanjiang), calcein (Sigma-Aldrich), 4% paraformaldehyde (Beijing Lei Gen Biotechnology, inc.), 75% ethanol (Shandong rilkang medical science and technology, inc.), tricaine (fluka), carboxymethylcellulose (Beijing Customs Biotechnology, inc.), trypsin (Promega), formic acid (Sigma-Aldrich), acetonitrile (Fisher Chemical) dithiothreitol (Sigma-Aldrich), iodoacetamide (Sigma-Aldrich), trypsin (Promega).
1.2 instruments and devices
A low-temperature high-speed centrifuge, a freeze dryer (Beijing Bo Yi kang laboratory instruments Co., ltd.), a turntable super-resolution confocal microscope (Olympus), an electric heating constant-temperature water tank (Shanghai-Hengxi laboratory instruments Co., ltd.), a low-temperature high-speed centrifuge (Hunan Hexi laboratory instruments Co., ltd.), and a biochemical incubator (Henan brother laboratory instruments Co., ltd.).
1.3 Experimental methods
1.3.1 extraction of the polypeptide
Removing viscera of fresh Sipunculus nudus, washing body wall with distilled water, draining, fully mincing the Sipunculus nudus, adjusting pH to 7.5, adding pure water according to a liquid-material ratio of 1.
1.3.2 Sipunculus nudus enzymolysis short peptide LC-MS/MS analysis and identification
1.3.2.1 reductive alkylation pretreatment
By ddH 2 O dissolving the short peptide, taking 10 mu g of short peptide and adding ddH 2 O to 100 mu L, adding Dithiothreitol (DTT) solution to make the final concentration to be 10mmol/L, and reducing in a water bath at 56 ℃ for 1h; adding Iodoacetamide (IAA) solution to make its final concentration be 50mmol/L, reacting for 40min in dark, desalting with C18 desalting column, and volatilizing solvent in vacuum centrifugal concentrator at 45 deg.C.
1.3.2.2 Trypsin (Trypsin) enzymolysis
By ddH 2 Dissolving the short peptide by O, taking a 10 mu g protein sample, and adding 50mmol/L NH 4 HCO 3 The solution was brought to 100. Mu.L, DTT solution was added to a final concentration of 10mmol/L, and the reaction solution was reduced in a 56 ℃ water bath for 1 hour. Adding IAA solution to make the final concentration 50mmol/L, and reacting for 40min in dark; adding trypsin according to the mass ratio of the trypsin to the substrate of 1; desalting the peptide fragment after enzyme digestion by using a C18 desalting column, and volatilizing the solvent in a vacuum centrifugal concentrator at 45 ℃.
1.3.2.3 capillary liquid chromatography conditions
Pre-column C18 (300. Mu.m i.d.. Times.5mm, 5. Mu.m,
) (ii) a Analytical column C18 (150. Mu. M i.d.. Times.150mm, 1.9. Mu.m,
) (ii) a Mobile phase A0.1% formic acid, 2% ACN; and (3) mobile phase B:0.1% formic acid, 80% acn; flow rate: 600nL/min; gradient elution conditions: 0-8min,6% by volume B;8-24min,9% by weight B;24-60,14% by weight B;60-75min,30% by weight B;75-78min,40% by weight B;78min,95% B.
1.3.2.4 Mass Spectrometry conditions
(1) Primary mass spectrum parameters: resolution setting: 70,000@ m/z; MS1 AGC:3e 6 (ii) a Maximum IT:40ms; ion scanning range: 300-1600m/z.
(2) Secondary mass spectrum parameters: resolution setting: 17,500; MS2 AGC:1e 5 ；Maximum IT：60ms；Top N：20；NCE/stepped NCE：27。
1.3.2.5 Mass Spectrometry data analysis and Activity screening
The mass spectrum original files respectively search the target protein databases by using Maxquant (1.6.2.10); the probability of PeptideRanker activity (http:// distilldeep. Ucd. Ie/PeptideRanker /); the cell penetration probability of PeptideRanker (http:// distilldeep. Ucd. Ie/CPPpred /) was used; water solubility assessment using Innovagen website (http:// www.innovagen.com/proteomics-tools); inputting amino acid sequence, analyzing by default parameters
1.3.3 Zebra fish rearing
With ABWild type zebra fish is used as a material, and embryos of the AB line zebra fish are obtained from natural mating and spawning of the wild type AB line zebra fish. Embryos and larvae of AB line were incubated in embryonic water (5 mmol/L NaCl, 0.17mmol/L KCl, 0.4mmol/L CaCl) under standard conditions 2 、0.16mmol/L MgSO 4 And 10ppm methylene blue) embryos were fed in water. The temperature of the breeding water is (28 +/-0.5) DEG C, the pH value is 7-7.2, and the dark/light cycle is 10h/14h.
1.3.4 administration and methods
1.3.5 calcein staining
Bone mineralization matrix deposition is an important indicator of bone formation and is assessed using calcein staining. Calcein is a living dye and is an effective fluorescent dye for detecting the development of the bones of zebra fish. Soaking zebra fish embryo in 0.2% calcein solution for 15min, and washing with embryo water for 10min for 3 times. After staining, the cells were anesthetized with tricaine, fixed with carboxymethyl cellulose, and fluorescence images were collected in confocal. Imaging is then performed on the confocal set. The fluorescence image excitation wavelength is 488nm, the confocal set is the same exposure time, exposure intensity and the same fixed conversion parameter. All images were acquired under the same conditions. The fluorescence image is converted into a grayscale image and the fluorescence intensity is converted into a grayscale value. The gray scale range is set to include all the gray scale localizations, and the area and density of the gray scale localization are calculated.
1.3.6 statistical analysis
Images were analyzed using Image-Pro Plus version 6.0 and calcein staining area and integrated optical density were quantitatively analyzed. 20 zebrafish larvae were analyzed per group.
2. Results and analysis
2.1 Sipunculus nudus enzymolysis short peptide LC-MS/MS analysis and identification result
The sipunculus nudus enzymolysis short peptide is subjected to analysis and detection after being pretreated, a total ion current chromatogram is shown in figure 1, and the total ion current chromatogram is shown in figure 1, so that in the whole chromatographic gradient, numerous chromatographic peaks are distributed uniformly, and the chromatographic separation effect is shown to be high.
2.2 active peptide screening based on an Online database
From table 1, peptides with a length of 9 amino acids can be selected: FPSLVGRP, GFAGDDAPR, GLGGLSPEK, LTEAPLNPK, PAVKPAP, SILSPSPR, TEAPLNPK, VAPEHPV, VVTTPKSG; the online database is used for analyzing the activity probability, the cell penetration probability and the water solubility of the 10 identified peptides, and 8 peptides (SEQ ID NO.1-SEQ ID NO. 8) with good water solubility can be screened from the table 2 based on the requirements of subsequent experiments on the water solubility of the peptides: GFAGDDAPR, GLGGLSPEK, LTEAPLNPK, PAVKKPAP, SILSPSPR, TEAPLNPK, VAPEEHPV, VVTTPKSG. By integrating the activity probability and the cell penetration probability, GFAGDDAPR, GLGGLSPEK has potential development and application values.
The polypeptide with the purity of more than 95 percent is synthesized by Beijing Baitai park biotechnology limited, the amino acid sequences of which are GFAGDDAPR, GLGGLSPEK and are respectively named as GSP1 and GSP2. The molecular structure of GSP1 is C 38 H 56 N 12 O 14 (FIG. 2), the amino acid sequence is GFAGDDAPR, the purity is more than 97%, and the molecular mass is 905.04Da; the molecular structure of GSP2 is C 37 H 64 N 10 O 13 (FIG. 3), the amino acid sequence is GLGGLSPEK, the purity is more than 97%, and the molecular mass is 857.06Da.
TABLE 1 LC-MS/MS relative quantification result of Sipunculus nudus enzymolysis oligopeptide
TABLE 2 Sipunculus nudus enzymolysis oligopeptide screening results
2.3 GSP1 and GSP2 for promoting bone development of zebra fish
Compared with the blank control group, the skull integrated optical density and the green fluorescence area of the AB zebra fish juvenile fish treated by the GSP1 solution concentration of 1-50 mu g/ml are increased (figure 4), and the effect of promoting bone development is best when the zebra fish concentration is 5 mu g/ml (figure 5). These results indicate that GSP1 can cause an increase in the mineralized matrix in zebrafish larvae.
Compared with a blank control group, the area of skull integrated optical density and green fluorescence of AB zebra fish juvenile fish treated by the GSP2 solution concentration of 1-50 mug/ml is increased (figure 6), and the effect of promoting bone development is best when the concentration of the zebra fish is 50 mug/ml (figure 7) along with the increase of the concentration. These results indicate that GSP2 can cause an increase in the mineralized matrix in zebrafish larvae.
The result shows that when the concentration of the sipunculus nudus oligopeptide GSP1 is 5 mu g/ml, the IOD of the zebra fish skull has a very significant difference with the value of the dyeing area, and when the concentration of the GSP2 is 50 mu g/ml, the IOD of the zebra fish skull has the maximum difference with the value of the dyeing area.
The change of the source of the Sipunculus nudus raw material, the production process and other conditions, the molecular weight of the Sipunculus nudus oligopeptide obtained by production and the structure of the obtained peptide fragment are different, thereby directly influencing the physicochemical property, the activity and the industrialized application of the enzymolysis oligopeptide. On the basis of a large amount of preliminary studies, the invention explores and summarizes a set of enzymolysis process and production flow, intercepts 2.5K fragment sample solution and obtains sample solution with optimal activityThe production process of sipunculus nudus oligopeptide. In order to better clarify the active peptide segment and the efficacy of the sipunculus nudus oligopeptide, the invention adopts LC-MS/MS to analyze and identify the sipunculus nudus oligopeptide, and 10 oligopeptides are identified in total; performing physical and biological activity analysis on the oligopeptide according to an analysis database, and screening out two oligopeptides GFAGDDAPR, GLGGLSPEK with excellent water solubility and activity, wherein the molecular structures of the two oligopeptides are respectively C 38 H 56 N 12 O 14 ，C 37 H 64 N 10 O 13 The molecular masses are 905.04Da and 857.06Da, respectively. In order to further understand the activities of the two oligopeptides, relevant analysis for efficiently evaluating bone development and component content of zebra fish is introduced on the basis.
Zebra fish is used as an excellent model animal to efficiently identify drug screening, overcomes the defects of low tracking efficiency, high cost and the like of the traditional animal experiment, and is widely applied to the fields of research inheritance, development, toxicology and the like. The zebra fish bone development evaluation is carried out in a 24-pore plate, the experimental period is 6 days, and the drug administration mode of drug soaking is simple and easy to operate. Under the CF488 laser, the calcein staining zebra fish bones are clearly visible, and according to the fluorescence intensity and the integral optical density, the GSP1 has the most obvious effect of promoting the development of the zebra fish bones when the concentration is 5 mu g/ml, and the GSP2 has the most obvious effect of promoting the development of the zebra fish bones when the concentration is 50 mu g/ml. The results show that the oligopeptide GSP1 and the oligopeptide GSP2 obviously promote the bone development of the zebra fish and lay a foundation for a zebra fish bone development model.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> southern China laboratory of Guangdong province in ocean science and engineering (Zhanjiang)
<120> Sipunculus nudus oligopeptide capable of promoting bone development and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Phe Ala Gly Asp Asp Ala Pro Arg
1 5
<210> 2
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Leu Gly Gly Leu Ser Pro Glu Lys
1 5
Claims (4)
1. The Sipunculus nudus oligopeptide composition for promoting bone development is characterized by consisting of a polypeptide shown as SEQ ID No.1 and a polypeptide shown as SEQ ID No.2 in amino acid sequence.
2. A method of preparing the osteogenesis-promoting sipunculus nudus oligopeptide composition of claim 1, comprising the steps of:
1) Preparing enzymolysis short peptide: carrying out enzymolysis on fresh Sipunculus nudus by using pancreatin, centrifuging an enzymolysis product, separating filtrate with a 2.5K ultrafiltration membrane to obtain filtrate with the pressure of below 2.5K, and freeze-drying to obtain enzymolysis short peptide;
2) Separation of Sipunculus nudus oligopeptide promoting bone development: after reductive alkylation pretreatment and trypsin enzymolysis are carried out on the enzymolysis short peptide, a Sipunculus nudus oligopeptide composition for promoting bone development is obtained by utilizing a liquid chromatography-mass spectrometry combined technology for separation;
the specific steps of the pancreatin enzymolysis of the sipunculus nudus in the step 1) are as follows: removing viscera of fresh Sipunculus nudus, mincing completely, adjusting pH to 7.5, adding pancreatin with a mass fraction of 0.5% of Sipunculus nudus, homogenizing completely, stirring continuously in 45 deg.C constant temperature water bath, performing enzymolysis for 5 hr, and inactivating enzyme in boiling water bath for 5min;
the reduction alkylation pretreatment in the step 2) comprises the following specific steps: by ddH 2 O dissolving the short peptide, taking 10 mu g of short peptide and adding ddH 2 O to 100 mu L, adding Dithiothreitol (DTT) solution to make the final concentration to be 10mmol/L, and reducing in a water bath at 56 ℃ for 1h; adding Iodoacetamide (IAA) solution to a final concentration of 50mmol/L, reacting in dark for 40min, desalting with C18 desalting column, and volatilizing solvent in 45 deg.C vacuum centrifugal concentrator;
the specific steps of the trypsin enzymolysis in the step 2) are as follows: by ddH 2 Dissolving the short peptide by O, taking a 10 mu g protein sample, and adding 50mmol/L NH 4 HCO 3 Adding DTT solution to 100 mu L, reducing in 56 ℃ water bath for 1h to make the final concentration of the solution be 10 mmol/L; adding IAA solution to make the final concentration 50mmol/L, and reacting for 40min in dark; adding trypsin according to the mass ratio of the trypsin to the substrate of 1; desalting the peptide fragment after enzyme digestion by using a C18 desalting column, and volatilizing the solvent in a vacuum centrifugal concentrator at 45 ℃;
the liquid chromatography conditions in the step 2) are as follows: c18, 150 μm.d. X150mm, 1.9 μm, 100A; mobile phase A0.1% formic acid, 2% ACN; mobile phase B:0.1% formic acid, 80% acn; flow rate: 600nL/min; gradient elution conditions: 0-8min,6% by volume B;8-24min,9% by weight B;24-60,14% by weight B;60-75min,30% by weight B;75-78min,40% by weight B;78min,95% by weight B;
the mass spectrum conditions in the step 2) are as follows:
1) Primary mass spectrum parameters: resolution setting: 70,000@ m/z; MS1 AGC:3e 6 (ii) a Maximum IT:40ms; ion scanning range: 300-1600m/z;
2) Secondary mass spectrum parameters: resolution setting: 17,500; MS2 AGC:1e 5 ；Maximum IT：60ms；Top N：20；NCE/stepped NCE：27。
3. Use of the skeletogenic sipunculus nudus oligopeptide composition of claim 1 in the preparation of a medicament for promoting bone development.
4. Use of the skeletogenic sipunculus nudus oligopeptide composition of claim 1 in the preparation of a medicament for treating osteoporosis.
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