CN107132360A - Active peptides high-throughput screening method based on tandem mass spectrum and molecular docking - Google Patents
Active peptides high-throughput screening method based on tandem mass spectrum and molecular docking Download PDFInfo
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Abstract
The present invention relates to field of medicaments, disclose a kind of based on active peptides high-throughput screening method associated with tandem mass spectrum and molecular docking, the present invention is using modern biochemical technology means, from ocean software class, extraction prepares mixed polypeptide in the animal bodies such as fish, active site/components group is determined by Preliminary activation separation, using the amino acid sequence of whole polypeptides in LC MS/MS methods identified activities position/components group, in molecular docking software, whole peptide sequences is subjected to Blast search, molecular docking software is then imported to be docked with target protein, analyze the Percentage bound of polypeptide and target protein, to evaluate the activity of its suppression/excitement, screen the active peptides determined and external activity rating is combined by synthesis in solid state, verify the accuracy of screening.The present invention has the advantages that quick, efficient searching Marine Bioactive Peptides, workable, with important application value.
Description
Technical field
The present invention relates to high flux screening field, and in particular to one kind is based on activity associated with tandem mass spectrum and molecular docking
Polypeptide high-throughput screening method, is a kind of with mixed polypeptide sequence in tandem mass spectrum identified activity position/components group, utilizes molecule
Docking screening active peptides simultaneously verify its active method.
Background technology
Active peptides refer to be made up of with amido link 2~20 amino acid residues, the material with certain physiologically active, lead to
Normal molecular weight is not higher than 2000Da.Modern study shows that active peptides have a variety of physiologically actives, and the activity of marine source is more
Peptide has abundance, and the features such as activity is notable is received significant attention in recent years.The bioactive peptide such as found from marine organisms
Material, such as Dolastatin-10, conotoxin, fish-skin Antihypertensive Peptides, show important market value and are imitated with social economy
Benefit, more and more scientists and Research Team turn to sight the searching and exploitation of active peptides in marine organisms.
System under the main guiding with activity of research of traditional Marine Bioactive Peptides is isolated and purified, based on Structural Identification,
Though this method is classical, there is time-consuming, laborious, the low shortcoming of flux.Active peptides in marine organisms are because species is abundant, structure
The low feature of special, content, still lacks effective separation, analysis, screening, identification solution, it is difficult to high flux at present
Completion active material screening and discovery.
Therefore, it is necessary on the basis of prior art, research and develop it is a kind of quick, efficiently find marine active
The method of polypeptide, the advantage that integrated LC-MS/MS Rapid identifications mixed polypeptide of the invention and computer high frequency zone optimize can be from
Fast searching active peptides in marine organisms (or enzymolysis product).
The content of the invention
Goal of the invention：The problem to be solved in the present invention is：Set up a kind of ocean based on tandem mass spectrum and molecular docking
Active peptides high-throughput screening method, this method can be not only used for the primary/secondary metabolite that itself is present in marine organisms
The discovery and screening of polypeptide, the also discovery and screening available for active peptides in the mixed polypeptide after cellulase treatment, this
Outside, this method is not limited to a kind of screening of the active peptides of action target spot, and the activity that can be applied to a variety of known target spots is more
The discovery and screening of peptide.
Technical scheme, to realize object above, the technical solution adopted by the present invention is：
Active peptides high-throughput screening method based on tandem mass spectrum and molecular docking, it is characterised in that including following step
Suddenly：
(1) logical to ocean clam class first to be extracted or biology enzyme enzymolysis, then separation prepares total polypeptide extract,
Then screening active ingredients are carried out in the case where activity is oriented to, determines active site；
(2) mass spectral analysis and then by LC-MS/MS to active site is carried out, by obtained mass spectrometric data by from the beginning surveying
Sequence is analyzed (de novo sequencing) and/or compared with Protein Data Bank, the quick amino for determining protein or polypeptide
Acid sequence；
(3) and then by computer virtual screen, drug target and active material are subjected to molecular docking, so as to seek
The best conformation for looking for acceptor to be acted on part, filters out the part optimal with receptor affinity, completes the activity of high-flux polypeptide
Screening, and pass through its activity of solid-phase synthetic peptide sequence verification.
The above-described active peptides high-throughput screening method based on tandem mass spectrum and molecular docking, described separation side
Method includes fractional precipitation, molecular exclusion, ion exchange, reversed phase column chromatography, hydrophobic chromatography etc..
Preparation method as preferred ocean clam class extract has：
Clam software is cleaned into silt, added water to cook, separation decoction liquor and meat slag are drained；Meat slag is taken, after the homogenate that adds water,
Biology enzyme enzymolysis is added, after enzymolysis terminates, in boiling water bath inactivation, is centrifuged, concentration obtains clam zymolyte.
Or clam software is cleaned into silt, add 3~8 times of 60~80% ethanol of amount and decoct 1~3 time, every time 30~60
Minute, merge extract solution, after concentration, freeze-drying obtains freeze-dried powder.
Clam extract is separated, different parts/components group is obtained, and evaluate different parts/components group that separation is obtained
Activity, determine active site/components group, concentrate, freeze-drying, obtain freeze-dried powder standby.
Wherein biology enzyme includes：Pepsin, trypsase, papain, neutral proteinase, alkali protease, wind
Taste protease etc..
The above-described active peptides high-throughput screening method based on tandem mass spectrum and molecular docking, described activity sieve
The target spot of choosing includes：Angiotensin converting enzyme (ACE), dipeptidyl peptidase-IV (DPP-IV), fibrin ferment (thrombin), second
Acetylcholinesterase (AChE) etc..
Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe activity rating method：
ACE and HHL solution preparation：Appropriate ACE is taken with 0.1mol/L borate buffers containing 0.3mol/LNaCl (pH8.3)
It is made into the ACE solution that concentration is 100mU/mL；Appropriate HHL is taken (to contain 0.3mol/LNaCl with 0.1mol/L borate buffers
PH8.3) it is made into the HHL solution that concentration is 5mmol/L；
Course of reaction：After 30 μ LHHL and 10 μ L samples (or cushioning liquid) are mixed, in preheating 5min under 37 DEG C of environment
(37 DEG C of water-baths), the ACE for adding 20 μ L starts reaction, is continued at after mixing and 1h (37 DEG C of water-baths) is reacted under 37 DEG C of environment, so
After be rapidly added 70 μ LHCl (1mol/L) terminating reactions, use efficient liquid phase chromatographic analysis result.Sample is replaced with borate buffer
Solution does blank control.
Chromatographic condition：SunFire-C18 posts (2.1 × 100mm, 3.5 μm, Waters, US)；Mobile phase：Acetonitrile-
0.5% aqueous formic acid, flow velocity：0.5mL/min；Double UV check：220nm、254nm；Column temperature：30℃；Sample size：10μL.
Condition of gradient elution：0~5min, 2% acetonitrile；5~25min, 2%~40% acetonitrile；25~30min, 40% acetonitrile.
Dipeptidyl peptidase-4 (DPP-IV) inhibitory activity evaluation method：
Using the activity of colorimetric method for determining dipeptidyl peptidase-4 peptide for inhibiting, comprise the following steps that：Example 1 and embodiment
3 peptide for inhibiting prepared respectively be dissolved in Tris-HCI buffer solutions (contain 20mmol/LTris, pH 8.0,0.1mol/LNaCl,
1mmol/L EDTA) corresponding sample liquid is made.Dipeptidyl peptidase-4 (DPP-4), Gly-Pro-PNA are slow with Tris-HCl respectively
Fliud flushing (Tris containing 20mmol/L, pH 8.0,0.1mol/L NaCl, 1mmol/L EDTA) is configured to 8U/L DPP-4 solution
With 1.6mmol/L Gly-Pro-PNA (substrate) solution.25 μ L samples and 25 μ L substrates are mixed, 37 DEG C of incubation 10min, then
50 μ L DPP-4 solution are added, are incubated 1 hour at 37 DEG C, it is rear to add 1mol/L sodium-acetate buffers (PH 4.0) 100 μ L,
Absorbance (A) is determined at 405nm, and calculates inhibiting rate.Sample is replaced to do blank control with Tris-HCI buffer solutions simultaneously.
DPP-4 inhibiting rates (%)=[(A negative control-A blank controls)-(A sample-A sample blanks)]/(A feminine genders are right
According to-A blank controls) × 100%
In formula：A is absorbance.
Thrombin-inhibiting activity evaluation method：
Common Carotid of-Rabbits take blood, and 3000rpm centrifugations 10min prepares test plasma.Test plasma is added in cup is determined
The 0.1mol/LpH 7.4Tris-HCl buffer solutions dilution of common pre-temperature certain time is added after 40 μ l, 37 DEG C of pre-temperature 3min
The μ l of the 15U/ml thrombin solutions 40 and μ l of solvent 20.Start clotting factor analyzer record while thrombin solution is added solidifying
Blood time (TT0).TT rate elongations (%)=(TT-TT0)/TT0× 100%.With the fibrin ferment of various concentrations, the clotting time is measured
TT.Calculate TT rate elongations (%).Concentration of thrombin (C) is mapped with lgTT rate elongations (%), C is obtained to lgTT rate elongations (%)
Influence standard curve.
After test plasma 40 μ l, 37 DEG C of pre-temperature 3min is added in determining cup, common pre-temperature 3min testing sample is added
The μ l of 20 μ l and 15U/ml fibrin ferment 40, record clotting time (TT), calculate lgTT rate elongations (%), according to standard curve, calculate
Blood coagulation enzyme activity, is compared with solvent.
Wherein separation method includes the methods such as ethanol precipitation, molecular exclusion, ion exchange, reversed phase column chromatography
Ethanol precipitation：Absolute ethyl alcohol is added into ocean clam class enzymolysis liquid, makes ethanol final concentration of 30% (v/v), 4
DEG C stand after 24h, centrifugation, lower sediment is alcohol precipitation position 1, after supernatant concentration, adds absolute ethyl alcohol, makes ethanol final concentration of
80% (v/v), 4 DEG C stand after 24h, centrifugation, and lower sediment is alcohol precipitation position 2, and supernatant is alcohol precipitation position 3.
Molecular exclusion chromatography：Ocean clam class enzymolysis liquid is splined on to the Sephadex gel chromatographies handled well in advance in batches
Post, purifies water elution, and flow velocity is 0.15ml/min, and auto partial sampler is collected, and 1 is collected per 2ml and is managed, ultraviolet specrophotometer
Often pipe 254nm absorbances are determined, elution curve is drawn, each absworption peak, which is collected, to be merged, and separation obtains different parts, cold after concentration
Lyophilized dry, -20 DEG C of preservations of dried frozen aquatic products are stand-by.Wherein Sephadex gel chromatographic columnses include Sephadex G-25, G-50, G-75
And G-100.
Ion-exchange chromatography：By ocean clam class zymolyte freeze-dried powder buffer solution, (pH of buffer scope is 6.0-
8.5), after loading, unadsorbed part is eluted into post with the buffer solution of about 5 times of column volumes.Then 0-0.5M NaCl buffer solutions rank
Section elution or gradient elution, flow velocity are 1ml/min, and auto partial sampler is collected, and 1 is collected per 3ml and is managed, ultraviolet specrophotometer
Often pipe 254nm absorbances are determined, elution curve is drawn, each absworption peak, which is collected, to be merged, and separation obtains different parts, cold after concentration
It is lyophilized dry, obtain dried frozen aquatic products.Wherein chromatograph packing material includes DEAE Sepharose, CM Sepharose, Q Sepharose etc..
Reversed phase column chromatography method：Using preparation HPLC system, binary gradient pump, UV, visible light spectrophotometric detector；
Chromatographic column selects anti-phase C18, T3, HILIC etc..Mobile phase A：0.1%TFA, Mobile phase B：Methanol；Elution requirement is：2%B, 0-
1.5min, 2%-60%B, flow velocity are 15ml/min.Applied sample amount is 1ml.Chromatographic peak is collected, separation obtains different parts, concentration
After be freeze-dried, -20 DEG C of preservations of dried frozen aquatic products are stand-by.
Active peptides Sequence Identification in active site of the present invention, using C18After solid phase extraction column desalination, enter LC-MS/MS
Analysis, the parent ion for selectively selecting ion band electric charge >=2 carries out being collisionally dissociated acquisition secondary fragment information (MS/MS), passes through
Software carries out de novo sequencing analyses, and conjugated protein database compares analysis and (is loaded under Protein Data Bank
Www.uniprot.org), the amino acid sequence of all polypeptides in active site is determined.
Active site is analyzed using nano LC-MS/MS, wears U3000NanoRSLC nanoliters of liquid phase systems of peace, chromatogram
Post is 5 μm of Reprosil C18AQ (75 μ m 150mm), LC-MS/MS network analyses cornu bubali difference sample.Applied sample amount is 5 μ
L, flow velocity 400nL/min, mobile phase A (acetonitrile/formic acid/water=2/0.2/98, v/v/v), Mobile phase B (acetonitrile/formic acid/water=
80/0.2/20, v/v/v), 2~30%B linear gradient elutions 60min.Thermo LTQ Orbitrap XL mass spectrographs be used for into
Row peptide piecewise analysis, spray voltage is 2.5kV, and ion transfer capillary temperature is 200 DEG C；Mass spectrum one-level full scan scope is m/
Z100~2000, separation width is 3Da；Tandem Mass Spectrometry Analysis uses the second order mses scan pattern of first mass spectrometric data dependence,
5 ions of first mass spectrometric ionic strength highest are chosen successively carries out collision induced dissociation (CID) two-stage tandem mass spectrum.Using
Xcalibur softwares carry out data analysis.Tandem mass spectrum data searches storehouse with the softwares of PEAKS 8.0, selects suitable database, inspection
Rope parameter is set to：Precursor ion error 10ppm；Daughter ion error 1Da；Cysteine residues fix modification (carbamoylmethyl
Change 57.0215Da)；The variable modification (oxidation+15.9949Da) of methionine residues；2 sites are allowed to cut by mistake, false positive rate
(FDR)≤1%；It is different according to cornu bubali Aqueous extracts sample, select different digestion modes：Non- digestion (No enzyme) or pancreas egg
White enzyme digestion (Trypsin)；Other specification is default parameters, and institute's score value has significant (P under above-mentioned search condition<
0.05) it is identified as effective qualification result.
So that it is determined that in active site/components group whole polypeptides amino acid sequence information.
As preferred：All active peptides sequences are carried out Blast search by the present invention, by all polypeptides (part) and target
Point protein (acceptor) is imported in molecular docking software, and setup parameter carries out molecular docking with CDOKER, determines part and acceptor
Combination situation and incorporating parametric, screening determines the part that can be combined with acceptor, the clearly peptide sequence with lateral reactivity；Enter
One step verifies the polypeptide active that molecular docking screening is determined by synthesis in solid state.
As preferred：Part construction method of the present invention is：
Blast search is carried out according to the amino acid sequence information of polypeptide：Use the softwares of Chem BioDraw Ultra 13.0
Each polypeptide structure is drawn, is then introduced into Discovery Studio 4.0 (DS 4.0) software, reuses CHARMm
Energy field optimizes and minimizes its energy, and part is prepared using the PREPAR LIGAND modules in DS 4.0.
(the http from the Protein Data Bank database://www.rcsb.org/pdb/) download protein
Crystal structure, as the receptor model of computer molecular docking, use the CDOCKER modules in software DS 4.0 to carry out molecule
Docking research, parameter is default setting.
The part that can be combined with acceptor is obtained by screening, the amino acid sequence of part is determined, using synthesis in solid state skill
Art synthesis polypeptide, its bioactivity is verified using external activity evaluation method.
Beneficial effect：Compared to the prior art, the present invention has the following advantages：
The present invention is screened by many experiments, researchs and develops a kind of quick, efficiently searching Marine Bioactive Peptides method,
The advantage that the integrated LC-MS/MS Rapid identifications mixed polypeptide of the present invention optimizes with computer high frequency zone, can from marine organisms (or
Enzymolysis product) in fast searching active peptides, it is workable, with important application value.
Brief description of the drawings
Fig. 1 is peptide fragment His-Asp-Gln-Leu-Pro-Gly-Tyr of the present invention de novo sequencing (de novo
Sequencing) result figure.
Fig. 2 is peptide fragment Thr-Pro-Glu-Lys-Glu-Asp-Glu-Leu-Arg de novo sequencing results
Figure.
Fig. 3 is peptide fragment Ala-Asp-Leu-Asp-Trp-Leu-Asp-Gly-Arg de novo sequencing results
Figure.
Embodiment
With reference to specific embodiment, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention, after the present invention has been read, various equivalences of the those skilled in the art to the present invention
The modification of form falls within the application appended claims limited range.
Embodiment 1
Dipeptidyl peptidase-IV (DPP-IV) inhibitor is screened in mottle clam：
1. the preparation of mottle clam zymolyte：
Mottle clam software is cleaned into silt, 3 times of amount decoctings is added and boils 2 times, 40 minutes every time, separate decoction liquor and meat slag,
Drain；Take after meat slag, plus 3 times of amount water homogenate, add the papain enzymolysis that enzymatic activity is 50000U/g, papain
It is the 1% of meat slag weight to add weight, and the temperature of enzymolysis is 55 DEG C, and the pH of enzymolysis is 7, and the enzyme digestion reaction time is 4h；Enzymolysis knot
Shu Hou, in boiling water bath inactivation, then adds a certain amount of absolute ethyl alcohol and carries out classification alcohol precipitation, final to prepare 30% ethanol precipitation portion
Position (alcohol precipitation position 1), 70% alcohol precipitation precipitation position (alcohol precipitation position 2) and alcohol precipitation supernatant (alcohol precipitation position 3), each position is through dense
Contracting, freeze-drying, obtains freeze-dried powder.
2.DPP-IV inhibitory activity is evaluated
Alcohol precipitation position 1,2,3 is dissolved in Tris-HCI buffer solutions (Tris containing 20mmol/L, pH 8.0,0.1mol/
LNaCl, 1mmol/L EDTA) corresponding sample liquid is made.Dipeptidyl peptidase-4 (DPP-4), Gly-Pro-PNA are used respectively
Tris-HCl buffer solutions (Tris containing 20mmol/L, pH 8.0,0.1mol/LNaCl, 1mmol/L EDTA) are configured to 8U/L's
DPP-4 solution and 1.6mmol/L Gly-Pro-PNA (substrate) solution.25 μ L samples and 25 μ L substrates are mixed, 37 DEG C of incubations
10min, adds 50 μ L DPP-4 solution, is incubated 1 hour at 37 DEG C, rear to add 1mol/L sodium-acetate buffers (PH 4.0)
Absorbance (A) is determined at 100 μ L, 405nm, and calculates inhibiting rate.Sample is replaced to do blank with Tris-HCI buffer solutions simultaneously
Control.
DPP-4 inhibiting rates (%)=[(A negative control-A blank controls)-(A sample-A sample blanks)]/(A feminine genders are right
According to-A blank controls) × 100%
In formula：A is absorbance.
It the results are shown in Table, the DPP-IV inhibitory activity at alcohol precipitation position 3 is optimal.
The DPP-IV inhibitory activity at the different alcohol precipitation positions of table 1 compares
3. the LC MS/MS analyses of active site 3
Active site 3 obtains MS/MS ms fragment information through mass spectral analysis, is carried out using non-digestion (No enzyme) pattern
Database compares analysis, and analysis is divided into therefrom and identifies 124 polypeptid acid sequences.With wherein peptide fragment His-Asp-Gln-
Exemplified by Leu-Pro-Gly-Tyr, MS/MS collection of illustrative plates is shown in Fig. 1, and molecular weight (MW) is 828.38Da, and patch information includes b1-b6, y1-
Y6, this peptide fragment is de novo sequencing analysis results, does not search derived protein in protein library.
The DPP-IV inhibitory activity molecular Docking Studies of 4.124 polypeptid acid sequences
By the homologous modeling of 124 polypeptid acid sequences, PREPARE LIGAND, DPP-IV target spots are carried out in DS 4.0
By http://www.rcsb.org/pdb/explore/explore.doStructureId=2P8S is downloaded, in DS 4.0
Progress goes water, hydrogenation to carry out PREPARE PROTEIN again, and part and DPP-IV target spots then are carried out into CDOCKER docks, parameter
Set as follows：Top Hits-10；RandomConformations-10；Orientations to Refine-10；Force
field-CHARMm；Use Full Potential-False, other specification selection default setting.
After docking, determine that 5 peptide sequences can well be docked with DPP-IV, it is potential DPP-IV to show it
Inhibitory activity polypeptide, 5 peptide sequences are shown in Table 2.
5 polypeptides are obtained by solid phase synthesis technique, purity is equal>98%, external 5 polypeptides of DPP-IV activity ratings, knot
Fruit is shown in Table 2.
The DPP-IV inhibitory activity of 25 polypeptides of table compares
Shown by the result of upper table 2, DPP-IV inhibitory activity polypeptides quickly are determined from mottle clam zymolyte.
Embodiment 2
Angiotensin converting enzyme (ACE) peptide for inhibiting is screened in clam extract：
1st, the preparation of clam extract
Clam software is cleaned into silt, 3 times of 60% ethanol of amount is added and decocts 2 times, 30 minutes every time, merging was extracted twice
Liquid, after concentration, freeze-drying obtains freeze-dried powder.
2nd, clam extract reversed phase column chromatography is separated
Clam extract freeze-dried powder is redissolved, using Waters preparation HPLCs system (2545-2489HPLC systems), point
From clam extract, using C18 chromatographic columns (5 μm, 19 × 150mm), binary gradient pump, Detection wavelength is 220nm；Mobile phase A：
0.1%TFA, Mobile phase B：Methanol；Elution requirement is：2%B, 0-3min, 2%-35%B, 3-9min, flow velocity are 10ml/min.
Applied sample amount is 500 μ l.Chromatographic peak is collected, separation obtains 5 different parts, is freeze-dried after concentration, and -20 DEG C of preservations of dried frozen aquatic products are treated
With.
3rd, ACE inhibitory activity evaluation is carried out to 5 positions
ACE and HHL solution preparation：Appropriate ACE is taken with 0.1mol/L borate buffers containing 0.3mol/LNaCl (pH8.3)
It is made into the ACE solution that concentration is 100mU/mL；Appropriate HHL is taken (to contain 0.3mol/LNaCl with 0.1mol/L borate buffers
PH8.3) it is made into the HHL solution that concentration is 5mmol/L；
Course of reaction：After 30 μ LHHL and 10 μ L samples (or cushioning liquid) are mixed, in preheating 5min under 37 DEG C of environment
(37 DEG C of water-baths), the ACE for adding 20 μ L starts reaction, is continued at after mixing and 1h (37 DEG C of water-baths) is reacted under 37 DEG C of environment, so
After be rapidly added 70 μ LHCl (1mol/L) terminating reactions, use efficient liquid phase chromatographic analysis result.Sample is replaced with borate buffer
Solution does blank control.
Chromatographic condition：SunFire-C18 posts (2.1 × 100mm, 3.5 μm, Waters, US)；Mobile phase：Acetonitrile-
0.5% aqueous formic acid, flow velocity：0.5mL/min；Double UV check：220nm、254nm；Column temperature：30℃；Sample size：10μL.
Condition of gradient elution：0~5min, 2% acetonitrile；5~25min, 2%~40% acetonitrile；25~30min, 40% acetonitrile.
ACE inhibitory activity evaluation result is shown in Table 3, and the wherein activity of position 3 is preferable.
The ACE inhibitory activity at 5 positions of clam extract of table 3 compares
The ACE inhibitory activity at wherein position 3 is optimal.
4th, the LC MS/MS analyses at ACE inhibitory activity position 3
Active site 3 obtains MS/MS ms fragment information through mass spectral analysis, is carried out using non-digestion (No enzyme) pattern
Database compares analysis, and analysis is divided into therefrom and identifies 131 polypeptid acid sequences.With wherein peptide fragment Thr-Pro-Glu-
Exemplified by Lys-Glu-Asp-Glu-Leu-Arg, MS/MS collection of illustrative plates is shown in Fig. 2, and molecular weight (MW) is 1115.55Da, and patch information includes
B2-b8, y1-y8, this peptide fragment are de novo sequencing analysis results, do not search derived Protein in protein library
Matter.
The ACE inhibitory activity molecular Docking Study of 5.131 polypeptid acid sequences
By the homologous modeling of 131 polypeptid acid sequences, in DS 4.0 carry out PREPARE LIGAND, ACE target spots by
http://www.rcsb.org/pdb/explore/explore.doStructureId=1O86 is downloaded, and is entered in DS 4.0
Row goes water, hydrogenation to carry out PREPARE PROTEIN again, and part and ACE target spots then are carried out into CDOCKER docks, parameter setting
It is as follows：Top Hits-10；RandomConformations-10；Orientations to Refine-10；Force
field-CHARMm；Use Full Potential-False, other specification selection default setting.
After docking, determine that 6 peptide sequences can well be docked with ACE, show that it suppresses to live for potential ACE
Property polypeptide, 6 peptide sequences are shown in Table 4.
6 polypeptides are obtained by solid phase synthesis technique, purity is equal>98%, as a result external 6 polypeptides of ACE activity ratings are shown in
Table 4.
The ACE inhibitory activity of 46 polypeptides of table compares
ACE inhibitory activity polypeptide quickly is determined from clam extract by upper table 4.
The screening and discovery of polypeptide with thrombin inhibitor activity in the blood clam of embodiment 3
1. the preparation of blood clam zymolyte：
Blood clam software is cleaned into silt, 3 times of amount decoctings is added and boils 2 times, 40 minutes every time, separate decoction liquor and meat slag, drip
It is dry；Take meat slag, plus after 3 times of amount water homogenate, add the neutral protease enzymolysis that enzymatic activity is 10000U/g, neutral proteinase plus
It is the 2% of meat slag weight to enter weight, and the temperature of enzymolysis is 45 DEG C, and the pH of enzymolysis is 7, and the enzyme digestion reaction time is 4h；Enzymolysis terminates
Afterwards, inactivated in boiling water bath, freeze-drying obtains freeze-dried powder.
2. blood clam zymolyte molecular exclusion is separated
Blood clam zymolyte freeze-dried powder is redissolved, supernatant is splined on Sephadex G-25 gel columns, purified water in batches after centrifugation
Elution, flow velocity is 0.15ml/min, and auto partial sampler is collected, and 1 is collected per 2ml and is managed, ultraviolet specrophotometer is determined and often managed
254nm absorbances, draw elution curve, and each absworption peak, which is collected, to be merged, and obtain G1, G3, G3 and G4 positions, after each position concentration
Freeze-drying, -20 DEG C of preservations of dried frozen aquatic products are stand-by.
3. pair 4 positions carry out thrombin-inhibiting activity evaluation
Common Carotid of-Rabbits take blood, and 3000rpm centrifugations 10min prepares test plasma.Test plasma is added in cup is determined
The 0.1mol/LpH 7.4Tris-HCl buffer solutions dilution of common pre-temperature certain time is added after 40 μ l, 37 DEG C of pre-temperature 3min
The μ l of the 15U/ml thrombin solutions 40 and μ l of solvent 20.Start clotting factor analyzer record while thrombin solution is added solidifying
Blood time (TT0).TT rate elongations (%)=(TT-TT0)/TT0× 100%.With the fibrin ferment of various concentrations, the clotting time is measured
TT.Calculate TT rate elongations (%).Concentration of thrombin (C) is mapped with lgTT rate elongations (%), C is obtained to lgTT rate elongations (%)
Influence standard curve.
After test plasma 40 μ l, 37 DEG C of pre-temperature 3min is added in determining cup, common pre-temperature 3min testing sample is added
The μ l of 20 μ l and 15U/ml fibrin ferment 40, record clotting time (TT), calculate lgTT rate elongations (%), according to standard curve, calculate
Blood coagulation enzyme activity, is compared with solvent.
Thrombin-inhibiting activity evaluation result is shown in Table 5, and wherein G3 positions activity is preferable.
The thrombin-inhibiting activity at 4 positions of blood clam zymolyte of table 5 compares
The LC MS/MS analyses at 4.G3 positions
Active site G3 obtains MS/MS ms fragment information through mass spectral analysis, is carried out using non-digestion (No enzyme) pattern
Database compares analysis, and analysis is divided into therefrom and identifies 87 polypeptid acid sequences.With wherein peptide fragment Ala-Asp-Leu-
Exemplified by Asp-Trp-Leu-Asp-Gly-Arg, MS/MS collection of illustrative plates is shown in Fig. 3, and molecular weight (MW) is 1059.50Da, and patch information includes
B2-b8, y1-y8, this peptide fragment are de novo sequencing analysis results, do not search derived Protein in protein library
Matter.
The thrombin-inhibiting activity molecular Docking Study of 5.87 polypeptid acid sequences
By the homologous modeling of 87 polypeptid acid sequences, PREPARE LIGAND, fibrin ferment target spot are carried out in DS 4.0
Byhttp://www.rcsb.org/pdb/explore/explore.doStructureId=3C1KDownload, in DS 4.0
Progress goes water, hydrogenation to carry out PREPARE PROTEIN again, and part and fibrin ferment target spot then are carried out into CDOCKER docks, parameter
Set as follows：Top Hits-10；RandomConformations-10；Orientations to Refine-10；Force
field-CHARMm；Use Full Potential-False, other specification selection default setting.
After docking, determine that 3 peptide sequences can well be docked with fibrin ferment, it is potential fibrin ferment to show it
Inhibitory activity polypeptide, 3 peptide sequences are shown in Table 6.
3 polypeptides are obtained by solid phase synthesis technique, purity is equal>98%, external thrombin activity evaluates 3 polypeptides, knot
Fruit is shown in Table 6.
The ACE inhibitory activity of 63 polypeptides of table compares
Shown by the result of upper table 6, thrombin-inhibiting activity polypeptide quickly is determined from blood clam zymolyte.
SEQUENCE LISTING
<110>Nanjing University of Traditional Chinese Medicine
<120>Active peptides high-throughput screening method based on tandem mass spectrum and molecular docking<130>
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 5
<212> PRT
<213>Artificial sequence
<400> 1
His Asp Gln Leu Pro Gly Tyr
1 5
<210> 2
<211> 7
<212> PRT
<213>Artificial sequence
<400> 2
Ala Leu Glu Asp Glu Ala Arg
1 5 7
<210> 3
<211> 7
<212> PRT
<213>Artificial sequence
<400> 3
His Leu Lys Asp Gly Val Met
1 5 7
<210> 4
<211> 8
<212> PRT
<213>Artificial sequence
<400> 4
Phe Ala Gly Asp Asp Ala Pro Arg
1 5 8
<210> 5
<211> 6
<212> PRT
<213>Artificial sequence
<400> 5
Phe Leu Met Glu Ser His
1 5 6
<210> 6
<211> 9
<212> PRT
<213>Artificial sequence
<400> 6
Thr Pro Glu Lys Glu Asp Glu Leu Arg
1 5 9
<210> 7
<211> 8
<212> PRT
<213>Artificial sequence
<400> 7
Thr Glu Glu Tyr Glu Asp Leu Arg
1 5 8
<210> 8
<211> 7
<212> PRT
<213>Artificial sequence
<400> 8
Trp Tyr Glu Glu Lys Met Glu
1 5 7
<210> 9
<211> 11
<212> PRT
<213>Artificial sequence
<400> 9
Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys
1 5 10 11
<210> 10
<211> 9
<212> PRT
<213>Artificial sequence
<400> 10
Asn Trp Asp Asp Met Glu Lys Ile Trp
1 5 9
<210> 11
<211> 9
<212> PRT
<213>Artificial sequence
<400> 11
Glu Phe Asp Asp Glu Trp Lys Ser Met
1 5 9
<210> 12
<211> 9
<212> PRT
<213>Artificial sequence
<400> 12
Ala Asp Leu Asp Trp Leu Asp Gly Arg
1 5 9
<210> 13
<211> 10
<212> PRT
<213>Artificial sequence
<400> 13
Glu Leu Arg Gln Glu Gln Glu Asn Tyr Lys
1 5 10
<210> 14
<211> 9
<212> PRT
<213>Artificial sequence
<400> 14
Glu Phe Asp Asp Glu Trp Lys Ser Met
1 5 9
Claims (6)
1. the active peptides high-throughput screening method based on tandem mass spectrum and molecular docking, it is characterised in that comprise the following steps：
(1) ocean clam class is extracted first or biology enzyme enzymolysis, then separation prepares total polypeptide extract, Ran Hou
Activity is oriented to lower progress screening active ingredients, determines active site；
(2) mass spectral analysis and then by LC-MS/MS to active site is carried out, obtained mass spectrometric data is passed through into de novo sequencing point
Analyse and/or compared with Protein Data Bank, the quick amino acid sequence for determining protein or polypeptide；
(3) and then by computer virtual screen, drug target and active material be subjected to molecular docking, thus find by
The best conformation that body is acted on part, filters out the part optimal with receptor affinity, completes the screening active ingredients of high-flux polypeptide,
And pass through its activity of solid-phase synthetic peptide sequence verification.
2. the active peptides high-throughput screening method according to claim 1 based on tandem mass spectrum and molecular docking, it is special
Levy and be, described separation method includes fractional precipitation, molecular exclusion, ion exchange, reversed phase column chromatography, hydrophobic chromatography.
3. the active peptides high-throughput screening method according to claim 1 based on tandem mass spectrum and molecular docking, it is special
Levy and be, the target spot of described screening active ingredients includes：Angiotensin converting enzyme, dipeptidyl peptidase-IV, fibrin ferment or acetyl courage
Alkali esterase.
4. the active peptides high-throughput screening method according to claim 1 based on tandem mass spectrum and molecular docking, it is special
Levy and be, described active site LC-MS/MS analysis methods include：Pacify U3000NanoRSLC nanoliters of liquid phase systems using wearing,
Chromatographic column is 5 μm of Reprosil C18AQ, and applied sample amount is 5 μ L, and flow velocity 400nL/min, mobile phase A is that volume ratio is 2:0.2:
98 acetonitrile-formic acid-water, Mobile phase B is acetonitrile-formic acid-water of volume ratio 80/0.2/20,2~30%B linear gradient elutions
60~90min；
Thermo LTQ Orbitrap XL mass spectrographs are used to carry out peptide piecewise analysis, and spray voltage is 2.5kV, ion transmission capillary
Pipe temperature is 200 DEG C；Mass spectrum one-level full scan scope is m/z 100~2000, and separation width is 3Da；Tandem Mass Spectrometry Analysis is adopted
With the second order mses scan pattern of first mass spectrometric data dependence, first mass spectrometric ionic strength 5 ions of highest are chosen successively
Collision induced dissociation two-stage tandem mass spectrum is carried out, and data analysis, tandem mass spectrum data selection are carried out using Xcalibur softwares
Suitable database, search argument is set to：Precursor ion error 10ppm；Daughter ion error 1Da；Cysteine residues are fixed
Modify (carbamoylmethyl 57.0215Da)；The variable modification (oxidation+15.9949Da) of methionine residues；Allow 2 sites
Cut by mistake, false positive rate≤1%；Then different digestion modes are selected：Non- digestion (No enzyme) or tryptic digestion；It is other
Parameter is default parameters, and institute's score value has significant under above-mentioned search condition, regards as effective qualification result, so that
Determine the amino acid sequence information of whole polypeptides in active site.
5. the active peptides high-throughput screening method according to claim 1 based on tandem mass spectrum and molecular docking, it is special
Levy and be, step (3) specific method is：
Blast search is carried out according to the amino acid sequence information of polypeptide：Use the Software on Drawing of Chem BioDraw Ultra 13.0
Each polypeptide structure, is then introduced into the softwares of Discovery Studio 4.0, reuses the optimization of CHARMm energy fields simultaneously
Its energy is minimized, part is prepared using the PREPAR LIGAND modules in DS 4.0.
The crystal structure of protein is downloaded from the Protein Data Bank database, computer molecular docking is used as
Receptor model, carry out molecular Docking Study using the CDOCKER modules in software Discovery Studio 4.0, parameter is
Default setting；The part that can be combined with acceptor is obtained by screening, the amino acid sequence of part is determined, using synthesis in solid state skill
Art synthesis polypeptide, its bioactivity is verified using external activity evaluation method.
6. the side based on dipeptidyl peptidase-iv inhibitor active peptides in tandem mass spectrum and molecular docking high flux screening mottle clam
Method, it is characterised in that comprise the following steps：
(1) preparation of mottle clam zymolyte：
Mottle clam software is cleaned into silt, 3 times of amount decoctings is added and boils 2 times, 40 minutes every time, separate decoction liquor and meat slag, drain；
Take after meat slag, plus 3 times of amount water homogenate, add the papain enzymolysis that enzymatic activity is 50000U/g, the addition of papain
Weight is the 1% of meat slag weight, and the temperature of enzymolysis is 55 DEG C, and the pH of enzymolysis is 7, and the enzyme digestion reaction time is 4h；After enzymolysis terminates,
In boiling water bath inactivation, then add a certain amount of absolute ethyl alcohol and carry out classification alcohol precipitation, it is final prepare 30% ethanol precipitation position 1,
The 70% heavy shallow lake position 2 of ethanol and the heavy supernatant position 3 of ethanol, each position are concentrated, and freeze-drying obtains freeze-dried powder.
(2) dipeptidyl peptidase-IV inhibitory activity is evaluated
Ethanol precipitation position 1, the heavy shallow lake position 2 of ethanol and ethanol alcohol precipitation supernatant position 3 are dissolved in Tris-HCI buffer solution systems
Into corresponding sample liquid；Dipeptidyl peptidase-4, Gly-Pro-PNA are configured to 8U/L DPP-4 with Tris-HCl buffer solutions respectively
Solution and 1.6mmol/L Gly-Pro-PNA (substrate) solution；
25 μ L samples and 25 μ L substrates are mixed, 37 DEG C of incubation 10min add 50 μ LDPP-4 solution, and it is small to be incubated 1 at 37 DEG C
When, rear add at 1mol/L sodium-acetate buffers 100 μ L, 405nm determines absorbance A, and calculates inhibiting rate；Use simultaneously
Tris-HCI buffer solutions are cooked blank control instead of sample；
Dipeptidyl peptidase-IV inhibiting rate (%)=[(A negative control-A blank controls)-(A sample-A sample blanks)]/(A is negative
Control-A blank controls) × 100%
(3) the LC MS/MS analyses of active site
Active site obtains MS/MS ms fragment information through mass spectral analysis, and carrying out database using non-enzyme cleavage patterns compares analysis, from
Wherein it is divided into analysis and identifies polypeptid acid sequence；
(4) the dipeptidyl peptidase-IV inhibitory activity molecular Docking Study of polypeptid acid sequence
By the homologous modeling of polypeptid acid sequence, PREPARE LIGAND are carried out in Discovery Studio 4.0,
Carry out going water, hydrogenation to carry out PREPARE PROTEIN again in Discovery Studio 4.0, then by part and dipeptidyl peptidase
Enzyme-IV target spots carry out CDOCKER docking, and parameter setting is as follows：Top Hits-10；RandomConformations-10；
Orientations to Refine-10；Force field-CHARMm；Use Full Potential-False, other ginsengs
Number selection default setting；
After docking, it is determined that the peptide sequence that can be docked very well with dipeptidyl peptidase-IV, it is potential two peptidyl to show it
Peptase-IV inhibitory activity polypeptides；Then polypeptide is obtained by solid phase synthesis technique, external dipeptidyl peptidase-IV activity rating is tested
Card.
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| CN110183512A (en) * | 2019-05-13 | 2019-08-30 | 大连工业大学 | A kind of preparation method of Patinopecten yessoensis dipeptides, its virtual screening method and its plural gel |
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| CN109187835A (en) * | 2018-09-17 | 2019-01-11 | 南京中医药大学 | A kind of discrimination method of the specificity peptide fragment containing protein-based Chinese medicine |
| CN109187835B (en) * | 2018-09-17 | 2021-03-23 | 南京中医药大学 | Method for identifying specific peptide fragment of protein-containing traditional Chinese medicine |
| CN109678928A (en) * | 2018-12-27 | 2019-04-26 | 渤海大学 | A kind of tripeptides with 1 inhibitory activity of cholinesterase and beta amyloid precursor protein cleavage enzyme |
| CN110156871A (en) * | 2019-05-13 | 2019-08-23 | 大连工业大学 | A kind of preparation method of Patinopecten yessoensis oligopeptides, its virtual screening method and its plural gel |
| CN110183512A (en) * | 2019-05-13 | 2019-08-30 | 大连工业大学 | A kind of preparation method of Patinopecten yessoensis dipeptides, its virtual screening method and its plural gel |
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| CN113252814A (en) * | 2021-05-25 | 2021-08-13 | 上海应用技术大学 | Method for identifying bitter peptides of soybean protein hydrolysate |
| CN114163516A (en) * | 2021-12-06 | 2022-03-11 | 中国水产科学研究院南海水产研究所 | Collagen source tyrosinase inhibitory peptide and preparation method and application thereof |
| CN114163516B (en) * | 2021-12-06 | 2022-12-30 | 中国水产科学研究院南海水产研究所 | Collagen source tyrosinase inhibitory peptide and preparation method and application thereof |
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