CN108659099A - A kind of MS probe and its application for hypertensin conversion enzyme activity detection - Google Patents
A kind of MS probe and its application for hypertensin conversion enzyme activity detection Download PDFInfo
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- CN108659099A CN108659099A CN201810488931.9A CN201810488931A CN108659099A CN 108659099 A CN108659099 A CN 108659099A CN 201810488931 A CN201810488931 A CN 201810488931A CN 108659099 A CN108659099 A CN 108659099A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The invention discloses a kind of MS probe for hypertensin conversion enzyme activity detection and its applications, belong to drug screening and assessment technique field.The MS probe, including polypeptide that amino acid sequence is Asp Ser Asp Lys Pro and modify piperazine compounds on the aspartic acid of the polypeptide N-terminal.MS probe provided by the invention by the polypeptide A sp Ser Asp Lys Pro of the angiotensin converting enzyme specific recognition small molecule piperazine compounds responded with high mass spectrum by can be formed by connecting, it can not only be by angiotensin converting enzyme specific recognition and digestion, there is high mass spectrum response simultaneously, Mass Spectrometer Method accuracy is high, can the activity of accurate response angiotensin converting enzyme or the inhibitory activity of angiotensin converting enzyme inhibitors, be also very applicable for from the complex systems such as Chinese medicine compound of the screening with angiotensin converting enzyme inhibition activity.
Description
Technical field
The present invention relates to drug screenings and assessment technique field, and in particular to one kind being used for hypertensin conversion enzyme activity
The MS probe of detection and its application.
Background technology
Angiotensin converting enzyme (Angiotensin-Converting Enzyme, ACE) is a kind of film of zinc dependent form
In conjunction with exopeptidase, it is distributed widely in the tissue of mammal.ACE can pass through renin-angiotensin system (Renin-
Angiotensin system, RAS) and kallikrein kinin system (Kallikrein-kinin system, KKS) catalysis
Angiotensin I converting is Angiotensin II, and bradykinin is made to inactivate, and blood pressure is caused to rise.In addition, the two systems are to electricity
Solution matter and isohydria, cardiovascular system development and Remolding of Functions also play important adjustment effect.Therefore, ACE, which has become, controls
Treat the important target spot of the diseases such as hypertension, acute myocardial infarction AMI and heart failure.
Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, such as captopril, enalapril, Benazepril, as treatment hypertension and heart failure
First-line drug is applied to clinic.However these drugs are used for a long time and are easy to cause the secondary work such as cough, albuminuria, renal failure
With.In recent years, find that the noval chemical compound with ACE inhibitory activity has become drug screening neck from natural products and traditional Chinese medicine
One research hotspot in domain.
The screening of Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe depends on corresponding method for detecting enzymatic activity.Currently, the active detection methods of ACE are main
Including fluorimetry, spectrophotometry, high performance liquid chromatography, enzyme coupling method etc..These methods are required for greatly by multiple
Step carries out sample process, cumbersome and there is a problem of that sensitivity is relatively low.In addition, the testing principle of these methods is substantially all
It is optical detection, is easy to be interfered by background.Natural products complicated component, in the detection wave band of substrate, there may be stronger
It absorbs, therefore the more difficult screening for directly applying to natural products of these methods.
The patent document of Publication No. CN1831529A discloses a kind of quickly to be sieved with high performance liquid chromatography and mass spectrometry
The method for selecting angiotensin converting enzyme inhibitors, using hippuric acid glycylglycine or hippuric acid histidine leucine as substrate, purifying
Rabbit Pulmonary Vascular angiotensin-converting enzyme or people's blank plasma be angiotensin converting enzyme source, in a water bath heating reaction,
Filter membrane or after centrifugation, with high performance liquid chromatography to substrate in angiotensin converting enzyme reaction mixture, product and
After added internal standard is detached, angiotensin converting enzyme catalysis substrate hydrolysis is detected under the negative ion mode of electrospray ionization mass spectrum
The hippuric acid generated.
Peptide fragment chemical derivatization be protein spectrum analysis in commonly use a kind of Sample Pretreatment Technique Used, by peptide fragment amino,
The small molecule label that easily ionizable is introduced on carboxyl or sulfydryl improves the sensitivity of peptide fragment and specificity and expands mass spectrographic detection model
It encloses.The technology provides a kind of new approaches to design the zymolyte of high sensitivity of mass spectrometry.
Invention content
The purpose of the present invention is to provide a kind of MS probes of high mass spectrum response, as zymolyte, by vasotonia
Plain invertase specific recognition detects vasotonia by the amount of probe or digestion products before and after mass spectroscopy endonuclease reaction
The activity of plain invertase.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of MS probe for hypertensin conversion enzyme activity detection, including amino acid sequence are Asp-Ser-
Piperazine compounds of the polypeptide and modification of Asp-Lys-Pro on the aspartic acid of the polypeptide N-terminal.
The present invention MS probe by can by the polypeptide of angiotensin converting enzyme specific recognition and high mass spectrum response
Small molecule is formed by connecting.The amino acid sequence of the polypeptide is:Asparate-serine-aspartic acid-Lys-Pro
The small molecule of (SEQ ID NO.1), high mass spectrum response are the piperazine compounds ,-NH and polypeptide of piperazine compounds
The carboxyl of the aspartic acid of chain N-terminal is condensed.
Amido bond of the angiotensin converting enzyme restriction enzyme site between aspartic acid-lysine.Therefore, mass spectrum of the present invention
The digestion products of probe are piperazine compounds-asparate-serine-aspartic acid and Lys-Pro.Due to piperazine
Class compound is responded with very strong mass spectrum, therefore reacts the front and back probe or digestion products piperazine compounds-by measuring
The amount of asparate-serine-aspartic acid, you can detect the activity of angiotensin converting enzyme.
Preferably, the piperazine compounds are 1- (2- pyrimidine radicals) piperazine, 1- (4- pyridyl groups) piperazines or 1- (1- first
Base -4- pyridyl groups) piperazine.
More preferably, the piperazine compounds are 1- (2- pyrimidine radicals) piperazine.
Shown in the molecular structural formula of the MS probe such as formula (I),
The present invention also provides a kind of preparation methods of the MS probe described in synthesis, including:
(1) using 2-Chlorotrityl Chloride Resin resins as carrier, fmoc-protected amino acid is raw material,
O- benzotriazole-tetramethylurea hexafluorophosphate is condensing agent, and synthetic amino acid array is Asp-Ser-Asp-Lys-Pro's
Polypeptide;
(2) according to the method for step (1), piperazine compounds are connected on the aspartic acid of the polypeptide N-terminal, matter is made
Probe crude product is composed, it is purified that the MS probe is made.
The present invention modifies piperazine compounds using solid-phase synthesis on the aspartic acid of polypeptide chain N-terminal.The asparagus fern of N-terminal
Propylhomoserin farther out from recognition site, piperazine compounds is modified on the site and do not interfere with ACE identifications.In addition, using synthesis in solid state
Method is easy to modify the carboxyl on Asp side chain, and the yield of probe is higher.
Specifically, the preparation method, including:
One, resin swellings
The 2-Chlorotrityl Chloride Resin resins that degree of substitution is 0.4mmol/g are weighed, resin is put into instead
Ying Guanzhong, add methylene chloride (DCM), vibrates 30min;
Two, connect first amino acid
Solvent is leached out, the Fmoc-L-Pro-OH of 3 times of molar excess is added, adds the diisopropyl second of 5 times of moles
Amine (DIEA) is eventually adding dimethylformamide (DMF) dissolving, vibrates 1h, with DMF and DCM alternately cleaning 6 times;
Three, are deprotected
20% Piperidine/DMF solution is added, reacts 5min, removes solvent, 20% Piperidine/DMF solution is added again, reacts
15min is cleaned twice after reaction with DMF, methanol and DMF;
Four, are condensed
O- benzotriazole-tetramethylurea hexafluoro the phosphorus of Fmoc-L-Lys (Boc)-OH and 3 times of mole of 3 times of moles
Hydrochlorate is dissolved with DMF, and reaction tube is added, and 5 times of mole DIEA are added immediately, reacts 60min;After reaction with DMF, methanol and
DMF is cleaned twice;Above step is repeated, is sequentially connected Asp, Ser, Asp and piperazine compounds from right to left, connection is completed
It is washed 4 times with methanol afterwards, drains 10min;
Five, are cut
Prepare cutting liquid (TFA:Water:EDT:TIS=95:2.5:2:0.5) polypeptide, is cut from resin, obtains crude product spy
Needle.
Six .HPLC purified polypeptides
Purify crude product probe with HPLC, solution after purification be lyophilized, -20 DEG C preserve it is for use.
It is a further object to provide the applications of the MS probe.The MS probe, can as zymolyte
By angiotensin converting enzyme specific recognition, digestion is at piperazine compounds-asparate-serine-aspartic acid and relies
Propylhomoserin-proline.Pass through MS probe or digestion products piperazines before and after mass spectrograph or LC-MS instrument measurement endonuclease reaction
The amount for closing object-asparate-serine-aspartic acid characterizes the activity of angiotensin converting enzyme using difference.
Therefore, the present invention provides the MS probes in the kit for preparing detection hypertensin conversion enzyme activity
In application.
The present invention also provides the MS probes in the kit for preparing screening angiotensin converting enzyme inhibitors
In application.
There is MS probe provided by the invention high mass spectrum to respond, and greatly improve the sensitivity of detection, in addition, by
It is to determine in the molecular weight of MS probe and piperazine compounds-asparate-serine-aspartic acid, the detection method
Selectivity and specificity it is very high, being highly suitable for from the complex systems such as Chinese medicine screening, there is angiotensin converting enzyme to inhibit
Active compound.
The present invention also provides the MS probes in the angiotensin converting enzyme inhibition activity of evaluation drug
Using.The angiotensin converting enzyme inhibition activity of different batches same drug is detected using the MS probe of the present invention, in turn
Evaluate the quality of different batches drug.
The advantageous effect that the present invention has:
MS probe provided by the invention is by can be by the polypeptide A sp-Ser- of angiotensin converting enzyme specific recognition
The small molecule piperazine compounds that Asp-Lys-Pro is responded with high mass spectrum are formed by connecting, can not only be by angiotensin converting enzyme
Specific recognition and digestion, while there is high mass spectrum response, Mass Spectrometer Method accuracy is high, being capable of accurate response vasotonia
The activity of plain invertase or the inhibitory activity of angiotensin converting enzyme inhibitors are also very applicable for from complex systems such as Chinese medicines
Middle compound of the screening with angiotensin converting enzyme inhibition activity.
Description of the drawings
Fig. 1 is the HPLC chromatogram of ACE MS probe purity analysis.
Fig. 2 is the HPLC-IT-MS base peak ion flow graphs of ACE MS probe structural identifications, and wherein upper right corner attached drawing is mass spectrum
Result figure.
Fig. 3 is the activity that ACE MS probes measure various concentration ACE.
Fig. 4 is the inhibiting rate that captopril is measured with ACE MS probes.
Specific implementation mode
The present invention is described further below by specific implementation case.
Embodiment 1
The synthesis of ACE MS probes
The structure of ACE probes is 1- (2- pyrimidine radicals) piperazines-aspartic acid-serine-aspartic acid-lysine-
Proline (PP-Asp-Ser-Asp-Lys-Pro), is synthesized by solid-phase synthesis, is mainly included the following steps that:
One, resin swellings
The 2-Chlorotrityl Chloride Resin resins that degree of substitution is 0.4mmol/g are weighed, resin is put into instead
Ying Guanzhong, add methylene chloride (DCM), vibrates 30min.
Two, connect first amino acid
Solvent is leached out, the Fmoc-L-Pro-OH of 3 times of molar excess is added, adds the diisopropyl of 5 times of molar excess
Ethamine (DIEA) is eventually adding a small amount of dimethylformamide (DMF) dissolving, vibrates 1h.With DMF and DCM alternately cleaning 6 times.
Three, are deprotected
20% Piperidine/DMF solution is added, reacts 5min, removes solvent, 20% Piperidine/DMF solution is added again, reacts
15min.It is cleaned twice with DMF, methanol and DMF after reaction.
Four, are condensed
The HBTU of Fmoc-L-Lys (Boc)-OH and 3 times of molar excess of 3 times of molar excess is dissolved with a small amount of DMF, is added
5 times of molar excess DIEA are added in reaction tube immediately, react 60min.It is cleaned twice with DMF, methanol and DMF after reaction.Repeat with
Upper step is sequentially connected Asp, Ser, Asp and 1- (2- pyrimidine radicals) piperazine, is washed 4 times with methanol after the completion of connection from right to left,
Drain 10min.
Five, are cut
Prepare cutting liquid (TFA:Water:EDT:TIS=95:2.5:2:0.5) polypeptide, is cut from resin, obtains crude product spy
Needle.
Six .HPLC purified polypeptides
Purify crude product probe with HPLC, solution after purification be lyophilized, -20 DEG C preserve it is for use.
Embodiment 2
The chemical characterization of ACE MS probes
ACE MS probes are synthesized using method described in embodiment 1, the purity of probe is analyzed with HPLC.Analysis
Condition is:Prompt human relations 1200HPLC chromatographic systems are equipped with variable-wavelenght detector (VWD), Detection wavelength 214nm;Chromatographic column,
Kromasil C18 (4.6mm × 150mm, 5 μm);Mobile phase is 0.1% trifluoroacetic acid-water (A) and 0.1% trifluoroacetic acid-second
Nitrile (B);Flow velocity 1.0mL/min;Isocratic elution, 0-25min, 5-70%B;Sample size, 10 μ L.
After ACE MS probes are dissolved with pure water, 10000rpm is centrifuged 5 minutes, sample introduction.HPLC chromatogram is as shown in Figure 1.
As can be seen that the ACE MS probe purity of synthesis is very high from figure, relative peak area accounting>95%, it can be used for
Subsequent experiment and research.
In addition, we further confirm the structure of probe with HPLC-MS.Analysis condition is:Agilent
1100HPLC chromatographic systems, series LC Q Deca XPplus ion trap mass spectrometries (HPLC-IT/MS);ESI ion sources;Cation mould
Formula;Mass-to-charge ratio (m/z), 100-1000;Capillary voltage, 15V;Source voltage, 3kV;350 DEG C of capillary temperature;Sheath gas (N2)
60arb;Assist gas (N2) 20arb;Chromatographic column, Zorbax SB C18 (4.6mm × 100mm, 1.8 μm);Mobile phase is 0.05%
Formic acid-water (A) and 0.05% formic acid-acetonitrile (B);Flow velocity 0.4mL/min;Gradient is 0-5min, 1%B;5-40min, 1-
30%B;40-45min, 30-100%B;45-50min, 100%B;Sample size, 5 μ L.
The HPLC-IT/MS chromatograms of ACE MS probes are as shown in Figure 2.It can be seen that quasi-molecular ion peak from mass spectrogram
[M+H]+(m/z 707.4) and double-charge ion [M+2H]2+(m/z354.6).These ions match with the structure of probe,
Also further confirm the structure of probe.
Embodiment 3
Application of the ACE MS probes in ACE Activity determinations
Using pH=8.3, the Tris-HCl of 10mM is as buffer solution.Take the ACE solution of ACE MS probes and various concentration
Each 20 μ L, 160 μ L of buffer solution, are placed in the centrifuge tube of 1.5mL, make the final concentration of 0.13mM of ACE MS probes, the end of ACE
A concentration of 0.005-0.06U/mL is incubated 2h at 37 DEG C.400 μ L methanol are added after reaction and terminate reaction.Solution vortex,
Centrifugation, the HPLC-IT/MS methods described in embodiment 2 are analyzed, and with the ACE MS probe enzymolysis products 1- that is generated after reaction, (2- is phonetic
Piperidinyl) piperazine-aspartic acid-serine-aspartic acid amount reflection ACE activity, the results are shown in Figure 3.
From figure as can be seen that in the concentration range, the ACE mass spectrums generated after the concentration (activity) of ACE and reaction are visited
The peak area of needle enzymolysis product has good linear relationship, i.e. ACE concentration is higher, and the probe being digested is more, digestion products
Peak area is also higher.Therefore, which can be used for the Activity determination of ACE.
Embodiment 4
Application of the ACE MS probes in Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe screening
Using pH=8.3, the Tris-HCl of 10mM is as buffer solution.ACE MS probes and each 20 μ L of ACE solution are taken, no
With the 50 μ L of captopril (Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) of concentration, 110 μ L of buffer solution are placed in the centrifuge tube of 1.5mL, make ACE MS probes
Final concentration of 0.13mM, the final concentration of 0.06U/mL of ACE, the final concentration of 0.08-327nM of captopril are incubated at 37 DEG C
Educate 2h.400 μ L methanol are added after reaction and terminate reaction.Solution is vortexed, centrifuges, the side HPLC-IT/MS described in embodiment 2
Method is analyzed, and enzyme inhibition rate is calculated with following formula:
ACE inhibiting rates (%)=[1- (Pinhibitor/Pblank)] × 100,
Wherein PinhibitorAnd PblankRespectively ACE MS probes after inhibitor group and control group (without inhibitor) reaction
The peak area of enzymolysis product.
Test results are shown in figure 4.From figure as can be seen that as captopril concentration rises, its inhibiting rate to ACE
Also it gradually increases.This shows the inhibiting effect that ACE probes can preferably reflect inhibitor to ACE, can be used for Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe
Screening.
Embodiment 5
Application of the ACE MS probes in the medicine quality evaluated with ACE inhibiting effect
Red rooted salvia powder each 0.5g of 5 different batches is taken, it is accurately weighed, add methanol-water (volume ratio 8:2) it mixes
Solution 50mL, ultrasonic extraction 30min, is dried with centrifugal concentrator, obtains Salvia root P.E powder, slow with Tris-HCl before experiment
Fliud flushing is configured to sample solution.
Using pH=8.3, the Tris-HCl of 10mM is as buffer solution.ACE MS probes and each 20 μ L of ACE solution are taken, it is red
Joining 50 μ L of sample solution, 110 μ L of buffer solution are placed in the centrifuge tube of 1.5mL, make the final concentration of 0.13mM of ACE MS probes,
The final concentration of 0.06U/mL of ACE, the final concentration of 2mg/mL of Radix Salviae Miltiorrhizae are incubated 2h at 37 DEG C.400 μ L first are added after reaction
Alcohol terminates reaction.Solution is vortexed, centrifuges, and the HPLC-IT/MS methods described in embodiment 2 are analyzed, and calculating enzyme with following formula inhibits
Rate:
ACE inhibiting rates (%)=[1- (Pinhibitor/Pblank)] × 100,
Wherein PinhibitorAnd PblankRespectively ACE mass spectrums are visited after Radix Salviae Miltiorrhizae sample sets and control group (without inhibitor) reaction
The peak area of needle enzymolysis product.
The result shows that the inhibiting rate of 5 batch Radix Salviae Miltiorrhizae ACE is respectively 23%, 29%, 19%, 25%, 24%.This shows
The effect of Radix Salviae Miltiorrhizae has ACE certain inhibiting effect, and ACE also invigorates blood circulation with Radix Salviae Miltiorrhizae matches.It therefore, can be compared with using this method
Reflect the bioactivity of Radix Salviae Miltiorrhizae well, it can also be used to the quality evaluation of Radix Salviae Miltiorrhizae.
The foregoing is merely the specific implementation cases of patent of the present invention, but the technical characteristic of patent of the present invention is not limited to
This, any those skilled in the relevant art in the field of the invention, made by changes or modifications all cover the present invention it is special
Among sharp range.
Sequence table
<110>Zhejiang University
<120>A kind of MS probe and its application for hypertensin conversion enzyme activity detection
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Asp Ser Asp Lys Pro
1 5
Claims (7)
1. a kind of MS probe for hypertensin conversion enzyme activity detection, which is characterized in that be including amino acid sequence
Piperazine compounds of the polypeptide and modification of Asp-Ser-Asp-Lys-Pro on the aspartic acid of the polypeptide N-terminal.
2. MS probe as described in claim 1, which is characterized in that the piperazine compounds are 1- (2- pyrimidine radicals) piperazine
Piperazine, 1- (4- pyridyl groups) piperazines or 1- (1- methyl -4- pyridyl groups) piperazine.
3. MS probe as described in claim 1, which is characterized in that the molecular structural formula of the MS probe such as formula (I) institute
Show,
4. the preparation method of MS probe as described in any one of claims 1-3, which is characterized in that including:
(1) using 2-Chlorotrityl Chloride Resin resins as carrier, fmoc-protected amino acid is raw material, O- benzene
And triazole-tetramethylurea hexafluorophosphate is condensing agent, synthetic amino acid array is the more of Asp-Ser-Asp-Lys-Pro
Peptide;
(2) according to the method for step (1), piperazine compounds are connected on the aspartic acid of the polypeptide N-terminal, mass spectrum is made and visits
Needle crude product, it is purified that the MS probe is made.
5. MS probe as described in any one of claims 1-3 is in the kit for preparing detection hypertensin conversion enzyme activity
In application.
6. MS probe as described in any one of claims 1-3 is in the reagent for preparing screening angiotensin converting enzyme inhibitors
Application in box.
7. MS probe as described in any one of claims 1-3 is in the angiotensin converting enzyme inhibition activity of evaluation drug
Application.
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CN111087445A (en) * | 2019-10-14 | 2020-05-01 | 浙江大学 | Mass spectrometry probe for ACE2 activity detection and preparation method and application thereof |
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