CN105987888B - A method of it fishes from mixture and screens active constituent monomer or active constituent group - Google Patents
A method of it fishes from mixture and screens active constituent monomer or active constituent group Download PDFInfo
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- CN105987888B CN105987888B CN201510067521.3A CN201510067521A CN105987888B CN 105987888 B CN105987888 B CN 105987888B CN 201510067521 A CN201510067521 A CN 201510067521A CN 105987888 B CN105987888 B CN 105987888B
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- 230000009758 senescence Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960001685 tacrine Drugs 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000018991 trans-resveratrol Nutrition 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to Pharmaceutical Analysis technical fields, are related to a kind of method fished from mixture and screen active constituent monomer or active constituent group.The present invention combination high-resolution tandem mass spectrometry HRMS and Applications of surface plasmon resonance SPR fish from mixed compound group screens small molecule active monomer or active constituent group based on target protein, HRMS including mixed compound group separates analysis, the chip coupling of receptor protein relevant to disease, enzyme, small molecule active monomer or the SPR of active constituent group targeting are fished, the HRMS of mixed compound group separates analysis after active constituent missing, protein binding sequence evaluation and high active ingredient monomer or active constituent group Structural Identification etc. based on each peak change；This method is simple and effective, quick primary dcreening operation effective component, and unknown active constituent monomer of the screening of fishing in advance from plant extracts based on target protein or at grouping for further separation and Extraction and can identify from mixing lead compound and provides the directive property of height.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, are related to a kind of fish from mixture and screen active constituent monomer or activity
At the method for grouping.
Background technique
Studies have shown that native compound has substantial amounts, and various structures, toxic side effect is small, and drug effect is definitely reliable etc. excellent
Thus gesture becomes the main source of new medicament screen.But its complicated component, mechanism of action is indefinite, makes the separation of its ingredient and activity
It screens time-consuming and laborious.Usually after completing cumbersome and arduous separation and Extraction, analyzing and identifying process, the lower natural production of content
Object need to also be screened on animal, organ (tissue) and cellular level model, and the method screening cost of the prior art is high, required
Sample size is big, experimental period is long, is not suitable for carrying out extensive, high-throughput screening.The SPR technique of Biacore is to utilize surface
Plasma resonance principle detects a kind of cutting edge technology of ligand and analyte effect on bio-sensing chip, has been widely used for point
Analyse the reaction power that biomolecular protein-protein, drug-protein, protein-nucleic acid, nucleic acid-nucleic acid interact
, binding site, reaction density etc..It is applied to molecular drug target identification and lead compound structure in terms of drug screening
Optimization has many advantages, such as real-time, high-throughput, specificity, without label.The powerful separation of high resolution mass spectrum and Structural Identification energy
Power allows the combination of two kinds of technologies quickly to search out the ligand of important albumen from multi-component complex system, there is document report
The combination of road SPR and MALDI-TOF are applied to the screening of high molecular weight protein ligand, but since small molecule compound is quick with albumen
The characteristics of dissociation, be difficult to obtain complex that is pure free and combining in recycling and dissociation solution, and due to binding force is weak,
Binding capacity is few, and the small molecule ingredient dissociated from target protein is caused to need the higher sensitivity of mass spectrum, and the combination of two kinds of technologies exists
Difficult aobvious advantage in small molecule complex system.There is patent CN 101339167A to the screening of small molecule compound using biological mass spectrometry
Method, separation system, pre-treating method is very loaded down with trivial details.
In view of the problems existing in the prior art, present inventor it is quasi- overcome it is fast after small molecule compound and protein binding
Speed dissociation and the problem that is not easily recycled, provide it is a kind of it is reliable, efficiently fish from mixture and screen active constituent monomer or work
Property at grouping method.
The prior art related to the present invention has:
1.Rich RL and Myszka DG:Advances in surface plasmon resonance
biosensor analysis.Curr Opin Biotechnol 11:54-61,2000.
2.Nedelkov D and Nelson RW:Analysis of native proteins from
biological fluids by biomolecular interaction analysis mass spectrometry(BIA/
MS):exploring the limit of detection,identification of non-specific binding
and detection of multi-protein complexes.Biosens Bioelectron 16:1071-1078,
2001.
3.Wang Q,Cheng XL,Li H,et al:Application of an efficient strategy for
discovery and purification of bioactive compounds from Chinese herbal
medicines,a case study on the Puerariae thomsonii Flos.J Pharm Biomed Anal
75:25-32,
4.Natsume T,Nakayama H and Isobe T:BIA-MS-MS:biomolecular interaction
analysis for functional proteomics.Trends Biotechnol 19:S28-33,2001.
5.Larsericsdotter H,Jansson O,Zhukov A,Areskoug D,Oscarsson S and
Buijs J:Optimizing the surface plasmon resonance/mass spectrometry interface
for functional proteomics applications:how to avoid and utilize nonspecific
adsorption.Proteomics 6:2355-2364,2006.
6.Heutmekers TH,Bremer MG,Haasnoot W and Nielen MW:A rapid surface
plasmon resonance(SPR)biosensor immunoassay for screening of somatotropins in
injection preparations.Anal Chim Acta 586:239-245,2007.
7.Heutmekers TH,Bremer MG,Haasnoot W and Nielen MW:A rapid surface
plasmon resonance(SPR)biosensor immunoassay for screening of somatotropins in
injection preparations.Anal Chim Acta 586:239-245,2007.
8.McDonnell JM:Surface plasmon resonance:towards an understanding of
the mechanisms of biological molecular recognition.Curr Opin Chem Biol 5:572-
577,2001.
9.Choulier L,Nomine Y,Zeder-Lutz G,et al:Chemical library screening
using a SPR-based inhibition in solution assay:simulations and experimental
validation.Anal Chem 85:8787-8795,2013.
10.Breault-Turcot J,Chaurand P and Masson JF:Unravelling nonspecific
adsorption of complex protein mixture on surfaces with SPR and MS.Anal Chem
86:9612-9619,2014.
11. old changqiang, progress laboratory medicine of the such as Gu Zhidong serum amyloid A protein in disease application,
2012,27(9):776-779.
12. Yao Qian member red wine protects cardiovascular effect food and drug, 2006,8 (01A): 72-73.
13. the shadow of anticancer active constituent trans-resveratrol content and its environmental factor in the grape wine such as Zhao Guang, LiJun
Ring Chinese medicine equipment, 2014,11 (3): 4-7.
14. Yan Rong, the pathogenesis and traditional Chinese medicine that the Alzheimer's disease amyloid beta such as normal Xiang is formed and deposited are dry
Pre- possible approaches new Chinese medicine and clinical pharmacology, 2013,24 (6): 629-632.
15. using amyloid A β as the progress Chinese Clinical pharmacology of target treatment Alzheimer disease with control
Iatreusiology, 2012,17 (10): 1172-1178.
16. the shadow that the such as Li Xuena, Li Xiangjun Cognex causes Alzheimer's Disease Rats learning memory disorder to A β 1-42
Ring Chinese patent drug, 2014,36 (11): 2233-2237.
17. the .HPLC-MS/MS method such as Wu Caisheng, Wang Yilin measure 7 kinds of effective components of ginkgo in dog plasma concentration and
The relative bioavailability Chinese Journal of New Drugs 2013,22 (4) of ginkgo leaf tincture: 93-98.
18. Ren Yanping, Chang Lu .HPLC-MS method measure containing for terpene lactone and flavonol glycosides in Shu Xuening injection simultaneously
Measure Pharmaceutical Analysis magazine, 2013,33 (2): 220-224.
19. Shen Tao, the intelligent equal .LC-MS/MS of a high man of virtue and ability are quickly measured in different plucking time Ginkgolides in Ginkgo biloba L. Leaves and gingko
The content Chinese Pharmaceutical Journal of ester, 2008,43 (5): 380-383..
Summary of the invention
The purpose of the present inventor be overcome the problems, such as it is of the existing technology, as after small molecule compound and protein binding quickly
The problem for dissociating and being not easily recycled, provide it is a kind of reliable, efficiently fish from mixture and screen active constituent monomer or activity
At the method for grouping.
The present invention provides a kind of high resolution mass spectrum technology HRMS and surface plasma resonance SPR Combined techniques from mixed
It fishes in polymerisable compounds group and filters out a kind of general key skill of the monomer of the small molecule active based on target protein or active constituent group
Art.The present invention solves Biacore/SPR system since small molecule compound is quickly dissociated with after protein binding, is difficult recycling
With pure problem that is free and combining the small molecule partner of state in two is obtained in dissociation solution, make the technology can be realized with
The combination of high-resolution tandem mass spectrometer, using its high sensitivity, amount of samples it is few, can be from complex system quick separating and structure elucidation
The advantages that ability, reaches the key technology and is not only suitable for structure it is known that being suitable for the complicated natural products that structural information lacks again
The high-throughput activated monomer or active constituent group of group's (such as Chinese medical extract) is fished the purpose of screening.
Specifically, a kind of method for screening active constituent monomer or active constituent group of fishing from mixture of the invention,
It fishes from small molecule mixed compound group in conjunction with two kinds of analytical technologies of HRMS and SPR and filters out the active constituent based on target protein
Monomer or active constituent group, comprising: small molecule mixed compound group is referred to as plant extraction liquid imports UPLC/QTOF system
The separation analysis of line map, obtains each chromatography or mass spectrum response at swarming；Receptor protein relevant to disease, the chip of enzyme are even
Connection；Small molecule mixed compound group imports Biacore/SPR system progress targeting proteins active constituent and fishes；Active constituent missing
Recovered liquid imports UPLC/QTOF system and carries out finger-print separation analysis afterwards, obtains each chromatography or mass spectrum response at swarming；
According to each peak change, carry out the protein binding sequence evaluation of each ingredient, further to high active ingredient monomer or activity at
Packet configuration identification.
In the present invention, using the kinetic parameter of the Biacore/SPR analysis single active constituent of determinand, its work is evaluated
Property, its consistency result finally verifies Conjoint Analysis technology simultaneously to the evaluation method of blending constituent.
In the present invention, to mix native compound component, candidate lead compound, the Chinese medicinal plant containing non-principal component are mixed
Extract, traditional Chinese medicine decoction of He Hanwei principal component etc. are determinand.
In the present invention, receptor protein relevant to disease, enzyme include such as serum amyloid A protein, beta-amyloid protein
Deng.
In the present invention, the analysis method and Biacore/SPR system for providing UPLC/QTOF are to small molecule compound and target
Binding characteristic that albumen, enzyme quickly dissociate and the composition proportion of specific sample reclaimer and recovered liquid, dissociation solution being arranged
With circulating collection number.
In the present invention, disclosing solubilizer is 5%DMSO or P20, occurs that target protein biological characteristics on chip not
Change, and all components are solvable in determinand；Buffer selects PBS, HBS-N, HBS-P, HBS-EP, the compatible elution of mass spectrum
Liquid, recovered liquid are 50mM NH4HCO3, 5% TFA.
More specifically, a kind of fish from mixture of the invention screens the side of active constituent monomer or active constituent group
Method, which is characterized in that itself comprising steps of
1) HRMS-SPR analytical technology method for combined use is established,
(1) mixture for determining albumen target spot and standard items is smaller ligand, establishes Biacore/SPR analysis, fishes,
The technology method for combined use and data statistical approach of UPLC/QTOF separation analysis；In the embodiment of the present invention, with serum amyloid sample
Albumin A (SAA) be target spot, piceatannol, caffeic acid, phloretin, four kinds of standard items of gibberellic acid mixture be smaller ligand,
It establishes Biacore/SPR analysis, fish, the technology method for combined use and data statistical approach of UPLC/QTOF separation analysis；
(2) using determining albumen as target spot, the mixture to standard items is smaller ligand, establishes Biacore/SPR points
It analyses, fish, the technology method for combined use and data statistical approach of UPLC/QTOF separation analysis；In the embodiment of the present invention, with
SAA is target spot, to beans fragrant acid, phloridzin, Hyperoside, Isorhamnetin-O-glycosides, rutin, resveratrol, polygonin, white skin
The mixture of eight kinds of standard items of China fir alcohol is smaller ligand, establishes Biacore/SPR analysis, fishes, UPLC/QTOF separation analysis
Technology method for combined use and data statistical approach；
2) HRMS-SPR analytical technology method for combined use is verified,
Biacore/SPR analytical procedure 1) (1) standard items mixture, in the mixture of the standard items of step 1) (2)
The kinetic parameter of single ingredient evaluates its activity, and its consistency result verification Conjoint Analysis technology is to blending constituent evaluation side
The feasibility and correctness of formula；In the embodiment of the present invention, piceatannol, caffeic acid, root skin are analyzed using Biacore/SPR
Element, gibberellic acid, to the fragrant acid of beans, phloridzin, Hyperoside, Isorhamnetin-O-glycosides, rutin, resveratrol, polygonin individually at
Point kinetic parameter, evaluate its activity, its consistency result verification Conjoint Analysis technology to blending constituent evaluation method can
Row and correctness.
3) HRMS-SPR analytical technology method for combined use, which is used to fish, screens the active constituent and analysis mirror of mixed compound group
It is fixed；In the embodiment of the present invention, active constituent and analysis mirror of the screening of fishing from French Vosne-Romanee red wine based on SAA
It is fixed；It active constituent of the screening based on beta-amyloid protein and is analyzed and identified with fishing from ginkgo-leaf oral liquor.
It filters out as a result, it was confirmed that this method can be fished by Biacore/SPR technology from complex mixture based on target egg
White small molecule active element monomers or active constituent group；It can residual components before fishing and after fishing using high-resolution mass spectrometer
In quickly, directional separation identify active constituent monomer or active constituent group in mixture.
The present invention changes the Loading sequence in BiacoreT200 recovery system, overcomes by using reverse thinking mode
The crucial problem for quickly dissociating and being not easily recycled after small molecule compound and protein binding, successful activity of fishing out from mixture
Ingredient；Mass spectrum separation identification in apply simple relative quantitation method, fish by comparing front and back data variation, judge with
The combination of the active constituent that protein-specific combines, Biacore/SPR therein and UPLC/QTOF technology is that quickly screening is candidate
Lead compound, medicinal herb components analysis, traditional Chinese medicine decoction Quality Control evaluation provide new means.
It fishes from known or unknown blending constituent sieve the invention discloses two kinds of analytical technologies of a kind of combination HRMS and SPR
The key technology method for selecting active constituent monomer or active constituent group based on target protein can be applied to containing in non-principal component
Medicine plant extracts active constituent monomer and fishing at grouping, and research is analyzed and identified to the directionality of active constituent；It answers
For synthesizing the high flux screening of candidate lead compound；It is evaluated applied to different sources plant extracts based on the Quality Control of potency
Wait the therapeutic evaluation etc. of group to the ill applied to the non-principal component of traditional Chinese medicine decoction and at grouping.
Detailed description of the invention
(A) and (B) UPLC/QTOF TIC (ESI-NEG mode) after fishing before tetra- kinds of standard items aggregate sample SPR of Fig. 1 fish
Analysis chart.
Tetra- kinds of standard items aggregate sample SPR of Fig. 2 front and back HRMS relative peak area change rate of fishing compares (n=10).
(A) and (B) UPLC/QTOF TIC (ESI-NEG mode) after fishing before eight kinds of standard items aggregate sample SPR of Fig. 3 fish
Analysis chart.
Eight kinds of standard items aggregate samples of Fig. 4 front and back relative peak area change rate of fishing compares (n=10).
A kind of standard items of Fig. 5 ten are compared with SAA protein binding force data.
The piceatannol of Fig. 6 various concentration and the kinetic curve of SAA albumen.
The matched curve of the piceatannol and SAA albumen of Fig. 7 various concentration.
The phloretin of Fig. 8 various concentration and the kinetic curve of SAA albumen.
The matched curve of the phloretin and SAA albumen of Fig. 9 various concentration.
The resveratrol of Figure 10 various concentration and the kinetic curve of SAA albumen.
The matched curve of the resveratrol and SAA albumen of Figure 11 various concentration.
(A) before Figure 12 France Vosne-Romanee red wine SPR fishes, fish after (B) UPLC/QTOF TIC (ESI-NEG mould
Formula) analysis chart (2.5-17.5min).
(A) before Figure 13 France Vosne-Romanee red wine SPR fishes, fish after (B) UPLC/QTOF TIC (ESI-NEG mould
Formula) analysis chart (17.5-30min).
Figure 14 France Vosne-Romanee red wine SPR each retention time base peak relative peak area change rate in front and back of fishing compares.
(A) before Figure 15 France Vosne-Romanee red wine SPR fishes, fish after (B) UPLC/QTOF XIC (ESI-NEG mould
Formula) analysis chart.
The ESI cleavage of mass spectrum figure for four kinds of active constituents that the combination of Figure 16 .HRMS-SPR analytical technology is fished out.
Figure 17 .4 kind active constituent resveratrol (M/Z=227.07137), piceatannol (M/Z=243.06628), Mongolian oak
The mass spectrum TIC analysis chart of the standard reference material of Pi Su (M/Z=301.03538) and phloretin (M/Z=273.07685).
(A) before Figure 18 ginkgo oral liquid SPR fishes, fish after (B) UPLC/QTOF TIC (ESI-NEG mode) analysis chart
(3-20min)。
(A) before Figure 19 ginkgo oral liquid SPR fishes, fish after (B) UPLC/QTOF TIC (ESI-NEG mode) analysis chart
(13-22min)。
Figure 20 ginkgo oral liquid SPR each retention time base peak relative peak area change rate in front and back of fishing compares.
Four kinds of active constituent Bilobalides (M/Z=325.1), ginkolide B (M/Z=in Figure 21 ginkgo oral liquid
423.1), the daughter ion map of ginkalide A (M/Z=407.1) and ginkalide C (M/Z=439.1).
Specific embodiment
Embodiment 1 establishes HRMS-SPR analytical technology method for combined use
1.1 equipment and material:
1.1.1Biacore T200,
1.1.1.1 equipment, Biacore T200 (GE Healthcare Life Sciences) are furnished with Control
Software, Evaluation Software software,
1.1.1.2 material and reagent, CM5 chip, difference PH sodium acetate, amino coupled kit, regenerative agent box, phosphoric acid
Salt buffer, P20 (tween), above-mentioned to be purchased from GE Health care, ammonium hydrogen carbonate, trifluoroacetic acid, dehydrated alcohol are above-mentioned equal
Purchased from Sinopharm Chemical Reagent Co., Ltd.；Serum amyloid A protein, beta-amyloid protein are purchased from Abcam company；France
Vosne-Romanee red wine is purchased from flagship store, Wang Jiuwang official, ginkgo oral liquid is purchased from Feng Delin nutrient health monopolized store；
1.1.2UPLC/QTOF
1.1.2.1 equipment: AB SCIEX Triple5600+Mass spectrograph, equipped with electric spray ion source and
Peakview2.0, Markview1.2.1 and Masterview1.0 data processing software, American AB SCIEX company；Agilent
1290Infinity liquid chromatographic system, including degasser, quaternary infusion pump, autosampler, column oven, U.S.'s Agilent are public
Department；
1.1.2.2 material and reagent, standard items: to the fragrant acid of beans, phloridzin, phloretin, that gibberellic acid is purchased from Sigma is limited
Company；Hyperoside, Isorhamnetin-O-glycosides, rutin are purchased from Shanghai Institute Center of Standardization for Traditional Chinese Medicine；Caffeic acid is purchased from me
Fourth reagent (Aladdin)；Resveratrol, polygonin are purchased from Beijing lark prestige Science and Technology Ltd.；Piceatannol is purchased from Tokyo
Chemical conversion industry Co., Ltd.；Purity is >=95%；Reagent: formic acid (purity >=98%), acetonitrile (chromatographically pure), dimethyl sulfoxide
(DMSO) it is purchased from Sigma Co., Ltd；
1.2 establish Biacore/SPR method:
1.2.1 coupling and the sample (standard items and its mixing sample) of target protein are prepared,
Target protein be SAA (for HRMS-SPR analytical technique establish, verifying, using) and beta-amyloid protein (use
In the application of HRMS-SPR analytical technique), it is fixed on four channels of CM5 sensing chip by amino coupled method,
The coupling amount in each channel will be close to or higher than 8000RU, and to improve efficiency of fishing, four channels require coupling target protein；And
When doing dynamics correlation Parameter analysis, it only need to be coupled a channel, if Fc2 is coupled target protein, Fc1 is as reference channel；Respectively
Standard items dehydrated alcohol or dimethyl sulfoxide are formulated as the stock solution of 1mg/ml, and working solution is diluted with buffer, make sample
The concentration of middle ethyl alcohol or dimethyl sulfoxide is lower than 5%；
It prepares four kinds of standard items aggregate samples: piceatannol, phloretin, gibberellic acid and caffeic acid respectively being taken respectively to take 20ul, be added
1520ul buffer makes the concentration 5% of solvent content such as ethyl alcohol and dimethyl sulfoxide, is computed, each standard items in mixture
Concentration is 12.5ug/ml；
It prepares eight kinds of standard items aggregate samples: respectively taking to beans fragrant acid, phloridzin, Hyperoside, Isorhamnetin-O-glycosides, reed
Fourth, resveratrol, polygonin, piceatannol respectively take 20ul, and 3042ul buffer, which is added, makes solvent content such as ethyl alcohol and dimethyl
The concentration of sulfoxide is 5%, is computed, and the concentration of each standard items is 6.25ug/ml in mixture；
1.2.2 sample feeding,
When above-mentioned working solution flows through chip surface with the speed of 20-30ul/min, certain ingredients and it is fixed on chip list
The target protein in face combines, and the quality of chip surface substance changes, and corresponding response variation, sample introduction terminate under instrument record
Afterwards, switching buffer flows through chip surface, and the spontaneous dissociation of the ingredient in conjunction with target protein, dissociation process is supervised in real time by response
Control；
1.2.3 sample recycles,
Execute " Inject and recover " step: with the NH of 50mM4HCO3For deposition solution,
0.5%TFA is washing lotion and recovered liquid, repeats to receive 10 times, incubation time 30 seconds, recovery time 28 seconds in a recycling circulation；It is right
The ingredient quickly dissociated after specifically binding with target protein, flows into waste liquid with buffer, can not be according to " Control
The method editted in Software " software is fished, and is needed to first pass through adjustment sample cell and is placed i.e. change Loading sequence,
It is recovered to and is not combined with target protein or the compound of non-specific binding, then with high resolution mass spectrum detection and data are analyzed, obtained
The low ingredient of relative amount is the ingredient in conjunction with protein-specific, that is, resulting ingredient of fishing；Method of adjustment is,
The position " sample " is put " running buffer ", and " sample " is put in the position " regeneration ", upper corresponding in sensing figure
" incubation start " indicates that sample introduction starts, and the sample of recovered is that do not have spy from incubation start and ending
The opposite sex combines the ingredient on chip；
1.3 establish fast performance liquid series connection high resolution mass spectrum method,
1.3.1 sample preparation:
Recovered liquid after taking the standard items aggregate sample solution of 1.2.1 preparation, fishing is vortexed after mixing and carries out UPLC/DAD/
QTOF analysis；
1.3.2 liquid phase and Mass Spectrometry Conditions,
1.3.2.1 chromatographic condition chromatographic column: Acquity UPLC HSS T3(100mm × 2.1mm I.D., 1.8 μm of grains
Diameter, waters)；Mobile phase: acetonitrile-water (contains 0.1% formic acid)；Flow velocity: 0.35mL/min；Column temperature: 30 DEG C；Sample volume: 5 μ L；
Gradient is as shown in table 1 below:
1. liquid chromatogram elution program of table
1.3.2.2 Mass Spectrometry Conditions, ion source: electric spray ion source；Negative-ion mode detection；Ion injection electric :-
4500V；Temperature: 550 DEG C；Gas 1 (GS1, N2) pressure in source: 50psi；Gas 2 (GS2, N2) pressure: 60psi；Curtain gas
(N2) pressure: 35psi；Scanning mode is in such a way that TOF-MS and Product Ion IDA is combined；TOF-MS scanning mode
In, DP:-80V, CE:-10eV, Mass Range:m/z 100~1000, Accumulation time:0.080035secs,
cycle time:0.6802secs；In Product Ion IDA scanning mode, DP:-80V；CE:-35eV；CES:15eV；
Mass Range:m/z 50~1000, Accumulation time:0.080035secs, cycle time:0.6802secs；
Experimental result is shown:
1, four kinds of standard items aggregate sample SPR fish front and back high resolution mass spectrum analysis and data processed result such as Fig. 1
It is shown, wherein piceatannol (M/Z=243.06628, RT=20.97,24.23,28.06), phloretin (M/Z=
273.07685, RT=28.33) peak height significantly reduces, peak area significantly reduces；Gibberellic acid (M/Z=345.13667, RT=
18.93) change smaller, Piceatannol (1) with caffeic acid (M/Z=179.03498, RT=12.78) peak height and peak area
(2) (3) are isomers through independent sample introduction confirmation；
Table 2 shows that four kinds of standard items aggregate sample SPR fish front and back HRMS data.
Table 2.
Wherein, C1、C2Respectively indicate the content before and after four kinds of standard items aggregate samples are fished, A1、A2Respectively indicate four kinds of standards
Product aggregate sample peak area corresponding to each standard items, O before and after fishingRPAIndicate each peak face before four kinds of standard items aggregate samples are fished
Relative peak area (relative peak area, RPA) before the ratio that product accounts for the aggregate sample gross area is fished, RRPAIndicate four kinds
Standard items aggregate sample after fishing each peak area account for relative peak area after aggregate sample gross area ratio is fished, (RRPA-ORPA)/ORPA
Indicate relative peak area change rate；Data are mean ± SD, n=10；Statistics display: piceatannol and caffeic acid, gibberellic acid ratio
Compared with phloretin all has extremely significant difference (p < 0.001 * * *) compared with caffeic acid, gibberellic acid；
The analysis of further progress data, the relative peak area of each standard items before and after being fished with four kinds of standard items aggregate sample SPR
Change rate characterizes the interactions of each standard items and serum amyloid A protein, and calculation formula is as follows: relative peak area variation
Rate=(RRPA-ORPA)/ORPA.It was found from table 2, Fig. 2 analysis: four kinds of pure and mild phloretins of standard items aggregate sample middle white skin China fir are with respect to peak
Area change rate is presented negative value, and absolute value is close to 1, respectively -1.0000 ± 1.934e-005, -0.9990 ± 0.0005；
Caffeic acid, gibberellic acid relative peak area change rate be positive value, respectively 1.7580 ± 0.2043,1.7830 ± 0.0525.It is white
Skin China fir alcohol compared with caffeic acid, gibberellic acid, phloretin compared with caffeic acid, gibberellic acid, all have extremely significant difference (* * * p <
0.001)。
Comprehensive analysis Fig. 1,2 and table 2, four kinds of standard items aggregate sample SPR front and back peak height of fishing is aobvious in UPLC/QTOF TIC figure
The ingredient one of negative value is presented in relative peak area change rate in the ingredient and data processed result that work reduces, peak area significantly reduces
It causes, is piceatannol, phloretin；In UPLC/QTOF TIC figure four kinds of standard items aggregate samples fish front and back peak height, peak area variation
The consistent of positive value is presented with relative peak area change rate in lesser ingredient, is gibberellic acid, caffeic acid.
2) eight kinds of standard items aggregate sample SPR fish front and back high resolution mass spectrum analysis and data processed result show (such as Fig. 3 institute
Show), fragrant to beans sour (M/Z=163.04007), polygonin (M/Z=389.12419), Hyperoside (M/Z=463.0882),
Rutin (M/Z=609.14611), piceatannol (M/Z=243.06628), Isorhamnetin-O-glycosides (M/Z=
477.10385), phloridzin (M/Z=435.12967), resveratrol (M/Z=227.07137), eight kinds of standard items aggregate samples
No. 5 peak piceatannols and No. 8 peak resveratrol peak areas are substantially reduced after SPR fishes；
From UPLC/QTOF TIC figure, it is evident that eight kinds of standard items aggregate sample SPR fish front and back piceatannol (No. 5),
The peak height of resveratrol phloretin (No. 8) significantly reduces, and peak area significantly reduces；To beans fragrant acid, polygonin, Hyperoside, reed
Fourth, Isorhamnetin-O-glycosides, the peak height of phloridzin, peak area variation are unobvious；
3. 8 kinds of standard items aggregate sample SPR of table fish front and back HRMS data
The result shows that: the pure and mild resveratrol relative peak area change rate of eight kinds of standard items aggregate sample middle white skin China firs presents negative
Value, respectively -0.7362 ± 0.0254, -0.2284 ± 0.0294；To beans fragrant acid, phloridzin, Hyperoside, Isorhamnetin-O-
Its relative peak area change rate of glucosides, rutin, polygonin be positive value, respectively 0.2505 ± 0.0065,0.2330 ± 0.0612,
0.2007±0.0316,0.2413±0.0660,0.1790±0.0840,0.1740±0.0904.Piceatannol and white black false hellebore
Alcohol statistically all has extremely significant difference (p < 0.001 * * *) compared with other six kinds；And between remaining six kinds of standard items
Compare, there was no significant difference；
The result shows that described four kinds and eight kinds of mixed samples are after in conjunction with target proteins, wherein piceatannol, root skin
Element, resveratrol peak area the largest loss, to beans fragrant acid, phloridzin, Hyperoside, Isorhamnetin-O-glycosides, rutin, polygonum cuspidate
Less, analysis knows piceatannol, phloretin, resveratrol and target proteins SAA for glycosides, caffeic acid, the variation of gibberellic acid peak area
Binding force is stronger, and remaining eight kinds of binding force is weaker.
Embodiment 2HRMS-SPR analytical technology, which is fished, screens active small molecular monomer or active constituent group based on target protein
Method validation
1, a kind of standard items of Biacore T200 analysis ten are compared with the binding force of SAA albumen, the results show that a kind of ten marks
Quasi- product middle white skin China fir alcohol, phloretin, the binding force of resveratrol and SAA albumen are strong, compared with other 8 kinds, statistically all
With extremely significant difference (p < 0.001 * * *), phosphate buffer as negative control, the binding force with SAA albumen is 0.4333 ±
0.1528, illustrating the solvent not influences the binding force of standard items and albumen.
A kind of standard items of table 4. ten are compared with SAA protein binding power
The kinetic parameter of three kinds of active constituents and SAA albumen that 2, Biacore T200 analysis is fished out, utilizes
Biacore T200 to piceatannol, phloretin, resveratrol carry out dynamic analysis, gained kinetic curve respectively as Fig. 6,
Shown in Fig. 8, Figure 10, as a result corresponding matched curve is fully demonstrated and is fished from aggregate sample as shown in Fig. 7, Fig. 9, Figure 11
Active constituent out has very strong affinity with target proteins SAA really；
The dynamic analysis of three kinds of active constituents and SAA albumen that table 5. is fished out from standard items aggregate sample
The dissociation of three kinds of active constituents and SAA albumen that the joint technology of the method for the present invention is fished out belongs to quick solution
From being difficult to complete intermolecular interaction according to the method setting in Bicore T200Control Software is quick solution
From small molecule compound targeting fish, the present invention by be inverted Loading sequence dexterously solve this technical barrier, recycle
To not combined with target protein or the compound of non-specific binding (i.e. bioactive molecule missing mixture), is then detected and counted with HRMS
According to analysis, peak area significantly reduces before relatively fishing, relative peak area change rate present negative value ingredient be and protein-specific
In conjunction with ingredient, that is, resulting active constituent of fishing.
In conclusion combination two kinds of analytical technologies of HRMS-SPR are fished in filtering out and are based on from small molecule mixed compound group
The active constituent monomer of target protein or the technical method of active constituent group are reasonable, feasible, correct, reliable.The technology can be efficient
Quickly just effective component rapidly is sifted out from mixing lead compound, fishing in advance from extract filters out based on target protein
Unknown active constituent monomer or at grouping, provide the directive property of height for further separation and Extraction and identification.
Embodiment 3
It fishes and active constituent of the screening based on SAA and analyzes and identifies in red wine
Red wine has rich in acids, phenolic compound etc. and delays senescence, prevents cardiovascular and cerebrovascular disease, pre- anti-cancer
Etc. functions.The present invention chooses French Vosne-Romanee red wine, therefrom fishes and filters out the active constituent based on SAA and analyze mirror
It is fixed；SAA is the precursor substance for leading to amyloidosis, is broken after SAA proteolysis, and amyloid filaments and deposition are generated
In tissue and organ, leads to secondary amyloidosis, there is no the side of specific recession amyloid deposition stove so far
Method, the emphasis of Clinical Processing are the supplies for reducing amyloid precusor protein；Red wine is generated with certain prevention disease
Function, filtered out from its complicated ingredient with amyloid precusor protein SAA specific binding active constituent, can be pointedly
The supply of SAA is reduced, deposition of the SAA in tissue and organ, the generation of prevention of amyloid degenerative disease are prevented；As a result, it was confirmed that
This method can be fished the small molecule active ingredient list filtered out based on target protein by Biacore/SPR technology from red wine
Body or active constituent group；Using high-resolution mass spectrometer can quick, directional separation be identified in residual components before fishing and after fishing
Active constituent monomer or active constituent group in red wine out；Attached drawing 14,15 is shown fish after M/Z=227.07137, M/Z
=243.06628, M/Z=301.03538, M/Z=273.07685 peak area significant changes prompt and target protein specificity knot
It closes；And M/Z=273.07685 due to before original solution is fished content it is lower, peak area is less than 100, to avoid error from disregarding
Enter statistics；Infer that these four ingredients are resveratrol (M/Z=227.07137), piceatannol (M/Z according to secondary fragment ions
=243.06628), Quercetin (M/Z=301.03538) and phloretin (M/Z=273.07685).
Embodiment 4 is fished from ginkgo-leaf oral liquor active constituent of the screening based on beta-amyloid protein and to be analyzed and identified
Alzheimer disease is a kind of neurodegenerative disease that can lead to the symptoms such as hypomnesia, cognition dysfunction,
Human health is seriously threatened, morbidity and beta-amyloid protein (β-amyloid, abbreviation A β) are closely related；A β is that have mind
Small molecular protein polypeptide through cytotoxicity, excess generation and aggregation can cause the extensive neuron of cerebral cortex, hippocampus
Dysfunction and death；Therefore, the accumulation of A β in brain is reduced, the progression of the disease of Alzheimer disease can be delayed；
Ginkgo oral liquid has the function of enhancing memory, improves heart and brain blood circulation, prevention Alzheimer disease, changes
Complicated component is learned, flavones, polysaccharide, lactone, phenolic acid class etc. are contained；The present embodiment application HRMS-SPR joint technology is therefrom fished sieve
It selects with the active constituent with A β specific binding；
Liquid phase and Mass Spectrometry Conditions:
Chromatographic condition chromatographic column: Acquity UPLC HSS T3(100mm × 2.1mm I.D., 1.8 μm of partial sizes,
waters)；Mobile phase: acetonitrile-water (contains 0.1% formic acid)；Flow velocity: 0.35mL/min；Column temperature: 30 DEG C；Sample volume: 4 μ L；Gradient
It is as shown in the table:
6. liquid chromatogram elution program of table
Mass Spectrometry Conditions: ion source: electric spray ion source；Negative-ion mode detection；Ion injection electric: -4500V；Temperature:
550℃；Gas 1 (GS1, N2) pressure in source: 50psi；Gas 2 (GS2, N2) pressure: 60psi；Curtain gas (N2) pressure:
35psi；Scanning mode is in such a way that TOF-MS and Product Ion IDA is combined；In TOF-MS scanning mode, DP:-
80V, CE:-10eV, Mass Range:m/z 100~1200, Accumulation time:0.080035secs, cycle
time:0.6802secs；In Product Ion IDA scanning mode, DP:-80V；CE:-35eV；CES:15eV；Mass
Range:m/z 50~1000, Accumulation time:0.080035secs, cycle time:0.6802secs.
Attached drawing 18,19,20 is shown fish after M/Z=325.0944 (t:10.92), M/Z=423.1333 (t:12.07)
M/Z=439.1279 (t:12.69) and M/Z=407.1369 (t:19.65) peak area are substantially reduced；
According to secondary fragment ions infer four kinds of ingredients be Bilobalide (M/Z=325.0944,325.0944 →
251.0920 → 193.1232), ginkolide B, M, J (M/Z=423.1333,423.1333 → 367.1439 →
125.0240), ginkalide A (M/Z=407.1369,407.1369 → 351.1486 → 172.0886) and ginkalide C (M/
Z=439.1279,439.1279 → 383.1375)；
Attached drawing 21 shows four kinds of active constituent Bilobalides (M/Z=325.1), ginkolide B (M/ in ginkgo oral liquid
Z=423.1), the daughter ion map of ginkalide A (M/Z=407.1) and ginkalide C (M/Z=439.1), the son marked
Ion is the daughter ion for the ingredient having been acknowledged in document)。
Claims (2)
1. a kind of method for screening active constituent monomer or active constituent group of fishing from mixture, which is characterized in that in conjunction with
HRMS and SPR analytical technology is fished the active constituent monomer filtered out based on target protein from small molecule mixed compound group
Or active constituent group, comprising: small molecule mixed compound group's extracting solution imports UPLC/QTOF system and carries out finger-print separation
Analysis obtains each chromatography or mass spectrum response at swarming；The chip of receptor protein relevant to disease is coupled；Small molecule mixing
Compound group imports Biacore/SPR system progress targeting proteins active constituent and fishes；Recovered liquid is led after active constituent missing
Enter UPLC/QTOF system and carry out finger-print separation analysis, obtains each chromatography or mass spectrum response at swarming；According to each peak
Value variation carries out the protein binding sequence evaluation of each ingredient, to high active ingredient monomer or active constituent group Structural Identification；
The method includes the steps:
1) HRMS-SPR analytical technology method for combined use is established,
Prepare coupling and the sample of target protein: target protein is serum amyloid A protein SAA, is fixed by amino coupled method
On four channels of CM5 sensing chip, the coupling amount in each channel reaches 8000RU, then four channels are all coupled the target egg
White, each standard items dehydrated alcohol or dimethyl sulfoxide are formulated as the stock solution of 1mg/ml, are diluted with buffer, make second in sample
The concentration of alcohol or dimethyl sulfoxide is lower than 5%；
Prepare the aggregate sample of four kinds of standard items caffeic acids, gibberellic acid, piceatannol and phloretin: each standard items is dense in mixture
Degree is 12.5 μ g/ml；
Prepare eight kinds of standard items beans fragrant acid, phloridzin, Hyperoside, Isorhamnetin-O-glycosides, rutin, resveratrol, polygonin
With piceatannol aggregate sample: the concentration of each standard items is 6.25 μ g/ml in mixture；
Sample feeding: when working solution flows through chip surface with the speed of 20-30 μ l/min, the quality of chip surface substance occurs
Change, corresponding response variation under instrument record, after sample introduction, switching buffer flows through chip surface, with target protein knot
The ingredient Auto-decomposition of conjunction, dissociation process are monitored in real time by response；
Sample recycling: " Inject and recover " step is executed: with the NH of 50mM4HCO3For deposition
Solution, 0.5% TFA are washing lotion and recovered liquid, repeated collection 10 times in recycling circulation, incubation time 30 seconds, time
28 seconds between time receiving；For the ingredient quickly dissociated after being specifically bound with target protein, flowed into waste liquid with buffer, it can not be by
It fishes according to the method editted in " ControlSoftware " software, needs to first pass through adjustment sample cell placement and change
Become Loading sequence, is recovered to and is not combined with target protein or the compound of non-specific binding, then detected and counted with high resolution mass spectrum
According to analysis, show that the low ingredient of relative amount is the ingredient in conjunction with protein-specific, that is, resulting ingredient of fishing；Adjustment
Method is that the position " sample " is put " running buffer ", and " sample " is put in the position " regeneration ", is schemed in sensing
Upper corresponding " incubation start " indicates that sample introduction starts, the sample of recovered be since incubation to
Terminate and does not specifically bind the ingredient on chip；
Establish fast performance liquid series connection high resolution mass spectrum analysis:
Recovered liquid after taking the coupling for preparing target protein and the standard items aggregate sample solution prepared in sample step and fishing, is vortexed
UPLC/DAD/QTOF analysis is carried out after uniformly；
2) verifying HRMS-SPR analytical technology combination,
Using Biacore/SPR analytical procedure 1) mixtures of standard items, to single ingredient in the mixture of step 1) standard items
Kinetic parameter, evaluate its activity, its consistency result verification Conjoint Analysis technology is to the feasible of blending constituent evaluation method
Property and correctness；Wherein:
Using Biacore/SPR analysis piceatannol, caffeic acid, phloretin, gibberellic acid, to beans fragrant acid, phloridzin, Hypericum Chinense
Glycosides, Isorhamnetin-O-glycosides, rutin, resveratrol, the single ingredient of polygonin kinetic parameter, evaluate its activity, it is consistent
Feasibility and correctness of the property result verification Conjoint Analysis technology to blending constituent evaluation method；
3) HRMS-SPR analytical technology method for combined use screens the active constituent of mixed compound group and analyzes and identifies for fishing,
Using SAA and beta-amyloid protein as target proteins, fish from French Vosne-Romanee red wine screening based on SAA activity at
Divide and analyzes and identifies；Or it fishes and active constituent of the screening based on beta-amyloid protein and analyzes and identifies from ginkgo-leaf oral liquor.
2. by the method for screening active constituent monomer or active constituent group of fishing in the slave mixture described in claim 1,
It is characterized in that, in this method, buffer selects PBS, HBS-N, HBS-P or HBS-EP.
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