CN108693354A - Flavone glycoside functional magnetic nanometer affinity probe and its preparation method and application and intracellular target proteins catching method - Google Patents
Flavone glycoside functional magnetic nanometer affinity probe and its preparation method and application and intracellular target proteins catching method Download PDFInfo
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Abstract
The present invention relates to drug targets protein-enriched, separation and detection fields, disclose a kind of flavone glycoside functional magnetic nanometer affinity probe;Application the present invention also provides the preparation method of the flavone glycoside functional magnetic nanometer affinity probe and its in the capture of drug targets albumen and/or identification of cell;And the method that intracellular target proteins are captured by flavone glycoside functional magnetic nanometer affinity probe.The affinity probe of the present invention includes magnetic nano-particle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface, wherein, one end of the coupled structures is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound.The affinity probe only needs a small amount of holoprotein extracting solution that high specific capture can be realized.
Description
Technical field
The present invention relates to drug targets protein-enriched, separation and detection fields, and in particular, to a kind of flavone glycoside work(
Magnetic Nano affinity probe and preparation method thereof and its answering in the capture of drug targets albumen and/or identification of cell can be changed
With;And the method that intracellular target proteins are captured by flavone glycoside functional magnetic nanometer affinity probe.
Background technology
Flavone compound is a kind of polyphenols being widely present in plant, it mostly from plant secondary generation
It thanks.Its basic parent nucleus is 2- phenyl chromones, is with C6-C3-C6Mode connect.It is distinguished according to link position and chemical property,
Mainly there are flavonoids, flavonols, flavanone, anthocyanidin, flavanonol, isoflavones, your ear ketone etc..Numerous studies display is yellow
Ketone compounds have Green Tea Extract, and analgesia is antipyretic, antitumor, mitigate cardiac-cerebral ischemia reperfusion injury, diuresis, and liver protection is immunized
It adjusts, the multiple functions such as antiviral, there is great exploitation potential quality.
With the extensive use of proteomics method, the method based on chemical proteomics confirms active ingredient of Chinese herbs
The research of target proteins identification is more and more in recent years.Presently, there are challenge mainly have two aspect:On the one hand there is pharmacology
Interaction itself between active molecule and protein is weaker, and conventional method is difficult to keep the interaction between them
It is not destroyed;Still further aspect is exactly the expression of drug targets albumen much in trace level, common ESI-MS or
MALDI-MS loses the information of many weight target proteins matters due to detection limit problem.And if in drug targets protein enrichment process
The concentration of target proteins can be improved, reduce the loss in preparation of samples, while life is combined using nanoliter Liquid Chromatography-Tandem Mass Spectrometry
It is identified out will to have more target proteins for object informatics technology.
Invention content
The purpose of the present invention is overcoming, the prior art needs a large amount of cell holoprotein extracting solutions and specificity is lower scarce
It falls into, provides a kind of flavone glycoside functional magnetic nanometer affinity probe, which only needs a small amount of holoprotein extracting solution i.e.
High specific capture can be achieved, the present invention also provides the preparation method of the flavone glycoside functional magnetic nanometer affinity probe and its
Application in the capture of drug targets albumen and/or identification of cell.
The present inventor is had found by in-depth study, is fixed on specific flavone glycoside molecule by chemical modification
Magnetic Nano material surface can utilize it to interact with target biomolecule directly from sample solution to protein/more
The target molecules such as peptide carry out enrichment and desalination, to eliminate the sample elution step for being easiest to cause damages;Also, pass through
Quick separating may be implemented using additional magnetic field, enormously simplify separation, shorten time for sample pretreatment;Simultaneously by building
The qualitative of the protein of target can be realized using a small amount of cell holoprotein extracting solution for the method for having found nano-LC-MS/MS
With quantitative analysis.
One aspect of the present invention provides a kind of flavone glycoside functional magnetic nanometer affinity probe as a result, wherein spy that this is affine
Needle includes magnetic nano-particle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface,
In, one end of the coupled structures is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound
It connects.
Second aspect of the present invention provides a kind of preparation method of flavone glycoside functional magnetic nanometer affinity probe, wherein
This approach includes the following steps,
1) compound of flavone glycoside compound and structure shown in formula (2) in the presence of an organic, is carried out first to connect
It touches, obtains the intermediate compound that one end of the compound of structure shown in formula (2) is mutually bonded with flavone glycoside compound;
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains knot shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of the compound of structure is connected with magnetic nano-particle;
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that amino or the amino of BOC protections, n are the whole of 2-10
Number.
Third aspect present invention provides a kind of method of the intracellular target proteins of capture, wherein this method includes following step
Suddenly:
1) make obtained by the flavone glycoside functional magnetic nanometer affinity probe of the present invention or the preparation method of the present invention
Ketoside functional magnetic nanometer affinity probe is contacted with cell holoprotein extracting solution, so that flavone glycoside functionalization magnetic
Property nanometer affinity probe is combined with intracellular target proteins;
2) unbonded albumen is removed, to isolate the albumen of capture;
3) albumen of capture is identified;
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside
The intracellular target proteins of compound specificity identification.
Fourth aspect present invention provides the flavone glycoside functional magnetic nanometer affinity probe of the present invention or the system of the present invention
Ketoside functional magnetic nanometer affinity probe obtained by Preparation Method is in the capture of drug targets albumen and/or identification of cell
Application.
Through the above technical solutions, the flavone glycoside functional magnetic nanometer affinity probe of the present invention only needs a small amount of cell
The capture of intracellular target proteins can be realized in holoprotein extracting solution, and albumen capture specificity is high.In addition, by the present invention's
Flavone glycoside functional magnetic nanometer affinity probe is for when capturing intracellular target proteins, passing through simple externally-applied magnetic field step
Target proteins that magnetic Nano affinity probe captures and other Protein Separations can be simplified operation.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, an and part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the ESI-MS collection of illustrative plates of radix scutellariae glycoside derivates Qz-1 in embodiment 1.
Fig. 2 is the infrared absorpting light spectra of radix scutellariae glycoside derivates Qz-1 in embodiment 1.
Fig. 3 is according to the four of the scutelloside functional magnetic nanometer affinity probe and alkynyl that are prepared in embodiment 2
The schematic diagram of Fe 3 O magnetic nano-particle.
Fig. 4 is the transmission electron microscope photo of the alkynyl ferriferrous oxide nano-particle in embodiment 2.
Fig. 5 is the TOF-SIMS mass spectrograms that the upper figure in embodiment 2 is MNPs@Qz-1 in embodiment 2, and figure below is MNPs's
TOF-SIMS mass spectrograms.
Fig. 6 is that scutelloside, the Qz-1 of various concentration in embodiment 6 are active to people's hepatomicrosome enzyme HLM fluorescence probes CDE
It influences.
It is special that the upper figure of Fig. 7 is that the positive control probe in embodiment 7 is captured from HEK293T cell holoprotein extracting solutions
Property target proteins matter GCYB1 one peptide fragment of trypsin digestion second order ms figure, figure below is that the peptide fragment is distinguished in the upper figure
First mass spectrometric figure after being marked by weight acetylation isotope reagent.
Specific implementation mode
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
Flavone glycoside functional magnetic nanometer affinity probe provided by the invention is characterized in that the affinity probe includes magnetic
Property nano-particle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface, wherein the idol
The one end for being coupled structure is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound.
According to the present invention, the magnetic nano-particle refers to the magnetic nano-particle of tool, such as can be four oxidations three
Fe nanometer particles.
In addition, the magnetic nano-particle may be nucleocapsid, for example, its kernel is magnetic particle, outer layer covers
SiO2.For example can be as such magnetic nano-particle:Its kernel is ferriferrous oxide nano-particle, outer layer covers SiO2
Magnetic nano-particle.
In accordance with the present invention it is preferred that the average grain diameter of the magnetic nano-particle is 100-400nm, preferably 100-
12nm。
Preferably, the group key that the magnetic nano-particle passes through the group of surface modification and one end of the coupled structures
It closes.
Preferably, the group of the magnetic nano-particle surface modification is alkynyl, the idol being mutually bonded with the alkynyl
The group for being coupled one end of structure is azido, alternatively, the group of the magnetic nano-particle surface modification is azido, and it is described
The group of the one end for the coupled structures that azido is mutually bonded is alkynyl, alternatively, the magnetic nano-particle surface modification
Group is n-hydroxysuccinimide, and one end group for the coupled structures being mutually bonded with the n-hydroxysuccinimide is ammonia
Base.
It is further preferred that the flavone glycoside compound is the compound of structure shown in following formula (1), and the formula
(1) base that the compound of structure shown in passes through the carboxyl and the coupled structures other end of 7 connected uronic acids of O of flavone glycoside
Group is mutually bonded.
It is further preferred that the coupled structures that the compound of the structure shown in the formula (1) is combined is another
The group at end is amino.
In accordance with the present invention it is preferred that the coupled structures are provided by following formula (2) compound represented,
Wherein, R1Indicate azido, R2Indicate that amino, n indicate the integer of 2-10, preferably 2,3,4 or 5.
In the present invention, the atom number of compound shown in formula (1) is as follows:
In a particularly preferred embodiment of the present, in formula (1), R1Indicate azido, R2Indicate amino, n 3;
And the group of the magnetic nano-particle surface modification is alkynyl;The compound of structure shown in formula (2) by azido with it is described
The alkynyl of magnetic nano-particle surface modification is mutually bonded, and is connected by 7 O of compound of structure shown in amino and the formula (1)
The carboxyl for connecing uronic acid is mutually bonded, also structure as shown in Fig. 2.
Above-mentioned alkynyl is preferably propargyl.
In the present invention, surface modification has the magnetic nano-particle of alkynyl that can be obtained by synthesizing, and can also lead to
Cross it is commercially available, such as can commercially available surface modification have the magnetic nano-particle of propargyl.
Flavone glycoside functional magnetic nanometer affinity probe provided by the invention, it is preferable that relative to 1 milligram of the magnetic
Property nano-particle, the load capacity of the flavone glycoside compound is 30-45nmol;It is highly preferred that relative to 1 milligram of the magnetic
Property nano-particle, the load capacity of the flavone glycoside compound is 32-41nmol.
The present invention also provides the preparation method of above-mentioned flavone glycoside functional magnetic nanometer affinity probe, this method include with
Lower step,
1) compound of flavone glycoside compound and structure shown in formula (2) in the presence of an organic, is carried out first to connect
It touches, obtains the intermediate compound that one end of the compound of structure shown in formula (2) is mutually bonded with flavone glycoside compound;
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains knot shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of the compound of structure is connected with magnetic nano-particle;
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that amino or the amino of BOC protections, n are the whole of 2-10
Number.
Preferably, in formula (2), R1Indicate azido, R2Indicate amino, n 2,3,4 or 5;It is highly preferred that in formula (2), R1
Indicate azido, R2Indicate amino, n 3.
According to the method for the present invention, the flavone glycoside compound is the compound of structure shown in following formula (1);
According to the method for the present invention, in step 1), in the presence of an organic, by flavone glycoside compound and formula (2) institute
Show that the compound of structure carries out the first contact, obtains one end of the compound of structure shown in formula (2) and flavone glycoside compound phase
The intermediate compound (also flavone glycoside derivative below) of bonding.
According to the method for the present invention, in step 1), the flavone glycoside compound and the compound of structure shown in formula (2)
Molar ratio is 1:0.9-1.3;Preferably 1:1-1.1.
Method in accordance with the invention it is preferred that first contact carries out in the presence of the first catalyst, the flavone sugar
The molar ratio of glycoside compound and first catalyst is 1:1-1.5 preferably 1:1.2-1.3.
As long as being capable of catalytic step 1 as first catalyst) connection reaction, it is preferable that described first urges
Agent is EDCI (1- ethyls -3- (3- dimethylamine propyls) carbodiimide hydrochloride) and HOBt (1- hydroxy benzo triazoles).
According to the method for the present invention, in step 1), the organic solvent can be n,N-Dimethylformamide (DMF),
It is one or more in methanol and dimethyl sulfoxide (DMSO), preferably n,N-Dimethylformamide.Dosage as organic solvent can be with
For the conventional amount used of this field, for example, relative to 1 mole of flavone glycoside compound, the organic solvent is preferably 0.05-5
Mole, more preferably 0.1-1 moles.
Preferably, the condition of first contact includes:The temperature of contact is 15-30 DEG C, and the time of contact is that 5-50 is small
When.It is highly preferred that the condition of first contact includes:The temperature of contact is 20-30 DEG C, and the time of contact is 20-30 hours.
According to the method for the present invention, in step 2), intermediate compound and magnetic nano-particle that step 1) is obtained carry out
Second contact, obtains the flavone glycoside function that the other end of the compound of structure shown in formula (2) is connected with magnetic nano-particle
Change magnetic Nano affinity probe;
According to the method for the present invention, the magnetic nano-particle refers to the magnetic nano-particle of tool, such as can be four
Fe 3 O nano-particle.
In addition, the magnetic nano-particle may be nucleocapsid, for example, its kernel is magnetic particle, outer layer covers
SiO2.For example can be as such magnetic nano-particle:Its kernel is ferriferrous oxide nano-particle, outer layer covers SiO2
Magnetic nano-particle.
According to the method for the present invention, have can be with the compound of structure shown in formula (2) for the magnetic nano-particle surface modification
One end the group that is mutually bonded of group.It can be according to the base of one end of the compound of structure shown in formula (2) as the group
It rolls into a ball to select, such as can be alkynyl, azido or amino.The group R of one end of the compound of structure shown in formula (2)1Table
Show that azido, the magnetic nano-particle surface are preferably modified with alkynyl, are more preferably modified with propargyl.
Method in accordance with the invention it is preferred that the average grain diameter of the magnetic nano-particle is 100-400nm, preferably
100-120nm。
According to the method for the present invention, in step 2), relative to magnetic nano-particle described in 1mg, the intermediate compound
Dosage is 40-80nmol.
Preferably, second contact carries out in the presence of the second catalyst, second catalyst and the flavone sugar
The molar ratio of glycoside compound is 1:8-15, preferably 1:9-11.
As long as being capable of catalytic step 2 as second catalyst) connection reaction, such as in R1Indicate nitrine
Base, when the magnetic nano-particle surface is preferably modified with alkynyl, second catalyst can be copper sulphate and vitamin C
Sodium.
Furthermore it is preferred that the second contact carries out in the presence of solvent, the solvent for example can be water and/or phosphate-buffered salt
Solution.The dosage of the solvent does not require particularly, can be conventional amount used.
Preferably, the condition of second contact includes:The temperature of contact is 15-30 DEG C, and the time of contact is 0.2-24
Hour.It is highly preferred that the condition of second contact includes:The temperature of contact is 20-30 DEG C, and the time of contact is 1-5 hours.
The present invention also provides a kind of methods of the intracellular target proteins of capture, wherein this approach includes the following steps:
1) make flavone glycoside functional magnetic nanometer affinity probe or the preparation method through the invention of the present invention made
The flavone glycoside functional magnetic nanometer affinity probe obtained is contacted with cell holoprotein extracting solution, so that flavone glycoside work(
Magnetic Nano affinity probe can be changed to be combined with intracellular target proteins;
2) unbonded albumen is removed, to isolate the albumen of capture;
3) albumen of capture is identified;
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside
The intracellular target proteins of compound specificity identification.
The method of the intracellular target proteins of capture according to the present invention, in step 1), it is preferable that the flavone glycoside function
Change the dosage that magnetic Nano affinity probe is guided and supported with cell holoprotein to change in wide range, it is preferable that the Huang
Ketoside functional magnetic nanometer affinity probe (in terms of the weight of surface group magnetic nano-particle) and cell holoprotein
The weight ratio for guiding and supporting middle total protein is 1:0.01-0.035, preferably 1:0.01-0.031.
As above-mentioned steps 1) in contact conditions may include:The temperature of contact is 3-5 DEG C, and the time of contact can be
1-8 hours, preferably 1-2 hours.
The method of the intracellular target proteins of capture according to the present invention in step (2), can abandon supernatant by using centrifugation
The mode of liquid removes unbonded albumen, and such as 14000-16000g centrifuges 10-15min, and uses phosphate buffered saline solution
(PBS), water washing magnetic bead removes non-specific adsorption protein on magnetic bead three times.
In step (3), trypsin digestion egg may be used in the method for the intracellular target proteins of capture according to the present invention
In vain, to pass through the albumen of nano-LC-MS/MS identification captures.Further, it is also possible to the method analyzed by Western Blot
Identify the albumen of capture, concrete operations are known to those skilled in the art, and details are not described herein.
The method of the intracellular target proteins of capture according to the present invention, can be to the intracellular target of capture by step (4)
It marks albumen and carries out specific analysis, so that it is determined that flavone glycoside compound specificity identifies intracellular target proteins.As step
4), for example, can the quantitative proteomics method based on stable isotope labeling quantitative comparison, row are carried out to the albumen of capture
Unless binding proteins specific, to obtain the intracellular target proteins of specific recognition.The concrete operation step of this method is this
Well known to field technology personnel, details are not described herein.
In addition, in order to accurately exclude nonspecific proteins, it is preferable that the magnetism by setting surface group is received
Rice corpuscles is control group, to exclude nonspecific magnetic nano-particle binding protein, with the flavone glycoside functional magnetic
Nanometer affinity probe is compared, and the surface group magnetic nano-particle is not bound with the target egg of flavone glycoside compound effects
White matter.
Preparation the present invention also provides the flavone glycoside functional magnetic nanometer affinity probe of the present invention or through the invention
Flavone glycoside functional magnetic nanometer affinity probe obtained by method is in the capture of drug targets albumen and/or identification of cell
Application.
The present invention will be described in detail by way of examples below.
In following embodiment, normal embryonic kidney cells (HEK293T) cell is purchased from Chinese Concord Hospital, deuterated-N- acetyl group
Succinimide is purchased from Sigma companies (Sigma-Aldrich) Aldrich);Mass spectrum grade trypsase is purchased from Promege companies;
Dithiothreitol (DTT) (DTT) is purchased from Pierce companies;Alkynyl ferroferric oxide magnetic nano magnetic bead is limited purchased from the cities Ying Rui science and technology
Company;Baicalin is purchased from Han Wei Science and Technology Ltd.s, 95% or more purity;Memebrane protein, holoprotein extracts kit are purchased from shellfish
Rich biotech firm;Grade trypsase is sequenced and is purchased from Hyclone companies;ZiptipC18 is purchased from Minipore companies;BCA albumen is fixed
It measures kit and is purchased from Tianjin biochemical technology Co., Ltd.DMEM culture mediums (are purchased from GIBCO companies), trypsase, fetal calf serum
(being purchased from Sigma companies), secondary deionized water is prepared through Millpore systems.Trifluoroacetic acid aqueous solution is purchased from Tedia companies.DMEM
(high sugar) culture medium (Invitrogen, USA), colourless DMEM culture mediums (Invitrogen, USA), pancreatin 0.25%
Trypsin-EDTA (Invitrogen, USA), fetal calf serum FBS (Invitrogen, USA);Sodium liter electron spray nozzle needle is purchased green
Continuous Science and Technology Ltd.'s (New Objective products).
Embodiment 1
The present embodiment 1 is used for illustrating the preparation and representation of the radix scutellariae glycoside derivates Qz-1 of the present invention.
Scutelloside (446.2mg, 1mmol, Baicalin, same as below shown in for example above-mentioned formula (1) of structure) is weighed,
HOBt (175.6mg, 1.3mmol) is placed in round-bottomed flask, and 6ml anhydrous DMFs are added, and stirs 30min.N is taken in refrigerator3-
PEG3-NH2(218.3mg, 1mmol (- 11 nitrine -3,6 of 1 amino, 9- trioxaundecanes) are dissolved in 2mlDMF, are uniformly mixed, by
It is added dropwise to flask, EDCI (249.2mg, 1.3mmol) is dissolved in 2ml anhydrous DMFs, is added dropwise, and reaction a period of time is added excessive
TEA (303.3mg, 3mmol), is stirred to react 72h at normal temperatures, adds water stopping reaction three times, CHCl3Extraction, anhydrous magnesium sulfate
It is dried overnight, silica gel column chromatography separating purification, baking oven drying, obtaining pale yellow powder, (radix scutellariae glycoside derivates Qz-1 is (also simple below
Referred to as Qz-1), shown in result such as formula (3)), yield 32.5%.
ESI-MS(m/z):647.2189([M+H+]),29H34N4O13.1H-NMR(DMSO-d6,400MHz)δH(ppm):
8.71 (s, 1H), 8.10 (d, J=8,2H), 8.04 (s, 1H), 7.61 (d, J=8,2H), 7.03 (d, 1H) 5.54 (d, 1H)
5.24 (d, J=8,1H), 5.10 (d, J=8,1H), 3.97 (d, J=8,1H), 3.45 (s, 4H), 3.42 (m, J=8,10H),
3.41(s,6H),1.23(s,1H);13C-NMR(150MHz,DMSO-d6)(ppm):183.13,168.62,164.09,
151.89,1489.77,147.29,132.65,131.41,131,2,129.71,126.98,106.77,106.31,101.11,
94.54,76.16,76.03,73.32,71.54,70.27,70.14,69.77,69.32,50.52,29.58。
The radix scutellariae glycoside derivates Qz-1 of preparation characterizes molecular weight by ESI-MS, and infrared absorption spectrum, nuclear-magnetism is respectively adopted
The methods of hydrogen spectrum, the carbon spectrum that resonate characterization molecular structure, can determine the knot of radix scutellariae glycoside derivates Qz-1 from the above spectrum analysis
Structure is the compound of structure shown in formula (3).Characterization result is as depicted in figs. 1 and 2, and wherein Fig. 1 is radix scutellariae glycoside derivates Qz-1's
ESI-MS schemes, wherein 647.2189 be the molecular ion peak of (M+1).Fig. 2 is the infrared absorption spectrum of radix scutellariae glycoside derivates Qz-1
Figure, wherein 2100-2120cm-1Position, 2959cm are absorbed for the azido of feature on Qz-1-1,2925cm-1It vibrates and inhales for-CH
It receives, 3430cm-1For-OH absorption of vibrations.
Embodiment 2
The present embodiment 2 be used for illustrate the present invention radix scutellariae glycoside derivates-ferroferric oxide magnetic nano affinity probe (namely
The present invention flavone glycoside functional magnetic nanometer affinity probe) preparation and representation.
A concentration of 30mg/ml of magnetic nano-particle of alkynyl functionalization, preservation liquid are 50% ethanol water, using preceding super
Sound makes it be uniformly dispersed.Radix scutellariae glycoside derivates Qz-1 is configured to 5mM using DMSO, is diluted to containing 1.25 weights with PBS using preceding
Measure %DMSO.Anhydrous cupric sulfate is configured to 5mM using water, and vitamine C sodium is configured to 50mM using preceding with water three times.Scutelloside spreads out
Biology-ferroso-ferric oxide is affine nano-probe (MNPs@Qz-1), and construction method is:Clean 1.5ml centrifuge tubes are taken, are added in pipe
100 μ L (3mg) magnetic nano-particles, Magnetic Isolation discards preservation liquid, while using water washing 3 times three times, passes through magnetic after washing
Property separation discard water.Then radix scutellariae glycoside derivates Qz-1 1.5m Μ 1ml, ultrasonic 1min are added in centrifuge tube makes magnetic bead again
Dispersion in the solution, sequentially adds 2 μ L 50mM vitamine C sodiums and 2 μ L 5mM anhydrous cupric sulfates, at room temperature, shakes and is incubated 1-
For 24 hours, Magnetic Isolation, it is MNPs@Qz-1 to abandon supernatant.
The structure of radix scutellariae glycoside derivates-ferriferrous oxide nano probe (MNPs Qz-1) is with four oxygen of alkynyl functionalization
Three Fe nanometer particles (MNPs) are control, and the structural schematic diagram of two kinds of probes is as shown in Figure 3.Wherein original alkynyl four aoxidizes three
Fe nanometer particles are a kind of nucleocapsid, and kernel is ferroso-ferric oxide, outer layer covers SiO2, surface bond has propargyl.
Ferroferric oxide magnetic nano-particles (MNPs) pattern and size of alkynyl are carried out by transmission electron microscope
Characterization, the results are shown in Figure 4, wherein Fig. 4 is transmission electron microscope photo, and transmission electron microscope results show the ferroso-ferric oxide of alkynyl
The average grain diameter (nm) of magnetic nano-particle is 100-120nm;It is characterized, is tied by Secondary Ion Mass Spectrometry MNPs@Qz-1
Fruit is as shown in figure 5, the TOF-SIMS mass spectrograms that the upper figure in Fig. 5 is MNPs Qz-1, and middle probe MNPs Qz-1 are because occur
Click chemistry reacts to form triazole ring, has C in arrow pointed location2N3 -66.043 (m/z) characterising mass spectrometry peaks, quality error
For the TOF-SIMS mass spectrograms that figure below in -44.7, Fig. 5 is MNPs, in arrow in the TOF-SIMS mass spectrograms of negative control MNPs
Head pointed location is without the C2N3 -Characterising mass spectrometry peak shows successfully to construct scutelloside functional magnetic nanometer affinity probe.
Embodiment 3
The Qz-1 contents that this example 3 is used for illustrating to load in scutelloside functional magnetic nanometer affinity probe of the invention.
Clean 1.5ml centrifuge tubes are taken, 100 μ L (3mg) alkynyl ferroferric oxide magnetic nano-particles are added in pipe, so
The volume for being separately added into 25 μ Μ afterwards is the radix scutellariae glycoside derivates Qz-1 of 200ul, 400ul, 600ul, 800ul, 1ml, 1.2ml, is surpassed
Sound 1min makes magnetic bead be dispersed again in solution, 2 μ L50mM vitamine C sodiums and 2 μ L5mM anhydrous cupric sulfates is sequentially added, in room
It under temperature, shakes and is incubated 2h, after reaction respectively by Magnetic Isolation probe with containing the reaction solution of Qz-1, pass through UV, visible light
The variation of Qz-1 absorption values may know that total in 3mg alkynyl ferroferric oxide magnetic nano-particles in spectrographic determination supernatant
The amount of the Qz-1 of valence modification.By can be calculated, 32.6- is covalently bonded in the alkynyl ferriferrous oxide nano-particle of 1mg
The radix scutellariae glycoside derivates of 40.8nmol.
Embodiment 4
The present embodiment 4 is used for illustrating that the scutelloside functional magnetic nanometer affinity probe of the present invention captures scutelloside in cell
The method and condition optimizing of interior target proteins.
(1) culture of HEK293 cells
Cell culture chooses HEK293 cells as experimental subjects.In 5%CO2 and 37 degree Celsius of insulating box culture,
Culture medium is:1%PS, 90% DMEM, 10% FBS.Contain antibiotic simultaneously.Embryonic kidney cells HEK293 is being added to 10%
It is cultivated in DMEM (high sugar) culture medium of fetal calf serum and 1% Pen .- Strep, contains 5% dioxy in 37 DEG C of incubators
Change carbon.Culture to cell covers with culture dish in 100mm culture dishes, removes culture medium, collects cell, in HEK293 cells
Holoprotein extraction.
(2) with the extraction of holoprotein
At 4 DEG C, 2500g pelleted by centrifugation 5min carefully suck culture medium, receive as far as possible the cell collected in above-mentioned (1)
Collect cell.Cell is washed with cold PBS twice, and supernatant is blotted as far as possible after washing twice.Every 5 × 106--1×107A cell is (big
About 50mg, 50ul cell volume) in the cold total protein extracting solutions of 500ul be added (come from BestBio holoprotein extracts reagents
Box), after blowing and beating mixing, shaken 20 minutes under the conditions of 4 DEG C, until cell fully cracks, without apparent cell precipitation.At 4 DEG C,
15min is centrifuged under the conditions of 14000g, supernatant is quickly sucked to the clean centrifuge tube of another precooling, you can obtain total protein.It will be upper
- 80 DEG C of refrigerators of packing save backup or are directly used in capture scutelloside specificity target egg after stating the extraction BCA standard measures of albumen
White matter.Its concrete operations is carried out with reference to BestBio holoprotein extracts kits.
(3) scutelloside specificity target proteins are captured
Positive control probe (the scutelloside functional magnetic nanometer affinity probe MNPs@that embodiment 2 obtains prepared by 10mg
Qz-1 it) is separately added into PBS buffer solution with equivalent control probe (ferroferric oxide magnetic nano-particles of alkynyl) and is diluted to
0.4mL, the two are mixed with the cell holoprotein extracting solution of equivalent respectively, and 2h is incubated at 4 DEG C.Magnetic Isolation, centrifugation
(14000g, 10min) removes the protein solution being not bound with, with PBS buffer solution and water washing capture protein nano spy three times
Needle for several times, removes non-specific adsorption protein, and the protein content combined on MNPs@Qz-1 probes is measured by BCA methods.
It can be calculated by BCA working curves, about 11.33 μ g/mg of control probe non-specific adsorption HEK293 protein contents, positive control probe
About 30.90 μ g/mg of (100nm) capture protein amount.It is covalently bonded with according to the alkynyl ferriferrous oxide nano-particle of 1mg
The radix scutellariae glycoside derivates of 32.6-40.8n mol, it is possible to calculate the binding capacity about 1.67- of MNPs Qz-1 and protein
2.04nmol Qz-1/ μ g albumen.
Embodiment 5
The present embodiment 5 is used for illustrating the scutelloside functional magnetic nanometer affinity probe of the different-grain diameter of the present invention to cell
The load efficiency of middle holoprotein.
According to the method for embodiment 2, by the commercialization alkynyl ferroso-ferric oxide functional magnetic nanoparticle of different-grain diameter
Son is reacted with Qz-1, to prepare the positive control probe of different-grain diameter.Wherein, dispersibility is selected preferably, grain size 100nm, 400nm alkynes
Base ferroferric oxide magnetic nano-particles are prepared for the scutelloside functional magnetic nanometer affinity probe of two kinds of different-grain diameters,
Confirmed by identical characterizing method in embodiment 2.The method according to embodiment 4 after probe has been prepared, albumen is carried out
Then the capture of matter removes the protein solution being not bound with and non-specific adsorption protein, measured in difference by BCA methods
The protein content combined on grain size probe is control with the ferroferric oxide magnetic nano-particles (MNPs) of alkynyl.As a result
It has been shown that, grain size are that the albumen quality that the MNPs@Qz-1 of 100nm are captured under the same conditions is more.
Embodiment 6
The present embodiment 6 is used for illustrating that radix scutellariae glycoside derivates Qz-1 does not make the bioactivity of radix scutellariae active constituent scutelloside
It reduces in other words without its physiological activity of large effect.It is intended to the affine spy of functional magnetic nanometer by scutelloside derivatization
Needle captures the target proteins that intracellular target proteins matter illustrates scutelloside.The front and back compound pair of modification is measured by 96 well plate methods
It is evaluated in the activity influence of people's hepatomicrosome HLM fluorescence probes DME.
The inhibitor of series concentration, specially 125nM, 250nM, 500nM are configured using the PBS containing 1.25%DMSO,
1.25 μM, 2.5 μM, 5 μM, 12.5 μM, 25 μM, 125 μM, 250 μM and 1Mm.It is sequentially added in 96 orifice plates by enzyme source and series
The inhibitor (scutelloside, Qz-1, MNPs@Qz-1) of concentration, enzyme and inhibitor are respectively 20 μ L, mixing, then in 37 DEG C of conditions
Lower preincubate 30min is then added 10 μ L DME (making final concentration of 3 μM) and originates reaction so that each enzymatic hydrolysis reaction exists
In PBS (100mM, pH=6.5) buffer system of 50 μ L systems, under the conditions of 37 DEG C after pre-reaction 30min, 50 μ L are added later
The detection reagent that shines stops reaction, and in 37 DEG C of conditioned response 20min, bioluminescence is detected with microplate reader.Depression effect passes through not
Luminous intensity with inhibitor concentration and control group is converted into the percentage of enzyme inhibition and indicates, is fitted IC50。
The results are shown in Figure 6, it can be seen from the figure that in the low concentration range, such as c<At 100 μM, scutelloside, Qz-1 pairs
People's hepatomicrosome enzymatic activity is suitable, and when compound concentration is higher, radix scutellariae glycoside derivates Qz-1 is more better compared to scutelloside.
Embodiment 7
The present embodiment 7 is used for illustrating that the scutelloside functional magnetic nanometer affinity probe of the present invention captures scutelloside in cell
The identification method of the nano-LC-MS/MS of interior target proteins.
(1) scutelloside specificity target proteins are captured
Positive control probe (the scutelloside functional magnetic nanometer affinity probe that embodiment 2 obtains) prepared by 3mg and control
Probe (ferroferric oxide magnetic nano-particles of alkynyl) is separately added into PBS buffer solution and is diluted to 0.4mL, the two respectively with etc.
Cell holoprotein extracting solution (containing 200 μ g total proteins) mixing of amount, 2h is incubated at 4 DEG C.Centrifugation (14000g, 10min) is gone
Except the protein solution being not bound with, PBS buffer solution is washed 3 times, washes 3 removal non-specific adsorption protein three times, you can
Obtain the specific protein of capture.
(2) capture protein pre-treatment
The albumen of capture is added 8M urea 200 μ L, 95 DEG C of denaturation 5min and excess 100Mm DTT is added after cooling at 37 DEG C
Insulating box reacts 4h, and appropriate IAA is added later, reacts 2h in 37 DEG C of insulating boxs, uses molecular cut off for 3K super filter tubes later
(14000g, 30min) desalination 3 times is centrifuged, 20mM NH are added4HCO3Weight is molten, according to 40:1 is added trypsase, is digested at 37 DEG C
Overnight.
(3) albumen of identification capture
The supernatant freeze-drying that the enzymolysis liquid centrifugation (14000g, 5min) that step (2) is obtained obtains, using containing 0.1%FA
After H2O (mass spectrum is pure) dissolvings Nano-LC-MS/MS analyses are carried out after 0.22um filter membranes, final 5 μ L.Chromatographic column used is:C18
Column (75 μ m 15cm, 3 μm, LC Packings).Liquid phase is Dionex Ultramate3000, mobile phase A:95%H2O, 5%
MeCN, 0.1%FA, Mobile phase B:95%MeCN, 5%H2O, 0.1%FA, flow velocity are:300nL/min, gradient:3%B-45%
B, 120min.Tryptic digestion peptide fragment first passes around trap column desalination 5min, subsequently into through C18After post separation, into EFI
Mist mass spectrum (Xevo G2Q-TOF) detects.Spray voltage+3.03kV, cone voltage+35V.Survey scan:350-1700,
MS/MS scan:50-2000.The parent ion charge number of selection is+2 ,+3 ,+4.
MASCOT v2.6.0 search engines are used to identify the albumen of capture.Quality error is set as:Parent ion 30ppm, son
Ion 0.8Da.Methionine is oxidized to variable modification.Trypsase leakage enzyme site maximum is set as 1, Expect threshold values and sets
It is set to 0.05, only with the peptide fragment (each most matched result of second order ms figure) to rank the first in qualification result, only confidence
Albumen (p < 0.05) within degree threshold is considered as identifying the albumen come.
According to the method for embodiment 6, in three parallel laboratory tests, the scutelloside work(compared with negative control probe capture protein
It is obviously more that magnetic Nano affinity probe capture protein can be changed, by protein functional assays, it is found that many protein participate in
Important metabolic process in cell, specially substance and energetic supersession (ATPB, CYC, GPR75 etc.), calcium adjust (CALL5),
The functions such as anti-oxidant (PRDX1), heat shock (HSP (30/60/70/90)) and active anticancer (Caspase14, RFA1) are related,
Action target albumen is mainly thanked to intracellular calcium ion, heat shock inhibition, Inflammatory Pathway and regulation and control energy related.
The amount of the most of target proteins matter captured as described in Example 4 is conventional enough in nanogram level range
NanoLC-MS/MS, which is analyzed and obtained, firmly believes reliable qualification result.
These results confirm that the scutelloside functional magnetic nanometer affinity probe of the present invention has stronger capture scutelloside
The ability of target proteins.
(4) specificity verification
The enzymolysis liquid centrifugation (17000g, 5min) that step (2) is obtained obtains supernatant, and positive control probe and control probe obtain
The supernatant arrived reacts 5 hours respectively with 100 times of excessive acetylation labelled reagents under 25 DEG C of stirring conditions, and positive control probe is caught
Peptide fragment of the albumen obtained through trypsin digestion is marked by heavy isotope (deuterated acetic acid-n-succinimidyl ester), control probe
Peptide fragment of the albumen of capture through trypsin digestion is marked by light isotope (acetic acid-n-succinimidyl ester).After label,
Isometric two parts of reaction solutions mixing, is added excessive azanol and reacts 20 minutes under conditions of pH 10-11 to consume
The labelled reagent of amount.The peptide fragment mixed liquor of isotope labelling is lyophilized, Ziptip C are used in combination18Desalination.Obtained peptide fragment solution into
Row Nano-LC-MS/MS analyses.Mass Spectrometry Conditions and the same step of parameter setting (3).
Quantitative analysis uses Mascot Distiller 2.6.0 softwares, the same peptide fragment of weight isotope labelling to own
Mass spectrogram is stacked to produce a total mass spectrogram, the peptide fragment and light isotope label peptide fragment of heavy label in total mass spectrogram
Ratio be by calculate isotopic peak peak height ratios obtain.The quantitative ratio of albumen is by calculating the same albumen
The intensity rate of all matching peptide fragments is averagely worth to.
The results show that 7 scutelloside functional magnetic nanometer affinity probe of the present embodiment has filtered out specific recognition target
Protein G CYB:Guanylate cyclase are guanylate cyclase, and the existing enzymatic activity of the albumen is also a kind of membrane receptor, are participated in
Intracellular biological chemical process, is mainly played a role by signal transduction process.Therefore the protein-specific combination scutelloside.
And negative probes binding capacity is not enriched to this protein, the quantitative result of representative trypsin digestion peptide fragment is as schemed
Shown in 7, illustrate that the catching method of the present invention can specifically capture scutelloside target proteins in the cell.
From above-described embodiment as can be seen that scutelloside functional magnetic nanometer affinity probe can be just made from the method for the present invention
Intracellular target proteins are specifically really captured, and the cell holoprotein extracting solution needed is less.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In the skill of the present invention
In art conception range, technical scheme of the present invention can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, belongs to
Protection scope of the present invention.
Claims (12)
1. a kind of flavone glycoside functional magnetic nanometer affinity probe, which is characterized in that the affinity probe includes magnetic nano particle
Son, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface, wherein the coupled structures
One end is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound.
2. affinity probe according to claim 1, wherein the magnetic nano-particle is nucleocapsid, kernel four
Fe 3 O nano-particle, outer layer covers SiO2;
Preferably, the average grain diameter of the magnetic nano-particle is 100-400nm;
Preferably, the group bonding that the magnetic nano-particle passes through the group of surface modification and one end of the coupled structures;
Preferably, the group of the magnetic nano-particle surface modification is alkynyl, the coupling knot being mutually bonded with the alkynyl
The group of one end of structure is azido, alternatively, the group of the magnetic nano-particle surface modification is azido, with the nitrine
The group of the one end for the coupled structures that base is mutually bonded is alkynyl, alternatively, the group of the magnetic nano-particle surface modification
For n-hydroxysuccinimide, one end group for the coupled structures being mutually bonded with the n-hydroxysuccinimide is amino.
3. affinity probe according to claim 1 or 2, wherein the flavone glycoside compound is knot shown in following formula (1)
The compound of structure, and carboxyl and institute of the compound of structure shown in the formula (1) by 7 connected uronic acids of O of flavone glycoside
The group for stating the coupled structures other end is mutually bonded;
Preferably, the group of the other end for the coupled structures that the compound of the structure shown in the formula (1) is combined is ammonia
Base;
4. according to the affinity probe described in any one of claim 1-3, wherein the coupled structures are by following formula (2) institute
The compound shown provides,
Wherein, R1Indicate azido, R2Indicate that amino, n indicate the integer of 2-10, preferably 2,3,4 or 5;
Preferably, R1Indicate azido, R2Indicate that amino, n 3, and the group of the magnetic nano-particle surface modification are alkynes
Base, the compound of structure shown in formula (2) is mutually bonded by azido with the alkynyl of the magnetic nano-particle surface modification, described
The compound of structure shown in formula (1) is mutually bonded by the carboxyl of 7 connected uronic acids of O of flavone glycoside with amino.
5. according to the affinity probe described in any one of claim 1-4, wherein relative to 1 milligram of the magnetic Nano
The load capacity of particle, the flavone glycoside compound is 30-45nmol;
Preferably, relative to 1 milligram of the magnetic nano-particle, the load capacity of the flavone glycoside compound is 32-
41nmol。
6. a kind of preparation method of flavone glycoside functional magnetic nanometer affinity probe, which is characterized in that this method includes following
Step,
1) flavone glycoside compound in the presence of an organic, is carried out first with the compound of structure shown in formula (2) to contact, is obtained
The intermediate compound being mutually bonded with flavone glycoside compound to one end of the compound of structure shown in formula (2);
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains structure shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of compound is connected with magnetic nano-particle;
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate amino or the amino of BOC protections, n is the integer of 2-10, excellent
It is selected as 2,3,4 or 5.
7. affinity probe according to claim 6, wherein the flavone glycoside compound is structure shown in following formula (1)
Compound;
Preferably, in formula (2), R1Indicate azido, R2Indicate amino, n 3.
8. affinity probe according to claim 6, wherein the magnetic nano-particle is nucleocapsid, kernel four
Fe 3 O nano-particle, outer layer covers SiO2;
Preferably, the magnetic nano-particle surface modification has alkynyl;
The average grain diameter of the magnetic nano-particle is 100-400nm.
9. according to the affinity probe described in any one of claim 6-8, wherein in step 1), the flavone glycoside chemical combination
The molar ratio of object and the compound of structure shown in formula (2) is 1:0.9-1.3;Preferably 1:1-1.1;
Preferably, first contact carries out in the presence of the first catalyst, and the flavone glycoside compound is urged with described first
The molar ratio of agent is 1:1-1.5 preferably 1:1.2-1.3;
Preferably, first catalyst is 1- ethyls -3- (3- dimethylamine propyls) carbodiimide hydrochlorides and 1- hydroxy benzos
Triazole;
Preferably, the condition of first contact includes:The temperature of contact is 20-30 DEG C, and the time of contact is 5-50 hours.
10. according to the affinity probe described in any one of claim 6-8, wherein in step 2), relative to magnetic described in 1mg
Property nano-particle, the dosage of the intermediate compound is 40-80nmol;
Preferably, second contact carries out in the presence of the second catalyst, second catalyst and the flavone sugar glycosidation
The molar ratio for closing object is 1:8-15, preferably 1:9-11;
Preferably, second catalyst is copper sulphate and vitamine C sodium;
Preferably, the condition of second contact includes:The temperature of contact is 20-30 DEG C, and the time of contact is 0.2-24 hours.
11. a kind of method of the intracellular target proteins of capture, which is characterized in that this approach includes the following steps:
1) make flavone glycoside functional magnetic nanometer affinity probe or the claim 6- described in any one of claim 1-5
Flavone glycoside functional magnetic nanometer affinity probe obtained by preparation method described in any one of 10 and cell holoprotein
Matter extracting solution is contacted, so that flavone glycoside functional magnetic nanometer affinity probe is combined with intracellular target proteins;
2) unbonded albumen is removed, to isolate the albumen of capture;
3) albumen of capture is identified;
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside chemical combination
The intracellular target proteins of object specific recognition.
12. the flavone glycoside functional magnetic nanometer affinity probe described in any one of claim 1-5 or claim 6-
The medicine target of ketoside functional magnetic nanometer affinity probe obtained by preparation method described in any one of 10 in cell
Mark the application in albumen capture and/or identification.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110627852A (en) * | 2019-10-12 | 2019-12-31 | 华中农业大学 | Biotin-labeled naringin, preparation method and application thereof |
| CN110658314A (en) * | 2019-10-12 | 2020-01-07 | 四川大学 | Method for identifying target of compound, method for detecting interaction between compound and target, and method for evaluating drug effect of compound |
| CN111812315A (en) * | 2019-04-12 | 2020-10-23 | 中国科学院化学研究所 | Drug damage DNA functionalized magnetic nano affinity probe and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102463102A (en) * | 2010-11-16 | 2012-05-23 | 中国科学院成都生物研究所 | Surface-bonded baicalin magnetic nano-particle, and preparation method and application thereof |
-
2017
- 2017-04-05 CN CN201710217509.5A patent/CN108693354B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102463102A (en) * | 2010-11-16 | 2012-05-23 | 中国科学院成都生物研究所 | Surface-bonded baicalin magnetic nano-particle, and preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| LIN-SEN QING等: "Rapid magnetic solid-phase extraction for the selective determination of isoflavones in soymilk using baicalin-functionalized magnetic nanoparticles", 《J AGRIC FOOD CHEM》 * |
| LIN-SEN QING等: "Using baicalin-functionalized magnetic nanoparticles for selectively extracting flavonoids from Rosa chinensis", 《J SEP SCI》 * |
| NANWEN LI: "Click-chemistry for nanoparticle-modification", 《J MATER CHEM》 * |
| XUE-LONG SUN等: "Carbohydrate and protein immobilization onto solid surfaces by sequential diels-alder and azide-alkyne cycloadditions", 《BIOCONJUGATE CHEM》 * |
| 张文晶: "基于链接化学策略的磁纳米粒子的功能化：制备、表征及其应用研究", 《中国博士学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111812315A (en) * | 2019-04-12 | 2020-10-23 | 中国科学院化学研究所 | Drug damage DNA functionalized magnetic nano affinity probe and preparation method and application thereof |
| CN110627852A (en) * | 2019-10-12 | 2019-12-31 | 华中农业大学 | Biotin-labeled naringin, preparation method and application thereof |
| CN110658314A (en) * | 2019-10-12 | 2020-01-07 | 四川大学 | Method for identifying target of compound, method for detecting interaction between compound and target, and method for evaluating drug effect of compound |
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