CN108491688B - Method for preprocessing FRET (fluorescence resonance energy transfer) double-hybridization detection data based on donor-acceptor concentration ratio - Google Patents
Method for preprocessing FRET (fluorescence resonance energy transfer) double-hybridization detection data based on donor-acceptor concentration ratio Download PDFInfo
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Abstract
The invention discloses a method for preprocessing FRET double-hybridization detection data based on a donor-acceptor concentration ratio. The method comprises the following steps: (1) measuring E of each live cell separatelyA、ED、DESTAnd AESTAnd according to the donor-acceptor concentration ratio RDA＝DEST/AESTThe data obtained for each living cell is based on RDASorting the numerical values; (2) will sort RDADividing the space according to equal intervals; (3) calculate each R separatelyDAInterval cell data EA、ED、DESTAnd AESTAverage value of (d); (4) calculation of free Donor concentration D Using the preprocessed datafreeAnd free acceptor concentration AfreeAnd used for fitting analysis of langmuir-type FRET binding curves. The method greatly improves the stability of FRET double-hybridization detection analysis and the reliability of analysis results.
Description
Technical Field
The invention belongs to the technical field of Fluorescence Resonance Energy Transfer (FRET) detection, and particularly relates to a method for preprocessing FRET double-hybridization detection data based on a donor-acceptor concentration ratio.
Background
Fluorescence Resonance Energy Transfer (FRET) refers to the Transfer of Energy in a nonradiative form by an excited donor fluorophore when the two fluorophores are sufficiently close togetherA process of giving a fluorophore to a neighboring acceptor. The strength of FRET between two fluorophores is usually characterized by using FRET efficiency (E) between the two (Zalt, Gascoigne N RJ. Photobalching-corrected FRET efficiency imaging of cells [ J]Biophysical journal,2004,6(6): 3923-. FRET microscopy based on Fluorescent Proteins (FPs) has been widely used to study protein-protein interactions in living cells. FRET efficiency (E) centered on the acceptorA) Can pass through 33FRET, et al, measurement of FRET efficiency (E) centered on the donorD) Can be measured by E-FRET or the like. FRET two-hybrid detection technology combines 33-FRET and E-FRET by simultaneous measurement of the maximum E between donor and acceptor in the biological complexAAnd EDTo determine the binding ratio of the two molecules in the complex (Bunz E S, Ben-Johny M, Shen M, et al.Quantifying macromolecular interactions inducing cells using FRET wo-hybrid assays [ J].Nature protocols,2016,11(12):2470-2498)。
FRET two-hybrid detection techniques by simultaneously measuring E between target-labeled donor and acceptor protein molecules in a large number of living cellsD、EAAnd the corresponding concentration of free donor molecules in the cell (D)free) And concentration of free acceptor molecules (A)free) Then separately for ED-AfreeAnd EA-DfreeData were as follows for Langmuir (Langmuir) type equation:
fitting to obtain the maximum ED(ED,maxE when all donors bind to the acceptorD) And maximum EA(EA,maxAll acceptors binding to donor EA) And characterization of dissociation constant (K) between donor and acceptord,EFF). FIG. 1 shows a schematic diagram of the fitting of the detection results. Donor-acceptor binding ratio (N) in composite moleculesD/NA) From EA,max/ED,maxAnd (4) calculating. As shown in FIG. 1, the FRET two-hybrid detection assay requires DfreeAnd AfreeThe range of (a) is relatively large, and the measurement value intervals are relatively dispersed.
In performing E-FRET and 33In the quantitative detection of FRET double hybridization, the E between donor and acceptor in each cell can be measuredD、EAAnd total donor concentration per cell (D)EST) And total receptor concentration (A)EST)。DESTAnd AESTThe ratio of (A) to (B) is the donor-acceptor concentration ratioDA)。
The live cell FRET two-hybrid experiment requires the co-transfection of plasmids expressing two target proteins labeled with FPs separately, followed by the selection of a series of different RsDAObtaining data from the cells of (1). Prior to fitting analysis, it was difficult to define each cell RDACan only be measured by a large number of cells to ensure that there is enough RDAThe different data thus obtained a complete binding curve. In actual measurement, R is sometimes presentDAThere are many cells with similar values, but their EAOr EDBut the difference is large, while the other RDAThe range of values has less cellular data. In the data shown in FIG. 3a, RDAThere are many cells with small and similar values, and their EAOr EDHowever, the difference is large, fitting the measured data of these cells directly results in a fitted curve with almost no curvature, as shown in FIGS. 3b and 3c, when K isd,EFFClose to 0 means that the two target proteins are completely bound together regardless of the concentration ratio, which is not in accordance with reality. The reason for this unrealistic fit analysis results is mainly due to the presence of a large number of RDAAre close in value and correspond to EAOr EDMeasurement data with large deviation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for preprocessing FRET double-hybridization detection data based on a donor-acceptor concentration ratio, so that the stability and reliability of fitting analysis of the FRET double-hybridization detection data are improved.
The purpose of the invention is realized by the following technical scheme: based on donor-acceptor concentration ratio (R)DA) A method for pre-processing FRET two-hybrid detection data comprisingThe method comprises the following steps:
(1) separately measuring the receptor-centered FRET efficiency E of each living cell by using the FRET double-hybridization detection technologyAFRET efficiency E centered on the donorDTotal donor concentration DESTAnd the total receptor concentration AESTAnd obtaining a donor-acceptor concentration ratio RDA＝DEST/AEST(ii) a Then according to RDAThe numerical values of (A) are sorted to obtain RDAA minimum value and a maximum value;
(2) dividing the space: according to the obtained RDAMinimum and maximum forming a range value, with RDADividing the interval with the minimum value as a starting point at equal intervals to obtain R at equal intervalsDAAn interval; wherein, the number of the divided intervals is not less than 25;
(3) data preprocessing: calculate each R separatelyDAInterval cell data EA、ED、DESTAnd AESTAverage value of (d);
(4) calculating the free donor concentration D by using the data preprocessed in the step (3)freeAnd free acceptor concentration AfreeTo obtain a concentration ratio (R) based on donor-acceptorDA) Pretreated FRET two-hybrid detection data.
The FRET double hybridization detection described in step (1) is E-FRET and 33Quantitative detection of FRET double hybridization.
The number of viable cells measured in step (1) is at least 25.
The living cells in the step (1) are transfected with donor marker plasmids and acceptor marker plasmids simultaneously.
The sorting according to the numerical value in the step (1) can be performed in a sequence from small to large or in a sequence from large to small.
The division of the compartment in the step (2) is preferably achieved by: observation of RDAWhen R is a density distribution ofDAWhen R is mostly distributed between 0 and 1, R isDAIs set to 0.05, and then with RDAIs taken as a starting point, each interval is divided by 0.05, and each interval contains RDAValue is atAll cell data within this interval (E)A、ED、DESTAnd AEST) (ii) a When R isDAWhen the data are mostly distributed above 1, the interval is set to be 0.2, the rest steps are the same, and the interval of the interval can be adjusted by self, but the data volume after final preprocessing is not less than 25 groups.
Calculating the concentration D of free donors according to the preprocessed data in the step (4)freeAnd free acceptor concentration AfreeCan be used for the fitting analysis of the Langmuir-type FRET binding curve.
The Langmuir (Langmuir) type equation is:
wherein E isA,maxAs E when all acceptors are bound to the donorA；ED,maxFor all donors bound to the acceptor ED；Kd,EFFIs the dissociation constant between donor and acceptor; n is a radical ofD/NAIs the donor-acceptor binding ratio.
The basic principle of the invention is as follows:
the donor-acceptor concentration ratio (R) of different cells under the same conditionsDA) Same, theoretically EAAnd EDShould be equal respectively. However, in actual measurement, R sometimes occurs due to various measurement errorsDASimilar cells EAOr EDBut vary widely. If a large amount of data with larger deviation is directly used for calculating DfreeAnd AfreeFitting analysis may yield results that do not match the facts. In the langmuir type FRET binding curve, if a large amount of data with large deviation occurs at the rising stage of the curve, the fitted curve will rise almost vertically. The use of this method to pre-process FRET double-hybridization detection data can greatly improve the stability and reliability of langmuir-type FRET fitting analysis.
Compared with the prior art, the invention has the following advantages and effects:
1. the method of the present invention utilizes FRET double-hybridization detection techniqueAll cells obtained were measured for RDAR is to beDADivided into intervals of a certain size, and measuring data E in each intervalD、EATotal donor concentration (D)EST) And total receptor concentration (A)EST) Carrying out data average pretreatment, namely, before the pretreatment, each living cell corresponds to a group of data, after the pretreatment, each interval corresponds to a group of data, and each interval respectively obtains an EA、ED、DESTAnd AEST. Computing D using preprocessed datafreeAnd AfreeFitting analysis of langmuir-type FRET binding curves was performed.
2. The method can reduce the influence of a large amount of data with larger deviation on the fitting result, thereby greatly improving the stability of FRET double-hybridization detection analysis and the reliability of the analysis result.
Drawings
Figure 1 is a schematic diagram of langmuir-type FRET binding curve fitting; wherein, the graph a shows that the square protein and the circular protein have interaction, and are respectively fused with a receptor YFP and a donor CFP; graphs b and c are idealized E, respectivelyA-DfreeAnd ED-AfreeAnd combining the curve fitting results.
FIG. 2 is acquisition IDD、IDAAnd IAAAnd a linear fitting result graph of the ratio of the systemic sensitization quenching factor (G) and the extinction coefficient of the acceptor-donor at the donor excitation wavelength by using 4 serial plasmid fitting, and a random collision test result graph; wherein, the diagram a is acquisition IDD、IDAAnd IAAThe schematic diagram is obtained by subtracting the average value of the fluorescence intensity of the pixels in the corresponding white frame from the average value of the fluorescence intensity of the pixels in the black circles in the DD, DA and AA images and dividing the difference by the respective exposure time; FIG. b shows the ratio of extinction coefficients of the systemic sensitization quenching factor (G) and the acceptor-donor at the donor excitation wavelength [ epsilon ] by fitting of YFP-G4-CFP, YFP-G10-CFP, YFP-G40-CFP and YFP-G80-CFP, 4 tandem plasmidsYFP(λex,D)/εCFP(λex,D) Linear fitting results of; graph c shows the results of the random collision test.
FIG. 3 is a comparison graph of the results of fitting analysis of raw data and pre-processed data thereof; wherein, the graphs a and d are E- -R before and after pretreatmentDAA scatter plot; plots b and c are fitting Langmuir-type FRET binding curves calculated directly on the raw data (plot a); plots e and f are the fitted langmuir-type FRET binding curves calculated for the pre-treatment data (plot d).
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
1. Source of plasmids
The donor fluorescent group is gene coding fluorescent protein CFP (C for short), and the acceptor is gene coding fluorescent protein YFP (Y for short). YFP-G4-CFP, YFP-G10-CFP, YFP-G40-CFP and YFP-G80-CFP are tandem plasmids formed by connecting peptide chains with different lengths to C and Y in series, wherein the number refers to the number of glycine. C-Bcl-xL is a fusion protein of Bcl-xL labeled by CFP, and Y-Beclin1mut is a mutant Beclin1 fusion protein labeled by YFP. Tandem plasmids are available in the literature: butz E S, Ben-Johny M, Shen M, et al.Quantifying macromolecular interactions inducing cells using FRET two-hybrid assays [ J ]. Nature protocols,2016,11(12): 2470-2498; Y-Beclin1mut is synthesized by Wuhan Kingrui bioengineering, Inc.; the remaining plasmids were purchased from the Addgene plasmid library, USA.
2. Double-channel wide-field fluorescence microscope system
The two-channel wide-field fluorescence microscope system was produced by carl zeiss, germany, and was model AxioObserver. The light source is an X-Cite 120Q series metal halide lamp of Lumen Dynamics company in America, the objective lens is an oil mirror with the magnification of 40 and the numerical aperture of 1.3(40 multiplied by 1.3NA), a three-hole excitation filter pulling plate, an attenuation piece rotating wheel, a cube (each cube can be provided with an excitation piece, a beam splitter and an emission piece), and the external connection of the three-hole excitation filter pulling plate and the three-hole excitation filter pulling plate is provided with two CCD cameras. The excitation light wavelength is selected by pushing and pulling an excitation optical filter pulling plate, and the excitation light intensity is adjusted by selecting different gears output by the light source and different attenuation degrees of the attenuation sheet rotating wheel. The donor channel (DD) is composed of 445/25nm excitation filter (emitting excitation light with 445nm as center and 25nm bandwidth) and 480/22nm emission filter (collecting emission light with 480nm as center and 22nm bandwidth); the FRET channel (DA) consisted of an 445/25nm excitation filter and a 530nm LP emission filter (collecting emission light greater than 530 nm); the receptor channel (AA) consisted of an 510/17nm excitation filter and a 530nm LP emission filter.
3. Cell culture and plasmid transfection
Hela cells were cultured in DMEM medium with 10% newborn calf serum in an incubator at 37 ℃ containing 5% carbon dioxide. Digesting the cells by trypsin, transferring the cells to a cell culture dish, culturing for 24 hours, and then using an in vitro transfection reagent Turbofect when the cells grow to 70-90 percentTMThe plasmid was transiently transferred into cells.
The specific steps of plasmid transfection: (1) to EP tubes, 100. mu.L of DMEM medium was added, followed by 2. mu.L of the transfection reagent TurbofectTMMixing with 500ng plasmid, mixing, and standing for 20 min; (2) after 20 minutes, washing cells, supplementing 100 mu of LDMEM culture medium to an EP tube, sucking all solutions in the EP tube out, uniformly adding the solutions into a culture dish, and putting the culture dish back into an incubator to incubate for 4-6 hours; (3) after 4-6 hours, the transfection solution is aspirated, and a DMEM medium containing 10% newborn calf serum is added into the culture dish and cultured for 24-48 hours, so that the culture dish can be used for experiments.
Measurement procedure of FRET sample
Step 1: measuring crosstalk coefficients
Four spectral crosstalk coefficients were measured with C and Y plasmid single transfer cells, respectively:
wherein d is a donor emission crosstalk coefficient, c is a donor excitation crosstalk coefficient, a is an acceptor excitation crosstalk coefficient, and b is an acceptor emission crosstalk coefficient; i isDD、IDAAnd IAAThe D in brackets indicates the donor sample and A indicates the acceptor sample, corresponding to the fluorescence intensities measured in the DD, DA and AA channels, respectively. The average of 10 or more cells was measured to obtain a ═ 0.2667, b ═ 0.0005, c ═ 0.0004, and d ═ 0.9324.
Step 2: correction factor of measurement system
Respectively transfecting the tandem plasmids YFP-G4-CFP, YFP-G10-CFP, YFP-G40-CFP and YFP-G80-CFP into cells, and measuring the extinction coefficient ratio epsilon of the system sensitization quenching factor G and the acceptor-donor at the excitation wavelength of the donorYFP(λex,D)/εCFP(λex,D)：
FC＝IDA-a·(IAA-c·IDD)-d·(IDD-b·IAA), (6)
For each tandem plasmid sample, the fluorescence intensity Fc of donor-sensitized to acceptor was calculated by measuring at least 15 cells by the formula (6) < CHEM >DD(DA)/a·IAA(DA) < average value (x) > and [ Fc/a.I >AA(DA) ] average value (y).
Obtaining one (x, y) for each tandem plasmid sample, and calculating YFP-G4-CFP as (1.3463, 3.7120), YFP-G10-CFP as (1.4570,2.9998), YFP-G40-CFP as (1.5545,2.0639), YFP-G80-CFP as (1.6832, 1.3769); the line of FIG. 2b is linearly fitted through these 4 points, obtained from the fitted line according to equation (7)G is 7.126 and εYFP(λex,D)/εCFP(λex,D) 0.0752; the ratio M of the intensity of the acceptor-donor fluorophore according to equation (8)A/MD0.5746, let MDWhen 1, then MA＝0.5746。
And step 3: measuring collision correction coefficient:
co-transfection of C-Bcl-xL and Y measurements 33-FRET collisional correction factor
And E-FRET collision correction coefficient mE-FRET：
First, the FRET efficiency E in the collided sample was directly calculated by the formula (9)AThen with IDDAs the abscissa, with EA,spurious＝EA(EA,spuriousE generated for random collisionAThe equation holds only for random collision samples) as the ordinate, a line through 0 points (fig. 2c) is fitted with data points obtained for at least 15 cells, the slope of the line according to equation (10)
Then m is obtained according to the formula (11)E-FRET＝0.000359。
And 4, step 4: live cell FRET two-hybrid assay
The co-transfected C-Bcl-xL and Y-Beclin1mut were subjected to a live cell FRET two-hybrid assay to obtain data for at least 25 cells, here 89 cells.
E of each cell was calculated according to equations (9) and (12), respectivelyAAnd EDAnd then calculating the corrected E according to the formula (13)A(EA，Corr) And ED(ED，Corr). The total donor concentration D per cell was then calculated by the following formulaESTAnd the total receptor concentration AEST：
4.1 according to the conventional steps:
by estimating the first hypothesis Kd,EFF＝0.01，ND/NA1 (donor-acceptor binding ratio), EA,max0.1 (all acceptors bound to donor E)A)，ED,max0.1 (E when all donors bound to the acceptorD) Then, the free donor concentration D was calculated by the following formulafreeAnd free acceptor concentration Afree：
The predicted value E for each cell was then obtained in the Langmuir equation under these estimatesA,predAnd ED,pred：
Then the variance of the predicted value and the measured value is obtained:
total bias was counted for all cells:
TotalError＝∑i(EA,Corr-EA,pred)2+∑i(ED,Corr-ED,pred)2. (20)
finally solving by planning, by automatically changing Kd,EFF、ND/NA、EA,maxAnd ED,maxLet TotalErrorMinimum, obtain Kd,EFF、ND/NA、EA,maxAnd ED,maxWas fitted to a binding curve (FIGS. 3b and c), at which time Kd,EFF0.0000007, close to 0, means that Bcl-xL and Beclin1mut are completely bound regardless of the concentration ratio, which is not in line with the reality.
4.2 pretreatment FRET two-hybrid data:
the donor-acceptor concentration ratio R was calculated for each cell separatelyDA＝DEST/AESTObtained RDAA minimum value of 0.9932 and a maximum value of 11.9242; the data corresponding to each living cell obtained are then obtained (E)A、ED、DEST、AESTAnd RDA) According to RDAThe numerical values of R are sorted and then R is observedDAThe density distribution of (A) was found to be RDAMost of them are distributed over 1 or more, so that the interval is set to 0.2 and then R is set from RDAIs divided into 29 intervals by 0.2 increment, and each interval contains RDAData E for all cells with values within this intervalA、ED、DESTAnd AEST(ii) a Calculate each R separatelyDAE within the intervalA、ED、DESTAnd AESTAverage value of data (average preprocessing) to obtain 29 sets of data, calculating the preprocessed data by using formulas (16) to (20), and then obtaining the average valueThe over-planning solution is solved by automatically changing Kd,EFF、ND/NA、EA,maxAnd ED,maxLet TotalErrorMinimum fit to the curves of FIGS. 3E and 3f, when EA,max＝0.2797，EA,max0.1232 and Kd,EFF0.2165, indicating strong affinity of Bcl-xL for Beclin1 mut; n is a radical ofD/NA2.2387, indicating that Bcl-xL and Beclin1mut may bind in a 2:1 fashion.
The interval between the divided areas can be adjusted by self, but the final data volume after preprocessing is not less than 25 groups: if R can be observed firstDAWhen R is a density distribution ofDAWhen the number of R is in the range of 0 to 1, R may beDAThe interval was set to 0.05 with RDACell data (E) sorted by dividing each interval by 0.05 from the minimum value of (D)A、ED、DESTAnd AEST) Partition equal RDAEach interval of the interval.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. A method for preprocessing FRET two-hybrid detection data based on donor-acceptor concentration ratio, comprising the steps of:
(1) separately measuring the receptor-centered FRET efficiency E of each living cell by using the FRET double-hybridization detection technologyAFRET efficiency E centered on the donorDTotal donor concentration DESTAnd the total receptor concentration AESTAnd obtaining a donor-acceptor concentration ratio RDA＝DEST/AEST(ii) a Then according to RDAThe numerical values of (A) are sorted to obtain RDAA minimum value and a maximum value;
(2) dividing the space: according to the obtained RDAMinimum and maximum forming a range value, with RDAMinimum sizeDividing the interval by the same interval with the value as the starting point to obtain R with the same intervalDAAn interval; wherein, the number of the divided intervals is not less than 25;
(3) data preprocessing: calculate each R separatelyDAInterval cell data EA、ED、DESTAnd AESTAverage value of (d);
(4) calculating the free donor concentration D by using the data preprocessed in the step (3)freeAnd free acceptor concentration AfreeAnd obtaining the FRET double-hybridization detection data after pretreatment based on the concentration ratio of the donor and the acceptor.
2. The method for preprocessing FRET two-hybrid assay data based on donor-acceptor concentration ratio according to claim 1, wherein:
the FRET double hybridization detection described in step (1) is E-FRET and 33Quantitative detection of FRET double hybridization.
3. The method for preprocessing FRET two-hybrid assay data based on donor-acceptor concentration ratio according to claim 1, wherein:
the number of viable cells measured in step (1) is at least 25.
4. The method for preprocessing FRET two-hybrid assay data based on donor-acceptor concentration ratio according to claim 1, wherein:
the living cells in the step (1) are transfected with donor marker plasmids and acceptor marker plasmids simultaneously.
5. The method for preprocessing FRET two-hybrid assay data based on donor-acceptor concentration ratio according to claim 1, wherein:
the concentration D of free donors calculated by using the preprocessed data in the step (4)freeAnd free acceptor concentration AfreeFitting analysis for langmuir type FRET binding curves.
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