CN108663428A - A method of based on mobility electrophoretic determination matter dimensions - Google Patents
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Abstract
The present invention relates to mobility electrophoretic techniques fields, disclose a kind of method based on mobility electrophoretic determination matter dimensions, buffer solution is injected to the sample introduction end of capillary and continue first time period including syringe pump, sampling pump injects sample solution to the sample introduction end of capillary later, syringe pump injects buffer solution to the sample introduction end of the capillary again after the completion of sample introduction, while applying separation voltage between the both ends of capillary；Detector is detected in the detection window of capillary, obtains the appearance time t of neutral markerMWith the appearance time t of sample；It obtains sample equivalent sphere radial features curve and and then obtains sample ions equivalent sphere radius R；Sample ellipse is obtained according to sample ions equivalent sphere radius R and reconstructs restrictive curve, and then obtains the conformation distribution of sample under experiment condition.The present invention is easy to operate, and analyze speed is fast, can be carried out at the same time with the separation of mixture.
Description
Technical field
The present invention relates to mobility electrophoretic techniques fields, specifically, being related to a kind of based on mobility electrophoretic determination matter dimensions
Method.
Background technology
Large biological molecule is closely bound up with vital movement, to the parsing of macromolecular substances structure help to understand its function and
Mechanism of action.Common structure elucidation technology includes X-ray crystal diffraction, nuclear magnetic resoance spectrum and cryo EM etc..Make
It needing to obtain the larger amount of sample of high-purity first when analyzing structure with above-mentioned kind of technology, initial processing steps are relatively complicated, and
And the reconstruct after data collection also faces the problems such as data volume is big.Therefore it needs a kind of sensitivity higher and there is complex component knot
The scheme of structure analysis ability measures the basic size of substance also with supplementary means and conformation can help to follow-up high score first
Distinguish prediction and the characterization of structure.
Invention content
In order to overcome above-mentioned technical problem, the present invention provides a kind of method based on mobility electrophoretic determination matter dimensions,
Easy to operate, analyze speed is fast, can be carried out at the same time with the separation of mixture.
To achieve the goals above, the present invention provides a kind of method based on mobility electrophoretic determination matter dimensions, packet
It includes：
Syringe pump is injected to the sample introduction end of capillary buffer solution and continues first time period, and sampling pump is to the capillary later
Sample solution is injected at the sample introduction end of pipe, and the syringe pump injects buffering to the sample introduction end of the capillary again after the completion of sample introduction
Liquid, while applying separation voltage between the both ends of the capillary；
Detection window of the detector on the capillary is detected, and obtains the appearance time t of neutral markerMWith sample
The appearance time t of product；
According toObtain sample equivalent sphere radial features curve；Wherein exist
Under experiment condition,U be separation voltage, L added by capillary both ends be the overall length of capillary, Lreal is capillary
Sample introduction end is to mole matter that the length of the detection window, q are ion band electricity, η is solution viscosity coefficient, m is sample material
Amount, R are sample ions equivalent sphere radius；
The appearance time t of the sample is matched to the sample equivalent sphere radial features curve, obtains corresponding institute
State sample ions equivalent sphere radius R；
According to the ion equivalent sphere radius R, obtains sample ellipse and reconstruct restrictive curve；
The possibility conformation library of the sample is obtained using molecular simulation and obtain in turn may the length-width ratio of conformation and equivalent
Radius windward；
It is bent that the length-width ratio of the possible conformation and the equivalent radius windward are matched to the sample ellipse reconstruct limitation
Line obtains the conformation distribution of sample under experiment condition.
In a kind of optional embodiment, the possibility conformation library and in turn that the sample is obtained using molecular simulation
The length-width ratio of possible conformation and equivalent radius windward are obtained, is specifically included：
Simulated conditions are set as consistent with the experiment condition, emulate to obtain the sample using molecular dynamics simulation
Possibility conformation library；
The ellipse that possible conformation is obtained according to the radius of gyration in three directions of x, y, z in the possibility conformation library of the sample is special
Levy parameter；
The length-width ratio of the possible conformation and the equivalent radius windward are obtained based on the oval feature parameter.
In a kind of optional embodiment, the method further includes：
Determine that arbitrary possible conformation reconstructs in y-direction with the sample ellipse at a distance between restrictive curveOr relative distance
Determine diOr Δ diMinimum possibility conformation is the conformation distribution of sample under experiment condition.
In a kind of optional embodiment, use the simulation time that molecular dynamics simulation emulates for 100ns.
In a kind of optional embodiment, the detector is fluorescence detector or mass detector.
Method of the present invention based on mobility electrophoretic determination matter dimensions, syringe pump are injected to the sample introduction end of capillary
Buffer solution simultaneously continues first time period, and sampling pump injects sample solution to the sample introduction end of capillary later, is injected after the completion of sample introduction
Pump injects buffer solution to the sample introduction end of the capillary again, while applying separation voltage between the both ends of capillary；Detection
Device is detected in the detection window of capillary, obtains the appearance time t of neutral markerMWith the appearance time t of sample；It obtains
Sample equivalent sphere radial features curve simultaneously and then obtains sample ions equivalent sphere radius R；According to sample ions equivalent sphere
Radius R obtains sample ellipse and reconstructs restrictive curve, and then obtains the conformation distribution of sample under experiment condition.The technical side of the present invention
The mixture in solution is separated by electrophoresis by liquid phase mobility for case, while being detected to get in solution in detection window
Fine size, the structural information of substance realize and synchronize matter dimensions knot in progress solution during complex sample detaches
The detection of structure.Technical scheme of the present invention accuracy of detection is high, and easy to operate, analyze speed is fast, realize the separation of mixing sample and
The disposable restructuring analysis of multiple component structure.
Description of the drawings
Fig. 1 is the flow chart of the method for the present invention based on mobility electrophoretic determination matter dimensions；
Fig. 2 is a kind of schematic device provided by the invention；
Fig. 3 is the relation curve of theoretical equivalent redius and transit time under three kinds of separation conditions；
Fig. 4 is the analysis chart that the method measures growth hormone release inhibiting hormone size through the invention；
Fig. 5 shows the marking precedence diagram by emulating obtained 10 kinds of conformations of growth hormone release inhibiting hormone；
Fig. 6 is the analysis chart that the method measures angiotensin I size through the invention；
Fig. 7 is the analysis chart that the method measures bradykinin size through the invention；
Fig. 8 is the analysis chart that the method measures mixed polypeptide size through the invention；
Fig. 9 is the molecular dynamics simulation figure that the method measures mixed polypeptide through the invention.
Specific implementation mode
Below with reference to the accompanying drawings illustrate the embodiment of the present invention.It is retouched in the attached drawing of the present invention or a kind of embodiment
The elements and features stated can be combined with elements and features shown in one or more other drawings or embodiments.It answers
When note that for purposes of clarity, being omitted known to unrelated to the invention, those of ordinary skill in the art in attached drawing and explanation
Component or processing expression and description.
Ion transfer mass spectrum is a kind of New Two Dimensional mass spectral analysis of ionic mobility isolation technics and mass spectrometry under gas phase
Technology.The measurement of ion mobility is using the buffer gas of electric-force gradient and lateral flow, and migration rate is dependent on ion
Collision cross-section and electriferous state.Complex sample quick separating in gas phase can be achieved to detect, also can get the object under the gas phase condition
The structural information of matter.Capillary electrophoresis technique be using capillary as split tunnel in liquid phase environment, ion under electric field driven,
According to its, respectively mobility (the average electrophoretic velocity of charged particle under unit electric field intensity) difference carries out being separated efficiently and rapidly skill
Art.
Liquid phase mobility theory is proposed in conjunction with electrophoretic separation technique on the basis of gaseous ion migration spectrum.This hair
In bright embodiment, based on the charge species in mobility electrophoretic separation solution and matter dimensions measurement is carried out, buffering liquid is by flowing
Syringe pump provides, and split tunnel is capillary, realizes and detaches by electric field interacts with fluid resistance, obtains mobility electrophoresis
Spectrogram.An embodiment of the present invention provides a kind of methods based on mobility electrophoretic determination matter dimensions, as shown in Figure 1, this method packet
It includes：
101, syringe pump is injected to the sample introduction end of capillary buffer solution and continues first time period, and hereafter sampling pump is to capillary
Sample solution is injected at the sample introduction end of pipe, and syringe pump injects buffer solution to the sample introduction end of capillary again after the completion of sample introduction, while
Apply separation voltage between the both ends of capillary.
Mobility electrophoresis experiment condition clear first.Mobility electrophoresis protocols can be in commercialization microflow analysis instrument (such as capillary
Electrophoresis apparatus) on realize, can also be realized on the simple split tunnel voluntarily built.Usually using separation capillary as separation
Channel.Can use peristaltic pump as syringe pump to the entrance of capillary inject constant speed buffering liquid stream, sampling pump to
The entrance of capillary injects sample solution.High voltage power supply receiving electrode provides separation voltage, and one inspection of setting on the capillary
Window is surveyed, detector determination sample appearance time etc. is placed.First time period is specially 0.1 minute to 10 minutes.
Various inside and outside diameter capillaries can be used in capillary.When protein isolate matter, coatings capillary pipe need to be used.High direct voltage
The output area 0 of power supply~+/- 30000V, high voltage power supply can be connected by conducting wire with electrode assembly.Syringe pump provides 0-
1000mbar constant pressures or equivalent flow.Detector can be fluorescence detector or mass detector etc..
The present invention a kind of mode of operation be：Buffer solution is injected capillary by syringe pump with constant pressure (1000mbar)
In, 5min is rinsed, after signal base line stabilization, pause is rinsed.Sampling pump uses a certain amount of sample of constant pressure (50mbar) sample introduction again
Product suspend sample introduction.Syringe pump applies separation voltage to use constant pressure to inject dissociating buffer.Using optical detection
Device detects signal, records neutral marker's appearance time t respectivelyMWith sample appearance time t, terminate mobility electrophoretic analysis.Herein
Used buffer solution can be certain volume than methanol aqueous solution (contain 0.1% formic acid).Fluorescence detector is specifically as follows
Ultraviolet specrophotometer.
102, detector is detected in the detection window of capillary, obtains the appearance time t of neutral markerMWith sample
Appearance time t.
103, basisObtain sample equivalent sphere radial features curve；
Wherein under experimental conditions,U is that separation voltage, L added by capillary both ends are the complete of capillary
Long, Lreal be capillary sample inlet end to the length of detection window (as shown in Figure 2), q be ion band electricity, η is solution viscosity
Coefficient, the molal weight that m is sample material, R are sample ions equivalent sphere radius.The detection window can be opened as shown in Figure 2
It is located on the tube body of capillary, can also be carried out in sample outlet end setting detector using the sample outlet end of capillary as detection window
Detection.
The mixing sample of (such as capillary) in microfluidic channels does with current-carrying under the traction of advection incompressible fluid and at the uniform velocity transports
It is dynamic, each group balance-dividing speed per hour degree and current-carrying speed vcarrierUnanimously.If mixing sample can dissociate as ion, by applying electricity
Field is allowed to by electric field force (FE) movement generated relatively with fluid is acted on, this process leads to dissociate component by resistance (Ff), then
Two kinds of effects can be expressed as：
FE=qE, Ff=6 π η rpV (formula 1)
Wherein E is electric field strength, and q is sample ions carried charge, and η is solution viscous drag coefficient, rpFor sample equivalent sphere
Body radius, v are sample fluid relative movement velocity.
(the i.e. F when electric field force and resistance reach balanceE=Ff), ion and current-carrying relative velocity v are constant, at this time ion table
See speed (vE) be：
vE=vcarrier+ v (formula 2)
Superficial velocity is related with equivalent sphere radius to ion live-wire property, the superficial velocities of different ions difference,
It is being time t differences used after L split tunnels, i.e. realization separation by a segment length.
According to Newtonian mechanics principle and principle of electrophoresis, sample ions migration distance y and sample ions transit time t, sample
There is correspondence between ion equivalent sphere radius R.
Specifically, ion motion meets the differential equation：
Y " [t]+hy ' [t]=k (formula 3)
Wherein：The π η R of ξ=6,
The differential equation of solution formula 3 can get ion motion analytical expression：
Wherein y is ion motion displacement, and U, L, Lreal, m, q, η are known quantity, t under experimental conditionsM, t can be from
It tests mobility electrophoretogram to obtain, will measure and be substituted into formula 4 above, sample equivalent sphere radial features curve, such as Fig. 3 can be obtained
It is shown.
104, the appearance time t of sample is matched to sample equivalent sphere radial features curve, obtain corresponding sample from
Sub- equivalent sphere radius R.
The sample equivalent sphere radial features curve of different samples is different.When the parameter of experiment condition changes, need
Reacquire sample equivalent sphere radial features curve.
Under specific condition of experiment, the effective range of sample equivalent sphere radial features curve is different, target sample ion position
Dynamic range in curve corner is wider, and resolution ratio is higher.
105, it according to sample ions equivalent sphere radius R, obtains sample ellipse and reconstructs restrictive curve.
Ellipsoid forms of motion in low reynolds number fluid is similar to sphere.If homogeneous ellipsoid in three directions of x, y, z half
Axial length is respectively a, b, c.It is that revolution is oval (such as long ball or oblate spheroid) to degenerate when ellipsoid, i.e., in a, b, c situations such as both arbitrary phase,
More biological sample structure information can be obtained under conditions of simple computation.
Such as assume that fluid is parallel to rotating shaft, and b=c, defines length-width ratioThen work as φ<It is oblate spheroid when 1, works as φ>1
When be long ball.At this point, the expression of oval feature parameter can be used in sample ions equivalent sphere radius R：
According to formula 5 and formula 6, the equivalent sphere radius R that each liquid phase mobility measures is brought into, it is ellipse thus to obtain sample
Circle reconstruct restrictive curve (such as Fig. 4 c), two dimension is extended to by sample structure resolution.
106, the conformation library of sample is obtained using molecular simulation and and then obtains the length-width ratio of conformation and equivalent radius windward.
The distribution on restrictive curve is reconstructed in ellipse for the conformation of sample under clear experiment condition, molecular simulation can be used
Obtain further data.
Specifically, simulated conditions are set as consistent with experiment condition, emulate to obtain sample using molecular dynamics simulation
Possibility conformation library.For example Gromacs program bags may be used and carry out molecular dynamics simulation.It is imitative using molecular dynamics simulation
Genuine simulation time can be 100ns.
Hereafter, the ellipse of possible conformation is obtained according to the radius of gyration in three directions of x, y, z in the possibility conformation library of sample
Characteristic parameter.
The length-width ratio of possible conformation and the equivalent radius windward are obtained based on oval feature parameter.
According to formula：
Wherein miIt is the quality of atom i, riIt is the location of atom i, rixIt is the position in the x direction of atom i, riy
It is the position in y-direction of atom i, rizIt is the position in a z-direction of atom i, can get the radius of gyration of these conformations
(radius of gyration, Rg) and all directions on the radius of gyration (RgX, RgY, RgZ)。
There is all directions upper rotary radius satisfaction again：
The ellipse of these possible conformations can be calculated by solving equation with three unknowns group (formula 11, formula 12, formula 13)
Characteristic parameter a, b, c further seek the length-width ratio for calculating possible conformationWith equivalent radius windward
107, the length-width ratio of conformation and equivalent radius windward are matched to sample ellipse and reconstruct restrictive curve, obtain experiment item
The conformation distribution of sample under part.
By φ and rpIt is brought into oval reconstruct restrictive curve, obtains the conformation distribution of sample under experiment condition.(as schemed
4g)
Further, the three-dimensional conformation more to tally with the actual situation in order to obtain, it is provided in an embodiment of the present invention to be based on mobility
The method of electrophoretic determination matter dimensions still further comprises：
Determine that arbitrary possible conformation reconstructs distance d between restrictive curve with sample ellipse in y-directionOr relative distanceWhereinFor y of i-th kind of conformation in figure
Direction distance, yiFor the directions y distance on the corresponding oval reconstruct restrictive curve of i-th kind of conformation x position.(shown in such as Fig. 4 (g))
Scoring criterion is set as when d or Δ d are smaller, corresponding conformation more tallies with the actual situation.
Determine that the possibility conformation of d or Δ d minimums is distributed for the conformation of sample under experiment condition.
The method based on mobility electrophoretic determination matter dimensions that technical solution of the present invention proposes passes through experiment and analogue simulation
The mode being combined realizes the prediction of the molecular conformation under solution condition.It can be widely applied to the survey of structure of biological macromolecule
In fixed, the assay method of structure of biological macromolecule has been expanded.This method can carry out on commercial wool capillary electrophoresis instrument, also can be certainly
Row is built to be realized in equipment.The embodiment of the present invention realizes the separation of mixing sample and the disposable restructuring analysis of multiple component structure, point
Analysis cost is relatively low, and analyze speed is fast, easily operated.The basic structure information that biomolecule mixing sample may be implemented in this method obtains
, guidance is provided with analysis for the prediction of follow-up high resolution structure, the time in fine structure parsing and fund cost can be saved.
The method based on mobility electrophoretic determination matter dimensions of the embodiment of the present invention is carried out with reference to specific embodiment
Explanation.
Embodiment 1
The structure of mobility electrophoretic determination growth hormone release inhibiting hormone (somatostatin).
Mobility electrophoresis experiment condition：Sample is the growth hormone release inhibiting hormone of 1mg/mL, and buffer solution is 50% methanol aqueous solution (w:W contains
0.1% formic acid, pH of buffer 3.0), 0.5% (w is added:W) phenol is uniformly mixed as neutral marker.Capillary pipe range
The distance of 50cm, sample introduction end to detection window are 40cm, 75 μm of bore, 214nm length ultraviolet spectrophotomelric assays.Operation side
Formula is：Buffer solution 180s is passed through using 1000mbar air pressures and rinses capillary interior conduit；Using 50mbar air pressure sample introductions 5s；
Capillary both ends apply air pressure 60mbar, and separation voltage is -20kV.Shown in experimental result such as Fig. 4 (a).According under experiment condition
Sample equivalent sphere radial features curve (formula 4) can obtain the equivalent sphere radius R of growth hormone release inhibiting hormone, as a result as shown in Fig. 4 (b).
Equivalent sphere radius R is substituted into formula 5, formula 6 can obtain growth hormone release inhibiting hormone ellipse under experiment condition and reconstruct restrictive curve, i.e.,
The relational expression of φ and c are obtained, as a result as shown in Fig. 4 (c).
Molecular simulation condition：Growth hormone release inhibiting hormone possible conformation under solution condition is obtained by database, as shown in Fig. 4 (d).
The radius of gyration of these conformations in three directions is calculated, result of calculation such as Fig. 4 (e) from which further follows that these possible conformations
Oval feature parameter a, b, c, result of calculation such as Fig. 4 (f).The possibility conformation parameter that molecular simulation is emulated substitutes into experiment meter
Obtained growth hormone release inhibiting hormone ellipse reconstruct restrictive curve, such as Fig. 4 (g).Further, as shown in figure 5, being according to growth with d
10 kinds of conformations of chalone are given a mark.D is smaller, and conformation more tallies with the actual situation.
Embodiment 2
The structure of mobility electrophoretic determination angiotensinⅠ (angiotensin I).
Mobility electrophoresis experiment condition：Sample is the angiotensinⅠ of 1mg/mL, and buffer solution is 50% methanol aqueous solution (w:w
Containing 0.1% formic acid, pH of buffer 3.0), 0.5% (w is added:W) phenol is uniformly mixed as neutral marker.Microcapillary tube
Long 50cm (distance at sample introduction end to detection window is 40cm), 75 μm of bore, 214nm length ultraviolet spectrophotomelric assays.Behaviour
It is as mode：Buffer solution 180s is passed through using 1000mbar air pressures and rinses split tunnel；Using 50mbar air pressure sample introductions 5s；
Split tunnel both ends apply air pressure 60mbar, and separation voltage is -20kV.Repeat this operation 5 times, experimental result such as Fig. 6 (c) institutes
Show, the repeatability of 5 groups of parallel tests is good.Blood vessel is calculated according to equivalent radius of sphericity indicatrix (formula 4) under experiment condition
The equivalent sphere radius R of Angiotensin Converting Enzyme I, result of calculation areEquivalent sphere radius R is substituted into formula 5
With formula 6, angiotensinⅠ ellipse reconstruct restrictive curve can be obtained under experiment condition to get to the relational expression of φ and c, as a result
As shown in Fig. 6 (d).
Molecular simulation condition：AngiotensinⅠ possible conformation under the solution condition is obtained by molecule simulation method,
Emulation experiment is carried out using 2016.1 program bags of GROMACS, the protein field of force uses AMBER99SB-ildn, AmberTools
16 for building polypeptide initial configuration, dissociated state when setting polypeptide as pH=3, and counter ion counterionsl gegenions are formic acid anion.Methanol-water
(1:1) as solvent box, solvent box size is aboutEnergy minimization 10ns at a temperature of 300K, dynamics calculation
100ns.The radius of gyration of obtained conformation library in three directions is calculated, shown in result of calculation such as Fig. 6 (a).By solution side
Journey group calculates oval feature parameter a, b, c of these possible conformations, shown in result of calculation such as Fig. 6 (b).Molecular simulation is emulated
Obtained possibility conformation substitutes into the angiotensin I ellipse reconstruct restrictive curve that experimental calculation obtains, as shown in Fig. 6 (d).Pass through
Marking (minimum range d is standard) obtains the possible three-dimensional conformation of angiotensin I under experiment condition, as shown in Fig. 6 (e).
Embodiment 3
The structure of mobility electrophoretic determination bradykinin (bradykinin).
Mobility electrophoresis experiment condition：Sample is the bradykinin of 1mg/mL, and buffer solution is 50% methanol aqueous solution (w:W contains
0.1% formic acid, pH of buffer 3.0), 0.5% (w is added:W) phenol is uniformly mixed as neutral marker.Capillary pipe range
50cm (distance at sample introduction end to detection window is 40cm), 75 μm of bore, 214nm length ultraviolet spectrophotomelric assays.Operation
Mode is：Buffer solution 180s is passed through using 1000mbar air pressures and rinses split tunnel；Using 50mbar air pressure sample introductions 5s；Dividing
Apply air pressure 60mbar from channel both ends, separation voltage is -20kV.Repeat this operation 5 times, shown in experimental result such as Fig. 7 (c), 5
The repeatability of group parallel test is good.According to equivalent radius of sphericity indicatrix (formula 4) under experiment condition calculate bradykinin etc.
Radius of sphericity R is imitated, result of calculation isEquivalent sphere radius R is substituted into formula 5, formula 6 can obtain
It obtains bradykinin ellipse under experiment condition and reconstructs restrictive curve to get to the relational expression of φ and c, as a result such as Fig. 7 (d) is shown.
Molecular simulation condition：Bradykinin possible conformation under the solution condition is obtained by molecule simulation method, is used
2016.1 program bags of GROMACS carry out emulation experiment, and the protein field of force uses AMBER99SB-ildn, AmberTools 16 to use
In structure polypeptide initial configuration, dissociated state when setting bradykinin as pH=3, counter ion counterionsl gegenions are formic acid anion.Methanol-water (1:
1) as solvent box, solvent box size is aboutEnergy minimization 10ns at a temperature of 300K, dynamics calculation
100ns.The radius of gyration of obtained conformation library in three directions is calculated, shown in result of calculation such as Fig. 7 (a).By solution side
Journey group calculates oval feature parameter a, b, c of these possible conformations, shown in result of calculation such as Fig. 7 (b).Molecular simulation is emulated
Obtained possibility conformation substitutes into the bradykinin ellipse reconstruct restrictive curve that experimental calculation obtains, as shown in Fig. 7 (d).Pass through marking
(minimum range d is standard) obtains the possible three-dimensional conformation of bradykinin under experiment condition, as shown in Fig. 7 (e).
Embodiment 4
The structure of mobility electrophoretic determination mixed polypeptide sample.
Mobility electrophoresis experiment condition：Sample is the bradykinin of 1mg/mL, the growth hormone release inhibiting hormone of 1mg/mL, buffer solution 2mM
0.5% (w is added in NaCl aqueous solutions (pH 7.0):W) phenol is uniformly mixed as neutral marker.Capillary pipe range 50cm
(distance at sample introduction end to detection window is 40cm), 75 μm of bore, 214nm length ultraviolet spectrophotomelric assays.Mode of operation
For：Buffer solution 180s is passed through using 1000mbar air pressures and rinses split tunnel；Using 50mbar air pressure sample introductions 5s；It is logical in separation
Road both ends apply air pressure 60mbar, and separation voltage is -25kV.This operation 5 times is repeated, shown in experimental result such as Fig. 8 (a), 5 groups flat
The repeatability of row experiment is good.The equivalent sphere of bradykinin is calculated according to equivalent radius of sphericity indicatrix (formula 4) under experiment condition
Body radius R, result of calculation areThe equivalent sphere radius R of growth hormone release inhibiting hormone is calculated, result of calculation isEquivalent sphere radius R is substituted into formula 5, formula 6 can obtain two kinds of polypeptides under experiment condition
Ellipse reconstruct restrictive curve is to get to the relational expression of φ and c, and as a result such as Fig. 8 (b) and 8 (c) are shown.
Molecular simulation condition：Bradykinin possible conformation under the solution condition is obtained by molecule simulation method, is used
2016.1 program bags of GROMACS carry out emulation experiment, and the protein field of force uses AMBER99SB-ildn, AmberTools 16 to use
In structure polypeptide initial configuration, dissociated state when setting bradykinin as pH=7, counter ion counterionsl gegenions Cl-Ion.Water is as solvent box
Son, solvent box size are aboutEnergy minimization 10ns at a temperature of 300K, dynamics calculation 100ns.Calculating is obtained
The radius of gyration of conformation library in three directions, result of calculation such as Fig. 9 (a) and 9 (c) are shown.It is calculated by solving equation group
Oval feature parameter a, b, c of these possible conformations, result of calculation such as Fig. 9 (b) and 9 (d) are shown.Molecular simulation is emulated to obtain
Possibility conformation substitute into the bradykinin ellipse reconstruct restrictive curve that experimental calculation obtains, as shown in Fig. 8 (b) and 8 (c).By beating
(minimum range d is standard) is divided to obtain the possible three-dimensional conformation of bradykinin under experiment condition, as shown in Fig. 8 (d).
Although the present invention and its advantage has been described in detail it should be appreciated that without departing from by the attached claims
Defined by can carry out various changes, replacement and transformation in the case of the spirit and scope of the present invention.Moreover, the model of the application
Enclose the specific embodiment for being not limited only to process, equipment, means, method and steps described in specification.In the art is common
Technical staff executes and corresponding reality described herein from the disclosure it will be readily understood that can be used according to the present invention
Apply the essentially identical function of example or obtain the result essentially identical with it, existing and process to be developed in future, equipment,
Means, method or step.Therefore, the attached claims are intended to include such process, equipment, hand in the range of them
Section, method or step.
Claims (5)
1. a kind of method based on mobility electrophoretic determination matter dimensions, which is characterized in that including：
Syringe pump is injected to the sample introduction end of capillary buffer solution and continues first time period, and sampling pump is to the capillary later
Sample solution is injected at sample introduction end, and the syringe pump injects buffer solution to the sample introduction end of the capillary again after the completion of sample introduction, together
When apply separation voltage between the both ends of the capillary；
Detection window of the detector on the capillary is detected, and obtains the appearance time t of neutral markerMWith sample
Appearance time t；
According toObtain sample equivalent sphere radial features curve；Wherein testing
Under the conditions of,U be separation voltage, L added by capillary both ends be the overall length of capillary, Lreal is capillary sample inlet
It is ion band electricity to hold the length of the detection window, q, η is solution viscosity coefficient, the molal weight that m is sample material, R
For sample ions equivalent sphere radius；
The appearance time t of the sample is matched to the sample equivalent sphere radial features curve, obtains the corresponding sample
Product ion equivalent sphere radius R；
According to the ion equivalent sphere radius R, obtains sample ellipse and reconstruct restrictive curve；
Using molecular simulation obtain the possibility conformation library of the sample and obtain in turn may conformation length-width ratio and it is equivalent windward
Radius；
The length-width ratio of the possible conformation and the equivalent radius windward are matched to the sample ellipse and reconstruct restrictive curve, is obtained
Obtain the conformation distribution of sample under experiment condition.
2. the method according to claim 1 based on mobility electrophoretic determination matter dimensions, which is characterized in that described use is divided
The quasi- possibility conformation library for obtaining the sample of submodule simultaneously obtains the length-width ratio of possible conformation and equivalent radius windward in turn, specific to wrap
It includes：
Simulated conditions are set as consistent with the experiment condition, using molecular dynamics simulation emulate to obtain the sample can
It can conformation library；
The oval feature ginseng of possible conformation is obtained according to the radius of gyration in three directions of x, y, z in the possibility conformation library of the sample
Number；
The length-width ratio of the possible conformation and the equivalent radius windward are obtained based on the oval feature parameter.
3. the method according to claim 1 based on mobility electrophoretic determination matter dimensions, which is characterized in that the method is also
Including：
Determine that arbitrary possible conformation reconstructs in y-direction with the sample ellipse at a distance between restrictive curveOr relative distance
Determine diOr Δ diMinimum possibility conformation is the conformation distribution of sample under experiment condition.
4. the method according to claim 2 based on mobility electrophoretic determination matter dimensions, which is characterized in that use Molecule Motion
The simulation time of mechanical simulation emulation is 100ns.
5. the method according to claim 1 based on mobility electrophoretic determination matter dimensions, which is characterized in that the detector
For fluorescence detector or mass detector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810535443.9A CN108663428A (en) | 2018-05-29 | 2018-05-29 | A method of based on mobility electrophoretic determination matter dimensions |
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| CN112147334A (en) * | 2019-06-26 | 2020-12-29 | 北京理工大学 | Method and system for characterizing protein shape |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050130157A1 (en) * | 2002-01-22 | 2005-06-16 | Philip Serwer | Electrophoretic ratchets and cyclic electrophoresis |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN103018317A (en) * | 2013-01-04 | 2013-04-03 | 中国药科大学 | Novel non-standard-dependence quantitative analysis method based on study on homologous/similar compound structure-mass-spectrum response relationship |
| WO2014026296A1 (en) * | 2012-08-17 | 2014-02-20 | Zymeworks Inc. | Systems and methods for sampling and analysis of polymer conformational dynamics |
| CN108008053A (en) * | 2016-12-05 | 2018-05-08 | 北京理工大学 | A kind of liquid phase mobility separator and control method and with liquid chromatogram and the interface of mass spectrometry |
| CN108007994A (en) * | 2017-06-27 | 2018-05-08 | 北京理工大学 | A kind of method for measuring Capillary Electrophoresis electroosmotic flow |
-
2018
- 2018-05-29 CN CN201810535443.9A patent/CN108663428A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050130157A1 (en) * | 2002-01-22 | 2005-06-16 | Philip Serwer | Electrophoretic ratchets and cyclic electrophoresis |
| WO2014026296A1 (en) * | 2012-08-17 | 2014-02-20 | Zymeworks Inc. | Systems and methods for sampling and analysis of polymer conformational dynamics |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN103018317A (en) * | 2013-01-04 | 2013-04-03 | 中国药科大学 | Novel non-standard-dependence quantitative analysis method based on study on homologous/similar compound structure-mass-spectrum response relationship |
| CN108008053A (en) * | 2016-12-05 | 2018-05-08 | 北京理工大学 | A kind of liquid phase mobility separator and control method and with liquid chromatogram and the interface of mass spectrometry |
| CN108007994A (en) * | 2017-06-27 | 2018-05-08 | 北京理工大学 | A kind of method for measuring Capillary Electrophoresis electroosmotic flow |
Non-Patent Citations (3)
| Title |
|---|
| CHANKVETADZE BEZHAN等: "Mechanistic study on the opposite migration orderof the enantiomers of ketamine witha- andb-cyclodextrin in capillary electrophoresis", 《JOURNAL OF SEPARATION SCIENCE》 * |
| O"BRIEN R. W.等: "Electrophoretic mobility of a spherical colloidal particle", 《JOURNAL OF THE CHEMICAL SOCIETY, FARADAY TRANSACTIONS 2: MOLECULAR AND CHEMICAL PHYSICS》 * |
| TISSOT B.等: "Differentiation of the fucoidan sulfated L-fucose isomers constituents by CE-ESIMS and molecular modeling", 《CARBOHYDRATE RESEARCH》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112147334A (en) * | 2019-06-26 | 2020-12-29 | 北京理工大学 | Method and system for characterizing protein shape |
| CN112147334B (en) * | 2019-06-26 | 2021-08-13 | 北京理工大学 | Method and system for characterizing protein shape |
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