CN108663428A - A method of based on mobility electrophoretic determination matter dimensions - Google Patents

Abstract

The invention relates to the technical field of mobility electrophoresis, and discloses a method for determining the size of a substance based on mobility electrophoresis, which includes injecting a buffer solution into the sampling end of a capillary with a syringe pump for a first period of time, and then injecting a buffer solution into the capillary from the sampling pump. The sample end is injected with the sample solution, and after the sample injection is completed, the syringe pump injects the buffer solution into the sample injection end of the capillary again, and at the same time, a separation voltage is applied between the two ends of the capillary; the detector detects at the detection window of the capillary to obtain neutrality. The peak time t _M of the marker and the peak time t of the sample; obtain the sample equivalent sphere radius characteristic curve and then obtain the sample ion equivalent sphere radius R; obtain the sample ellipse reconstruction limit curve according to the sample ion equivalent sphere radius R , and then obtain the conformational distribution of the sample under the experimental conditions. The invention is easy to operate, fast in analysis speed and can be carried out simultaneously with the separation of the mixture.

Description

A method for determining the size of substances based on mobility electrophoresis

technical field

The invention relates to the technical field of mobility electrophoresis, in particular to a method for determining the size of a substance based on mobility electrophoresis.

Background technique

Biological macromolecules are closely related to life activities, and the analysis of the structure of macromolecules is helpful to understand their functions and mechanisms. Common structural elucidation techniques include X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. When using the above-mentioned techniques to analyze the structure, it is necessary to obtain high-purity and large-scale samples first, the pre-processing steps are relatively cumbersome, and the reconstruction after data collection also faces problems such as a large amount of data. Therefore, there is a need for a solution with high sensitivity and the ability to analyze the structure of complex components. In addition, the use of auxiliary means to first determine the basic size and conformation of substances can help the prediction and characterization of subsequent high-resolution structures.

Contents of the invention

In order to overcome the above technical problems, the present invention provides a method for determining the size of substances based on mobility electrophoresis, which is easy to operate, fast in analysis speed, and can be carried out simultaneously with the separation of mixtures.

In order to achieve the above object, the present invention provides a method for determining the size of a substance based on mobility electrophoresis, including:

The syringe pump injects the buffer solution into the sampling end of the capillary for a first period of time, and then the sampling pump injects the sample solution into the sampling end of the capillary. After the sampling is completed, the syringe pump injects the sample into the capillary again. Inject a buffer into the capillary while applying a separation voltage between the two ends of the capillary;

The detector detects at the detection window on the capillary, and obtains the peak time t _M of the neutral marker and the peak time t of the sample;

according to Obtain the sample equivalent spherical radius characteristic curve; where under the experimental conditions, U is the separation voltage applied at both ends of the capillary, L is the full length of the capillary, Lreal is the length from the sampling end of the capillary to the detection window, q is the ion charge, η is the viscosity coefficient of the solution, and m is the molar mass of the sample substance , R is the radius of the sample ion equivalent sphere;

Matching the peak-exit time t of the sample to the sample equivalent sphere radius characteristic curve to obtain the corresponding ion equivalent sphere radius R of the sample;

Obtain a sample ellipse reconstruction limit curve according to the ion equivalent sphere radius R;

Using molecular simulation to obtain the possible conformation library of the sample and then obtain the aspect ratio and equivalent windward radius of the possible conformation;

The aspect ratio of the possible conformation and the equivalent windward radius are matched to the ellipse reconstruction limit curve of the sample to obtain the conformational distribution of the sample under the experimental conditions.

In an optional embodiment, the molecular simulation is used to obtain the possible conformation library of the sample, and then the aspect ratio and the equivalent windward radius of the possible conformation are obtained, specifically including:

Setting the simulation conditions to be consistent with the experimental conditions, and using molecular dynamics simulation to obtain a possible conformation library of the sample;

According to the radius of gyration in the three directions of x, y, and z of the possible conformation library of the sample, the ellipse characteristic parameters of the possible conformation are obtained;

The aspect ratio of the possible conformation and the equivalent windward radius are obtained based on the ellipse characteristic parameters.

In an optional embodiment, the method also includes:

Determine the distance between any possible conformation and the ellipse reconstruction limit curve of the sample in the y direction or relative distance

Determine the smallest possible conformation of d _i or Δd _i as the conformation distribution of the sample under the experimental conditions.

In an optional implementation manner, the simulation time of molecular dynamics simulation is 100 ns.

In an optional embodiment, the detector is an optical detector or a mass spectrometer detector.

In the method for determining the size of a substance based on mobility electrophoresis described in the present invention, the syringe pump injects the buffer solution into the sampling end of the capillary for a first period of time, and then the sampling pump injects the sample solution into the sampling end of the capillary, and the sampling is completed After that, the syringe pump injects the buffer solution into the sampling end of the capillary again, and simultaneously applies a separation voltage between the two ends of the capillary; the detector detects at the detection window of the capillary, and obtains the peak time t _M of the neutral marker and The peak time t of the sample; obtain the characteristic curve of the sample equivalent sphere radius and then obtain the sample ion equivalent sphere radius R; obtain the sample ellipse reconstruction limit curve according to the sample ion equivalent sphere radius R, and then obtain the conformation of the sample under the experimental conditions distributed. The technical scheme of the present invention separates the mixture in the solution through liquid-phase mobility electrophoresis, and at the same time detects in the detection window to obtain the fine size and structure information of the substance in the solution, and realizes the simultaneous separation of the substance in the solution during the separation of complex samples. Inspection of dimensional structures. The technical scheme of the invention has high detection accuracy, simple operation and fast analysis speed, and realizes the separation of mixed samples and one-time reconstruction analysis of multi-component structures.

Description of drawings

Fig. 1 is the flowchart of the method for determining the size of a substance based on mobility electrophoresis according to the present invention;

Fig. 2 is a kind of device schematic diagram provided by the present invention;

Fig. 3 is the relationship curve of theoretical equivalent radius and migration time under three kinds of separation conditions;

Fig. 4 is the analytical diagram of determining the size of somatostatin by the method of the present invention;

Figure 5 shows the scoring sequence diagram of 10 conformations of somatostatin obtained by simulation;

Fig. 6 is the analytical diagram of determining the size of angiotensin I by the method of the present invention;

Fig. 7 is the analytical diagram of determining the size of bradykinin by the method of the present invention;

Figure 8 is an analysis diagram for determining the size of mixed polypeptides by the method of the present invention;

Fig. 9 is a molecular dynamics simulation diagram of determining mixed polypeptides by the method of the present invention.

Detailed ways

Embodiments of the present invention are described below with reference to the drawings. Elements and features described in one drawing or one embodiment of the present invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that representation and description of components or processes that are not relevant to the present invention and known to those of ordinary skill in the art are omitted from the drawings and descriptions for the purpose of clarity.

Ion mobility mass spectrometry is a new type of two-dimensional mass spectrometry analysis technology that combines ion mobility separation technology with mass spectrometry in the gas phase. The ion migration rate is measured using an electric field gradient and a laterally flowing buffer gas, and the migration rate depends on the ion's collision cross section and charge state. It can realize the rapid separation and detection of complex samples in the gas phase, and can also obtain the structural information of substances under the gas phase conditions. Capillary electrophoresis technology uses a capillary as a separation channel in a liquid phase environment. Under the drive of an electric field, ions are efficiently and quickly separated according to their respective mobility (the average electrophoretic velocity of charged particles under a unit electric field strength).

The theory of liquid phase mobility is proposed on the basis of gas phase ion mobility spectrometry combined with electrophoretic separation technology. In the embodiment of the present invention, the charged substances in the solution are separated based on mobility electrophoresis and the size of the substances is measured. The buffer liquid is provided by a flow syringe pump, and the separation channel is a capillary. The separation is realized through the interaction between the electric field and the fluid resistance, and the mobility electrophoresis spectrogram. The embodiment of the present invention provides a method for determining the size of a substance based on mobility electrophoresis, as shown in Figure 1, the method includes:

101. The syringe pump injects the buffer solution into the sampling end of the capillary for the first period of time, after which the sampling pump injects the sample solution into the sampling end of the capillary, and the syringe pump injects the buffer solution into the sampling end of the capillary again after the injection is completed , while applying a separation voltage between the two ends of the capillary.

First, clarify the experimental conditions of mobility electrophoresis. The mobility electrophoresis scheme can be implemented on commercial microflow analysis instruments (such as capillary electrophoresis apparatus), or on a simple separation channel built by oneself. Typically a separation capillary is used as the separation channel. A peristaltic pump can be used as a syringe pump to inject a constant velocity buffer flow into the inlet section of the capillary, and a sample pump to inject the sample solution into the inlet section of the capillary. The high-voltage power supply is connected to the electrode to provide the separation voltage, and a detection window is set on the capillary, and a detector is placed to measure the peak time of the sample, etc. The first period of time is specifically 0.1 minutes to 10 minutes.

Various inner and outer diameter capillaries can be used for the capillary. When separating proteins, use coated capillaries. The output range of the DC high-voltage power supply is 0~+/-30000V, and the high-voltage power supply can be connected with the electrode device through wires. Syringe pump, providing 0-1000mbar constant pressure or equivalent flow. The detector can be an optical detector or a mass spectrometer, etc.

One operation mode of the present invention is: the syringe pump injects the buffer solution into the capillary at a constant pressure (1000 mbar), flushes for 5 minutes, and suspends flushing after the signal baseline is stable. The injection pump injects a certain amount of sample with a constant pressure (50mbar), and then pauses the injection. The syringe pump injects the separation buffer using a constant pressure while applying the separation voltage. The optical detector is used to detect the signal, and the peak time t _M of the neutral marker and the peak time t of the sample are recorded respectively, and the mobility electrophoresis analysis is ended. The buffer used here can be methanol aqueous solution (containing 0.1% formic acid) in a certain volume ratio. Specifically, the optical detector can be an ultraviolet spectrophotometer.

102. The detector performs detection in the detection window of the capillary, and obtains the peak time t _M of the neutral marker and the peak time t of the sample.

103. According to Obtain the sample equivalent spherical radius characteristic curve;

Among them, under the experimental conditions, U is the separation voltage applied at both ends of the capillary, L is the full length of the capillary, Lreal is the length from the sampling end of the capillary to the detection window (as shown in Figure 2), q is the ion charge, η is the viscosity coefficient of the solution, and m is The molar mass of the sample substance, R is the radius of the equivalent sphere of the sample ion. The detection window can be set on the tube body of the capillary as shown in Figure 2, or the sample outlet end of the capillary can be used as the detection window, and a detector can be arranged at the sample outlet for detection.

The mixed sample in the microfluidic flow channel (such as a capillary) moves at a uniform speed with the carrier current under the traction of the advective incompressible fluid, and the velocity of each component is consistent with the carrier velocity v _carrier when the components are balanced. If the mixed sample can be dissociated into ions, by applying an electric field to make it subject to the electric field force (F _E ) to generate relative motion with the fluid, this process causes the dissociated components to be resisted (F _f ), then the two effects can be expressed respectively for:

F _E =qE, F _f =6πηr _p v (Formula 1)

Where E is the electric field strength, q is the ion charge of the sample, η is the solution viscous resistance coefficient, r _p is the radius of the equivalent sphere of the sample, and v is the velocity of the relative fluid movement of the sample.

When the electric field force and the resistance are in balance (ie F _E =F _f ), the relative velocity v between the ion and the carrier current is constant, and the apparent velocity of the ion (v _E ) at this time is:

v _E = v _carrier + v (Formula 2)

The superficial velocity and ion charging properties are related to the radius of the equivalent sphere. The superficial velocity of different ions is different. After passing through a separation channel with a length of L, the time t used is different, that is, the separation is achieved.

According to the principle of Newtonian mechanics and electrophoresis, there is a corresponding relationship between the migration distance y of the sample ion, the migration time t of the sample ion, and the radius R of the equivalent sphere of the sample ion.

Specifically, the ion motion satisfies the differential equation:

y″[t]+hy′[t]=k (Formula 3)

in: ξ=6πηR,

Solving the differential equation of Equation 3, the analytical expression of ion motion can be obtained:

Wherein y is ion movement displacement, under experimental conditions U, L, Lreal, m, q, η are all known quantities, t _M , t can be obtained from experimental mobility electrophoresis diagram, and above quantity is substituted in the formula 4, can The characteristic curve of the equivalent spherical radius of the sample is obtained, as shown in Figure 3.

104. Match the peak exit time t of the sample to the characteristic curve of the sample equivalent sphere radius to obtain the corresponding sample ion equivalent sphere radius R.

The sample equivalent spherical radius characteristic curves of different samples are different. When the parameters of the experimental conditions change, the characteristic curve of the equivalent spherical radius of the sample needs to be obtained again.

Under specific experimental conditions, the effective range of the sample equivalent spherical radius characteristic curve is different, and the wider the dynamic range of the target sample ion at the corner of the curve, the higher the resolution.

105. Obtain the sample ellipse reconstruction limit curve according to the radius R of the sample ion equivalent sphere.

Ellipsoids behave similarly to spheres in low Reynolds number fluids. Let the semi-axis lengths of the uniform ellipsoid in the x, y, and z directions be a, b, and c, respectively. When the ellipsoid degenerates into an ellipse of revolution (such as a prolate sphere or an oblate sphere), that is, when any two of a, b, and c are equal, more structural information of biological samples can be obtained under simple calculation conditions.

Assuming that the fluid is parallel to the axis of rotation, and b=c, define the aspect ratio Then when φ<1, it is an oblate sphere, and when φ>1, it is a prolate sphere. At this time, the radius R of the sample ion equivalent sphere can be expressed by the ellipse characteristic parameters:

According to Equation 5 and Equation 6, the equivalent spherical radius R of each liquid-phase mobility measurement is brought in, thereby obtaining the sample ellipse reconstruction limit curve (as shown in Figure 4c), and expanding the resolution of the sample structure to two dimensions.

106. Use molecular simulation to obtain the conformation library of the sample, and then obtain the aspect ratio and equivalent windward radius of the conformation.

In order to clarify the distribution of the conformation of the sample on the elliptical reconstruction limit curve under the experimental conditions, further data can be obtained by molecular simulation.

Specifically, the simulation conditions are set to be consistent with the experimental conditions, and molecular dynamics simulation is used to obtain the possible conformation library of the sample. For example, the Gromacs program package can be used for molecular dynamics simulation. The simulation time of the molecular dynamics simulation can be 100 ns.

Thereafter, according to the radius of gyration in the x, y, and z directions of the possible conformation library of the sample, the ellipse characteristic parameters of the possible conformation are obtained.

The aspect ratio of the possible conformation and the equivalent windward radius are obtained based on the characteristic parameters of the ellipse.

According to the formula:

where m _i is the mass of atom i, r _i is the position of atom i, r _ix is the position of atom i in the x direction, r _iy is the position of atom i in the y direction, r _iz is the position of atom i According to the position in the z direction, the radius of gyration (radius of gyration, R _g ) of these conformations and the radius of gyration in each direction (R _gX , R _gY , R _gZ ) can be obtained.

And the radius of gyration in each direction satisfies:

By solving the ternary equations (Formula 11, Formula 12, Formula 13), the ellipse characteristic parameters a, b, and c of these possible conformations can be calculated, and the aspect ratio of the possible conformation can be further calculated and equivalent windward radius

107. Match the aspect ratio and equivalent windward radius of the conformation to the sample ellipse reconstruction limit curve to obtain the conformational distribution of the sample under the experimental conditions.

Bring φ and r _p into the elliptic reconstruction limit curve to obtain the conformational distribution of the sample under the experimental conditions. (as shown in Figure 4g)

Further, in order to obtain a more realistic three-dimensional conformation, the method for determining the size of a substance based on mobility electrophoresis provided by the embodiment of the present invention further includes:

Determine the distance d between any possible conformation and the sample ellipse reconstruction limit curve in the y direction or relative distance in is the distance in the y-direction of the i-th conformation in the figure, and y _i is the distance in the y-direction on the ellipse reconstruction limit curve corresponding to the x-position of the i-th conformation. (as shown in Figure 4(g))

The scoring standard is set to be that when d or Δd is smaller, the corresponding conformation is more in line with the actual situation.

Determine the possible conformation with the smallest d or Δd as the conformational distribution of the sample under the experimental conditions.

The method for measuring the size of a substance based on mobility electrophoresis proposed by the technical solution of the present invention realizes the prediction of molecular conformation under solution conditions through a combination of experiment and simulation. It can be widely applied to the determination of the structure of biological macromolecules, and expands the determination method of the structure of biological macromolecules. This method can be carried out on a commercial capillary electrophoresis instrument, and can also be realized on self-built equipment. The embodiment of the present invention realizes the separation of mixed samples and the one-time reconstruction analysis of multi-component structures, and the analysis cost is low, the analysis speed is fast, and the operation is easy. This method can realize the acquisition of basic structure information of biomolecular mixed samples, provide guidance for subsequent high-resolution structure prediction and analysis, and save time and capital costs in fine structure analysis.

The method for determining the size of a substance based on mobility electrophoresis in an embodiment of the present invention will be described below in conjunction with specific examples.

Example 1

The structure of somatostatin was determined by mobility electrophoresis.

Mobility electrophoresis experiment conditions: the sample is 1mg/mL somatostatin, the buffer is 50% aqueous methanol (w:w contains 0.1% formic acid, buffer pH 3.0), and 0.5% (w:w) phenol is added as neutral Marker, mix well. The length of the capillary tube is 50cm, the distance from the injection end to the detection window is 40cm, the inner diameter of the tube is 75μm, and the wavelength of 214nm is detected by ultraviolet spectrophotometry. The operation method is as follows: use 1000mbar air pressure to feed the buffer solution for 180s to flush the inner pipe of the capillary; use 50mbar air pressure to inject the sample for 5s; apply an air pressure of 60mbar at both ends of the capillary, and the separation voltage is -20kV. The experimental results are shown in Fig. 4(a). According to the sample equivalent spherical radius characteristic curve (Formula 4) under the experimental conditions, the equivalent spherical radius R of somatostatin can be obtained, and the results are shown in Figure 4(b). Substituting the equivalent spherical radius R into Equation 5 and Equation 6, the somatostatin ellipse reconstruction limit curve under experimental conditions can be obtained, that is, the relationship between φ and c can be obtained, and the result is shown in Figure 4(c).

Molecular simulation conditions: The possible conformation of somatostatin under solution conditions was obtained through the database, as shown in Figure 4(d). Calculate the radius of gyration of these conformations in three directions, the calculation results are shown in Figure 4(e), and further obtain the ellipse characteristic parameters a, b, c of these possible conformations, the calculation results are shown in Figure 4(f). Substitute the possible conformational parameters obtained from the molecular simulation into the somatostatin elliptical reconstruction limit curve obtained from the experimental calculation, as shown in Figure 4(g). Further, as shown in FIG. 5 , the 10 conformations of somatostatin were scored on the basis of d. The smaller d is, the more realistic the conformation is.

Example 2

The structure of angiotensin I was determined by mobility electrophoresis.

Mobility electrophoresis experiment conditions: the sample is 1 mg/mL angiotensin Ⅰ, the buffer is 50% methanol aqueous solution (w:w contains 0.1% formic acid, buffer pH 3.0), and 0.5% (w:w) phenol is added as the medium Sex markers, mix well. The length of the capillary tube is 50 cm (the distance from the injection end to the detection window is 40 cm), the inner diameter of the tube is 75 μm, and the wavelength of 214 nm is detected by ultraviolet spectrophotometry. The operation method is: use 1000mbar air pressure to inject buffer solution for 180s to flush the separation channel; use 50mbar air pressure to inject samples for 5s; apply air pressure at both ends of the separation channel to 60mbar, and the separation voltage is -20kV. Repeat this operation 5 times, the experimental results are shown in Figure 6(c), and the repeatability of the 5 parallel experiments is good. The equivalent spherical radius R of angiotensin I was calculated according to the equivalent spherical radius characteristic curve (formula 4) under the experimental conditions, and the calculation result was Substituting the equivalent spherical radius R into Equation 5 and Equation 6, the angiotensin I ellipse remodeling limit curve under experimental conditions can be obtained, that is, the relationship between φ and c can be obtained, and the result is shown in Figure 6(d).

Molecular simulation conditions: The possible conformation of angiotensin Ⅰ in this solution condition was obtained by molecular simulation method, and the GROMACS 2016.1 program package was used for simulation experiments. The protein force field was AMBER99SB-ildn, and AmberTools16 was used to construct the initial structure of the polypeptide and set the polypeptide It is in the dissociated state at pH=3, and the counter ion is formic acid anion. Methanol water (1:1) is used as the solvent box, and the size of the solvent box is about At 300K temperature, the energy minimization takes 10ns, and the kinetic calculation takes 100ns. Calculate the radius of gyration of the obtained conformation library in three directions, and the calculation results are shown in Fig. 6(a). The ellipse characteristic parameters a, b, and c of these possible conformations are calculated by solving the equations, and the calculation results are shown in Fig. 6(b). Substitute the possible conformation obtained by molecular simulation into the angiotensin I ellipse reconstruction limit curve obtained by experiment, as shown in Fig. 6(d). The possible three-dimensional conformation of angiotensin I under the experimental conditions was obtained by scoring (the minimum distance d was the standard), as shown in FIG. 6( e ).

Example 3

The structure of bradykinin was determined by mobility electrophoresis.

Mobility electrophoresis experimental conditions: the sample is 1mg/mL bradykinin, the buffer is 50% aqueous methanol (w:w contains 0.1% formic acid, buffer pH 3.0), and 0.5% (w:w) phenol is added as neutral Marker, mix well. The length of the capillary tube is 50 cm (the distance from the injection end to the detection window is 40 cm), the inner diameter of the tube is 75 μm, and the wavelength of 214 nm is detected by ultraviolet spectrophotometry. The operation method is: use 1000mbar air pressure to inject buffer solution for 180s to flush the separation channel; use 50mbar air pressure to inject samples for 5s; apply air pressure at both ends of the separation channel to 60mbar, and the separation voltage is -20kV. Repeat this operation 5 times, the experimental results are shown in Figure 7(c), and the repeatability of the 5 parallel experiments is good. Calculate the equivalent spherical radius R of bradykinin according to the equivalent spherical radius characteristic curve (Formula 4) under the experimental conditions, and the calculation result is Substituting the equivalent spherical radius R into Equation 5 and Equation 6, the bradykinin ellipse remodeling limit curve under experimental conditions can be obtained, that is, the relationship between φ and c can be obtained, and the result is shown in Figure 7(d).

Molecular simulation conditions: The possible conformation of bradykinin in this solution condition was obtained by molecular simulation method, and the GROMACS 2016.1 program package was used for simulation experiments. The protein force field was AMBER99SB-ildn, and AmberTools 16 was used to construct the initial structure of the polypeptide. The kinin is dissociated at pH=3, and the counter ion is formate anion. Methanol water (1:1) is used as the solvent box, and the size of the solvent box is about At 300K temperature, the energy minimization takes 10ns, and the kinetic calculation takes 100ns. Calculate the radius of gyration of the obtained conformation library in three directions, and the calculation results are shown in Fig. 7(a). The ellipse characteristic parameters a, b, c of these possible conformations are calculated by solving the equations, and the calculation results are shown in Fig. 7(b). Substitute the possible conformation obtained by molecular simulation into the elliptical reconstruction limit curve of bradykinin obtained by experiment, as shown in Fig. 7(d). The possible three-dimensional conformation of bradykinin under the experimental conditions was obtained by scoring (the minimum distance d was the standard), as shown in Figure 7(e).

Example 4

Mobility electrophoresis to determine the structure of mixed peptide samples.

Mobility electrophoresis experimental conditions: the sample is 1mg/mL bradykinin, 1mg/mL somatostatin, the buffer is 2mM NaCl aqueous solution (pH 7.0), add 0.5% (w:w) phenol as a neutral marker, mix uniform. The length of the capillary tube is 50 cm (the distance from the injection end to the detection window is 40 cm), the inner diameter of the tube is 75 μm, and the wavelength of 214 nm is detected by ultraviolet spectrophotometry. The operation method is: use 1000mbar air pressure to feed buffer solution for 180s to flush the separation channel; use 50mbar air pressure to inject samples for 5s; apply air pressure at both ends of the separation channel to 60mbar, and the separation voltage is -25kV. Repeat this operation 5 times, the experimental results are shown in Figure 8(a), and the repeatability of the 5 parallel experiments is good. Calculate the equivalent spherical radius R of bradykinin according to the equivalent spherical radius characteristic curve (Formula 4) under the experimental conditions, and the calculation result is Calculate the equivalent spherical radius R of somatostatin, and the calculation result is Substituting the equivalent sphere radius R into Equation 5 and Equation 6, the two polypeptide elliptic reconstruction limit curves under the experimental conditions can be obtained, that is, the relationship between φ and c can be obtained, and the results are shown in Figures 8(b) and 8(c) .

Molecular simulation conditions: The possible conformation of bradykinin in this solution condition was obtained by molecular simulation method, and the GROMACS 2016.1 program package was used for simulation experiments. The protein force field was AMBER99SB-ildn, and AmberTools 16 was used to construct the initial structure of the polypeptide. The kinin is dissociated at pH=7, and the counter ion is Cl ^- ion. Water serves as the solvent box, which measures approximately At 300K temperature, the energy minimization takes 10ns, and the kinetic calculation takes 100ns. Calculate the radius of gyration of the obtained conformation library in three directions, and the calculation results are shown in Figures 9(a) and 9(c). The ellipse characteristic parameters a, b, and c of these possible conformations are calculated by solving the equations, and the calculation results are shown in Figures 9(b) and 9(d). Substitute the possible conformations obtained from the molecular simulation into the elliptical reconstruction limit curve of bradykinin obtained from the experiment, as shown in Figures 8(b) and 8(c). The possible three-dimensional conformation of bradykinin under the experimental conditions is obtained by scoring (the minimum distance d is the standard), as shown in FIG. 8( d ).

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made hereto without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not limited to the specific embodiments of the procedures, devices, means, methods and steps described in the specification. Those of ordinary skill in the art will readily appreciate from the disclosure of the present invention that existing and future devices that perform substantially the same function or obtain substantially the same results as the corresponding embodiments described herein can be used in accordance with the present invention. The developed process, device, means, method or steps. Accordingly, the appended claims are intended to include within their scope such processes, means, means, methods or steps.

Claims (5)

1. A method based on mobility electrophoresis to determine the size of a substance, characterized in that, comprising: The syringe pump injects the buffer solution into the sampling end of the capillary for a first period of time, and then the sampling pump injects the sample solution into the sampling end of the capillary. After the sampling is completed, the syringe pump injects the sample into the capillary again. Inject a buffer into the capillary while applying a separation voltage between the two ends of the capillary; The detector detects at the detection window on the capillary, and obtains the peak time t _M of the neutral marker and the peak time t of the sample; according to Obtain the sample equivalent spherical radius characteristic curve; where under the experimental conditions, U is the separation voltage applied at both ends of the capillary, L is the full length of the capillary, Lreal is the length from the sampling end of the capillary to the detection window, q is the ion charge, η is the viscosity coefficient of the solution, and m is the molar mass of the sample substance , R is the radius of the sample ion equivalent sphere; Matching the peak-exit time t of the sample to the sample equivalent sphere radius characteristic curve to obtain the corresponding ion equivalent sphere radius R of the sample; Obtain a sample ellipse reconstruction limit curve according to the ion equivalent sphere radius R; Using molecular simulation to obtain the possible conformation library of the sample and then obtain the aspect ratio and equivalent windward radius of the possible conformation; The aspect ratio of the possible conformation and the equivalent windward radius are matched to the ellipse reconstruction limit curve of the sample to obtain the conformational distribution of the sample under the experimental conditions. 2. The method for determining the size of a substance based on mobility electrophoresis according to claim 1, wherein the molecular simulation is used to obtain the possible conformation library of the sample and then obtain the aspect ratio and the equivalent windward radius of the possible conformation , including: Setting the simulation conditions to be consistent with the experimental conditions, and using molecular dynamics simulation to obtain a possible conformation library of the sample; According to the radius of gyration in the three directions of x, y, and z of the possible conformation library of the sample, the ellipse characteristic parameters of the possible conformation are obtained; The aspect ratio of the possible conformation and the equivalent windward radius are obtained based on the ellipse characteristic parameters. 3. the method for measuring material size based on mobility electrophoresis according to claim 1, is characterized in that, described method also comprises: Determine the distance between any possible conformation and the ellipse reconstruction limit curve of the sample in the y direction or relative distance Determine the smallest possible conformation of d _i or Δd _i as the conformation distribution of the sample under the experimental conditions. 4. The method for determining the size of a substance based on mobility electrophoresis according to claim 2, wherein the simulation time of the molecular dynamics simulation is 100 ns. 5. The method for determining the size of a substance based on mobility electrophoresis according to claim 1, wherein the detector is an optical detector or a mass spectrometer.