Protein solution conformation change analysis system

Abstract

The invention provides a protein solution conformation change analysis system. The system comprises a synchrotron radiation infrared light source module, a Fourier transform infrared spectrometer, an infrared microscope, a high voltage generation module, a data processing module and a conformation analysis and two-dimensional space distribution analysis module. The protein solution conformational change analysis system analyzes the protein conformational change in the solution in a high-pressure environment based on the synchrotron radiation infrared light source, and meets the requirements of practical application.

Description

Technical Field

[0001] The invention belongs to the technical field of protein analysis and relates to a protein solution conformation change analysis system. Background Art

[0002] High pressure, as an external disturbance factor, can cause changes in the weak interactions that maintain the conformation of biological macromolecules, leading to the occurrence of protein unfolding. Misfolding of proteins can make them easily aggregate and affect their normal physiological functions, thereby inducing the occurrence of various diseases. Therefore, it is of great significance to study the changes in protein conformation in solution under pressure. As a traditional and powerful technology, Fourier transform infrared spectroscopy can study the changes in protein secondary structure under high pressure from the perspective of molecular vibration.

[0003] The common high-pressure generating device consists of a pair of diamond anvils, and the size of the anvil surface and the thickness of the gasket determine the pressure that the device can generate. Usually, an anvil surface of several hundred microns can generate the pressure required for protein conformational changes. The spot size of a traditional infrared spectrometer is about 5mm, which is much larger than the size of the diamond anvil observation window. It is necessary to install optical components such as focusing mirrors in the main sample compartment of the spectrometer to condense the infrared spot in order to obtain sufficient infrared light energy. This method is inconvenient to operate and cannot observe the changes in the protein solution in real time. The infrared microscope can detect the changes in its infrared signal while observing the changes in the protein solution in the anvil pressure chamber.

[0004] However, if a traditional thermal radiation light source is used, when the microscope lens is 15x and the aperture is less than 30×30μm, the signal-to-noise ratio of the spectrum is poor and cannot provide effective signals. Protein solutions can form aggregated particles smaller than 30μm after folding under high pressure, so the use of traditional infrared spectroscopy microscopy to study the structural changes of protein particles is limited. Summary of the invention

[0005] The present invention provides a protein solution conformation change analysis system, which analyzes protein conformation changes in a solution in a high-pressure environment based on a synchrotron radiation infrared light source to meet the needs of practical applications.

[0006] The invention provides a protein solution conformation change analysis system, comprising a synchrotron radiation infrared light source module, a Fourier transform infrared spectrometer, an infrared microscope and a high voltage generation module, a data processing module and a conformation analysis and two-dimensional spatial distribution analysis module; the synchrotron radiation infrared light source module is used to provide a synchrotron radiation infrared light source; the Fourier transform infrared spectrometer is used to convert a parallel incident synchrotron radiation infrared light source into interference infrared light; the infrared microscope and high voltage generation module comprises an infrared microscope and a high voltage generation module, which are used to apply high voltage to a protein solution based on the high voltage generation module, convert the interference infrared light into a focused light spot based on the infrared microscope, and collect infrared signals after the focused light spot is projected onto the protein solution to which high voltage is applied; the data processing module is used to obtain a single-point infrared spectrum and two-dimensional infrared spectrum imaging of the protein solution based on the infrared signal; the conformation analysis and two-dimensional spatial distribution analysis module is used to perform conformation analysis and two-dimensional spatial distribution analysis on the protein solution based on the single-point infrared spectrum and two-dimensional infrared spectrum imaging.

[0007] In one embodiment of the present invention, the high-voltage generating module is arranged on the sample stage of the infrared microscope. Two diamond anvils in the high-voltage generating module are arranged on a sleeve opposite to each other and separated by a gasket in the middle. The hole wall of the gasket and the anvil surfaces of the two diamond anvils constitute a closed space. The closed space contains a pressure transmission medium, a standard pressure substance and the protein solution.

[0008] In one embodiment of the present invention, the anvil is made of IIa type diamond, the gasket is made of stainless steel or rhenium, and the pressure transmission medium is D 2 O, the standard pressure material is barium sulfate or α-quartz.

[0009] In one embodiment of the present invention, the resolution of the single-point infrared spectrum is 4 cm -1 , the scanning number is 64 times, the single-point scanning aperture size is 30x30μm; the scanning aperture size of two-dimensional infrared spectral imaging is 20x20μm, and the step size is 3x3μm.

[0010] In one embodiment of the present invention, the conformation analysis and two-dimensional spatial distribution analysis module includes a solution system pressure calibration module, a second-order derivative spectrum calculation module, a peak fitting module, a two-dimensional correlation analysis and a two-dimensional spatial distribution analysis module;

[0011] The solution system pressure calibration module is used to calculate the system pressure of the protein solution based on the peak shift of the standard pressure substance;

[0012] The second-order derivative spectrum calculation module is used to calculate the second-order derivative spectrum of the protein solution based on the single-point infrared spectrum under different pressures to analyze the changes in the secondary structure of the protein in the protein solution;

[0013] The peak fitting module is used to perform baseline correction and peak fitting based on the single-point infrared spectrum amide I' band to obtain the relative content changes of each secondary structure of the protein in the protein solution;

[0014] The two-dimensional correlation analysis and two-dimensional spatial distribution analysis module is used to analyze the movement sequence of the secondary structure of the protein in the protein solution under the action of pressure based on the two-dimensional correlation calculation of the single-point infrared spectrum.

[0015] In one embodiment of the present invention, the system pressure P = aΔv 2 +bΔv+c, wherein Δv is the peak displacement of the standard pressure substance in the pressure sensitive spectral band, and a, b and c are preset coefficients.

[0016] In one embodiment of the present invention, the second-order derivative spectrum calculation module first performs a smoothing process based on the Savitzky-Golay smoothing algorithm on the single-point infrared spectrum to obtain a smoothed spectrum group; and then performs a second-order derivative spectrum calculation on the smoothed spectrum group to obtain the second-order derivative spectrum.

[0017] In one embodiment of the present invention, the peak fitting module performs baseline correction and peak fitting on the single-point infrared spectrum amide I' band, and then calculates the ratio of each secondary structure peak area to the total peak area to obtain the relative content change of each secondary structure.

[0018] In one embodiment of the present invention, the two-dimensional correlation analysis and two-dimensional spatial distribution analysis module first performs a smoothing process on the single-point infrared spectrum based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group, then performs a two-dimensional correlation calculation on the smoothed spectrum group, and finally analyzes the movement order of the protein secondary structure under pressure through the results of synchronous and asynchronous two-dimensional correlation spectra.

[0019] In one embodiment of the present invention, the two-dimensional correlation analysis and two-dimensional spatial distribution analysis module adds the relative content of the protein secondary structure at each pixel point in the infrared spectrum imaging image according to the pixel position to obtain a protein secondary structure content distribution map.

[0020] As described above, the protein solution conformation change analysis system of the present invention has the following beneficial effects:

[0021] (1) Synchrotron radiation spectrum acquisition fully combines the advantages of the high brightness of synchrotron radiation light. The minimum spot size at the protein solution sample can reach 5x5μm. At the same time, the spectrum has a high signal-to-noise ratio and its spatial resolution can reach the diffraction limit of infrared light. It has good secondary structure analysis capabilities for protein aggregates larger than 5μm in size, and can realize real-time observation of changes in protein solutions while collecting infrared spectra, and can achieve high-precision, high-spatial-resolution infrared spectral imaging;

[0022] (2) The overall assembly height of the high-pressure generating module is controlled below 22 mm, which is suitable for the working distance of the 15x lens of the infrared microscope; when the diamond anvil surface is 400 μm and the gasket thickness is 100 μm, the maximum pressure of the high-pressure module can reach 10 GPa, which fully meets the pressure range of protein conformational transition;

[0023] (3) By calculating the second-order derivative spectra under different pressures, we can obtain information about the changes in the peak position and intensity of each secondary structure of the protein with pressure; by fitting the peaks of the amide I' band, we can obtain the changes in the relative content of each secondary structure of the protein; by calculating the two-dimensional correlation spectrum, we can obtain the order of movement of each secondary structure of the protein under pressure induction; by analyzing the two-dimensional spatial imaging of protein aggregates, we can obtain the changes in the distribution of the content and secondary structure of the protein in the bulk solution and aggregates. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a flow chart of a protein solution conformational change analysis system in one embodiment of the present invention;

[0025] Figure 2 Shown is a schematic diagram of a single-point infrared spectrum under different pressures in one embodiment of the present invention;

[0026] Figure 3 Display as Figure 2 Schematic diagram of the second-order derivative spectrum corresponding to a single-point infrared spectrum;

[0027] Figure 4 It is shown as 1700-1600cm during the pressure increase process of the present invention. -1 A schematic diagram of a synchronous two-dimensional correlation spectrum at in one embodiment;

[0028] Figure 5 It is shown as 1700-1600cm during the pressure increase process of the present invention. -1 A schematic diagram of an asynchronous two-dimensional correlation spectrum at in one embodiment;

[0029] Figure 6 Shown is a two-dimensional spatial imaging diagram of protein aggregated particles under high pressure in the present invention. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0031] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0032] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0033] like Figure 1 As shown, in one embodiment, the protein solution conformational change analysis system of the present invention includes a synchrotron radiation infrared light source module 1, a Fourier transform infrared spectrometer 2, an infrared microscope and high voltage generation module 3, a data processing module 4 and a conformation analysis and two-dimensional spatial distribution analysis module 5.

[0034] The synchrotron radiation infrared light source module 1 is used to provide a synchrotron radiation infrared light source. The synchrotron radiation infrared light source has the characteristics of high brightness, and its spatial resolution can reach the diffraction limit of infrared light. Therefore, the protein solution conformation change analysis system of the present invention is a system for in-situ studying the changes of the secondary structure of proteins in the solution under pressure induction based on synchrotron radiation infrared microscopy technology, and analyzing the protein conformation change.

[0035] The Fourier transform infrared spectrometer 2 is used to convert the parallel incident synchrotron radiation infrared light source into interference infrared light. The high brightness synchrotron radiation light source is parallel incident on the reflector of the Fourier transform infrared spectrometer 2 and reflected to the spectrometer interferometer, thereby obtaining the interference infrared light.

[0036] The infrared microscope and high-voltage generating module 3 include an infrared microscope and a high-voltage generating module, which are used to apply high voltage to the protein solution based on the high-voltage generating module, convert the interfering infrared light into a focused light spot based on the infrared microscope, and collect the infrared signal after the focused light spot is projected onto the protein solution to which high voltage is applied. Among them, the interfering infrared light passes through the focusing lens, diaphragm, objective lens and other optical elements of the infrared microscope in sequence to generate a micron-level high-brightness focused light spot at the sample stage. The high-voltage generating module applies high voltage to the protein solution; the focused light spot passes through the high-voltage generating module, and the infrared light carrying the sample information is collected by the condenser of the microscope, and is incident on the infrared light detector after being focused by optical elements such as the diaphragm and focusing lens, thereby obtaining the infrared signal.

[0037] In one embodiment, the high-pressure generating module adopts a diamond anvil structure design. Two pads inlaid with diamond anvils are fixed to the upper and lower sleeves with screws respectively. After the upper and lower sleeves are aligned and assembled, they are tightened with four screws. The pressure of the pressure chamber can be controlled according to the tightness of the screws. The two diamond anvils are against each other, and the middle is separated by a gasket, which plays a buffering role and prevents the anvil from being broken due to uneven stress during the high-pressure generation process. The hole wall of the gasket and the anvil surface of the upper and lower diamond anvils form a closed space, and the pressure transmission medium, the standard pressure substance and the protein solution sample are placed in the closed space. Preferably, the diamond anvil used for the high-pressure generating module uses IIa type diamond, which has good transmittance and fewer impurity peaks in the mid-infrared band and is transparent to visible light. During infrared spectrum acquisition and infrared imaging, the aggregation behavior of the protein solution can be observed in real time through the camera of the infrared microscope. The gasket can be made of stainless steel, rhenium, etc.; the protein solution system can directly use water solvent as the pressure transmission medium. Most pressure-induced conformational changes of proteins occur in the pressure range of 500-600 MPa. The standard pressure material is barium sulfate or α-quartz. The pressure response ranges of these two materials cover the pressure range of protein conformational changes.

[0038] Since accurate two-dimensional infrared spectrum imaging is required, the high-voltage generating module needs to be placed on the high-precision automatic sample stage of the infrared microscope to achieve automatic point-by-point scanning imaging. Therefore, a rectangular sample holder with a groove and a circular light hole at the bottom is fixed on the automatic sample stage of the infrared microscope by four screws. The width of the groove is equivalent to the outer diameter of the sleeve of the high-voltage generating module, and the diameter of the circular light hole is slightly smaller than the outer diameter of the sleeve of the high-voltage module, so that the high-voltage generating module can be quickly and accurately reset to the sample stage after each pressurization and decompression; at the same time, the overall loading height of the high-voltage generating module at the sample stage of the infrared microscope can be reduced. In addition, in order to adapt to the working distance of the infrared microscope objective, it is necessary to assemble a sleeve, a gasket and a sealing gasket of appropriate height to control the vertical height of the high-voltage generating module below 22mm.

[0039] In one embodiment, when preparing the protein solution, due to H 2 O at 1700-1600cm -1 There is strong absorption at the amide I band. To eliminate its influence on the amide I band, D 2 O was used as solvent. The protein to be tested was required to undergo sufficient hydrogen-deuterium exchange, and the deuterated amide I band was labeled as amide I'. 2 O as a solvent can also increase the optical path length and thus reduce the concentration of the protein to prevent the occurrence of irreversible aggregation. Preferably, the prepared protein solution is loaded into the high pressure generating module together with the standard pressure substance using a syringe needle or a tungsten needle.

[0040] The data processing module 4 is used to obtain the single-point infrared spectrum and two-dimensional infrared spectrum imaging of the protein solution based on the infrared signal. The data processing module 4 performs Fourier transform and other processing on the obtained infrared signal to obtain the single-point infrared spectrum and two-dimensional infrared spectrum imaging of the protein solution.

[0041] The conformation analysis and two-dimensional spatial distribution analysis module 5 is used to perform conformation analysis and two-dimensional spatial distribution analysis on the protein solution based on the single-point infrared spectrum and the two-dimensional infrared spectrum imaging. In one embodiment, the resolution of the single-point infrared spectrum is 4 cm -1 , the scanning number is 64 times, the single-point scanning aperture size is 30x30μm; the scanning aperture size of two-dimensional infrared spectral imaging is 20x20μm, and the step size is 3x3μm.

[0042] In one embodiment, the conformation analysis and two-dimensional spatial distribution analysis module 5 includes a solution system pressure calibration module, a second-order derivative spectrum calculation module, a peak fitting module, a two-dimensional correlation analysis and a two-dimensional spatial distribution analysis module.

[0043] The solution system pressure calibration module is used to calculate the system pressure of the protein solution based on the peak displacement of the standard pressure substance. Wherein, the system pressure P = aΔv 2 +bΔv+c, where Δv is the peak displacement of the pressure-sensitive band of the standard pressure substance, and a, b and c are preset coefficients. For example, when the standard pressure substance is barium sulfate, the pressure-sensitive band of barium sulfate is located at 983 cm -1 The barium sulfate and protein solution samples were simultaneously loaded into the high pressure generation module, and the barium sulfate was measured at 983 cm -1 The corresponding system pressure is calculated by the peak shift at , where a = 0.031, b = 2.097, and c = 0.0.

[0044] The second-order derivative spectrum calculation module is used to calculate the second-order derivative spectrum of the protein solution based on the single-point infrared spectrum under different pressures to analyze the changes in the secondary structure of the protein in the protein solution. First, the single-point infrared spectrum obtained under different pressures is smoothed based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group; then the second-order derivative spectrum of the smoothed spectrum group is calculated to obtain the second-order derivative spectrum at different pressures. The number of smoothing points can be 5 points, and the second-order derivative calculation can use a third-order polynomial and 9-point smoothing. The main secondary structure peak position of the protein: α-helix 1654cm -1 , β-folding 1637cm -1 , antiparallel β fold 1615cm -1 and 1685cm -1 , random curl 1645cm -1 , corner 1683-1662cm -1 According to the protein amide I' band (1700-1600cm -1 ) can be used to analyze the increase or decrease of the secondary structure content in the system by the enhancement and weakening of the characteristic peaks of each secondary structure in the second-order derivative spectrum; the transition between the secondary structures in the system can be judged according to the wavenumber shift of the characteristic peaks of the secondary structure in the second-order derivative spectrum.

[0045] The peak fitting module is used to perform baseline correction and peak fitting based on the amide I' band of the single-point infrared spectrum to obtain the relative content changes of each secondary structure of the protein in the protein solution. The peak fitting module selects the amide I' band (1700-1600cm -1 ), perform baseline correction, and then perform peak fitting. Then calculate the ratio of each secondary structure peak area to the total peak area, and the ratio is the relative content of the secondary structure. The change in the relative content of each secondary structure is obtained by changing the ratio.

[0046] The two-dimensional correlation analysis and two-dimensional spatial distribution analysis module is used to analyze the movement order of the secondary structure of the protein in the protein solution under pressure based on the two-dimensional correlation calculation of the single-point infrared spectrum. Among them, the two-dimensional correlation analysis and two-dimensional spatial distribution analysis module first performs a smoothing process based on the Savitzky-Golay smoothing algorithm on the single-point infrared spectrum to obtain a smoothed spectrum group, then performs a two-dimensional correlation calculation on the smoothed spectrum group, and finally analyzes the movement order of the secondary structure of the protein under pressure through the results of synchronous and asynchronous two-dimensional correlation spectra. In one embodiment, in the change interval P of the protein solution system disturbed by pressure P min and P max Select m dynamic spectra at equal intervals within max -p min ) / (m-1). The intensity of the synchronous two-dimensional correlation spectrum can be estimated as where v 1 and v 2 are infrared optical variables at different wave numbers, is the pressure disturbance p j The infrared light intensity of the point, The intensity of the asynchronous two-dimensional correlation spectrum can be estimated as Where N jk represents the element in the jth row and kth column of the Hilbert-Noda transformation matrix,

[0047] The rules for determining the movement order of different secondary structures under pressure disturbance are as follows:

[0048] (1) If the synchronous correlation peak φ(v 1 , v 2 )>0, at v 1 >v 2 In the region of asynchronous cross peak ψ(v 1 , v 2 )>0, it means that the spectral intensity changes at high wavenumber v 1 Prior to low wave number v 2 occurs at v 1 The movement of the relevant secondary structure represented by v takes precedence over 2 The motion of the secondary structure represented by the asynchronous cross peak ψ(v 1 , v 2 )<0, it means that the spectral intensity changes at high wavenumber v 1 Lagging behind low wavenumber v 2 At v 1 The movement of the related secondary structure represented by v 2 The movement of the secondary structure represented by .

[0049] (2) If the synchronous correlation peak φ(v 1 , v 2 )<0, the above rules for determining the movement order of the secondary structure are opposite.

[0050] (3) If there is only a synchronous correlation peak, the asynchronous cross peak ψ(v 1 , v 2 )=0, then v 1 , v 2 The changes in the secondary structure represented by the residues occur simultaneously.

[0051] (4) If the synchronous correlation peak φ(v 1 , v 2 )=0, then v 1 , v 2 The change order of the secondary structure represented by the two-dimensional infrared correlation spectrum cannot be determined. The two-dimensional infrared correlation spectrum has a high resolution and can distinguish the small peaks and weak peaks covered by the one-dimensional spectrum; at the same time, the movement order of each secondary structure relative to the pressure disturbance can be effectively determined by analyzing the synchronous correlation peaks and asynchronous cross peaks.

[0052] In addition, the two-dimensional correlation analysis and two-dimensional spatial distribution analysis modules add the relative content of the secondary structure of the protein at each pixel point in the infrared spectrum imaging diagram according to the pixel position to obtain the distribution diagram of the secondary structure content of the protein. The infrared spectrum information of each pixel point in the selected area is obtained by automatic point-by-point scanning of the microscope stage; the infrared spectrum diagram of each pixel point is integrated with the peak area in the band of 1700-1600cm-1 using Cytospec software, and then assembled into a two-dimensional infrared spectrum imaging according to the spatial position of each pixel point. The infrared spectrum imaging diagram of each pixel point is subjected to peak fitting to obtain the relative content of the secondary structure of the protein at each pixel point, and then the distribution diagrams of different secondary structure contents are obtained according to the pixel position combination, so as to analyze the difference in the secondary structure of the protein in the bulk solution and the aggregate.

[0053] The protein solution conformational change analysis system of the present invention is further described below through specific examples.

[0054] In this embodiment, a beam of high-brightness synchrotron radiation infrared light source is incident on the Fourier transform infrared spectrometer in parallel, and a beam of interference infrared light is generated by the Fourier transform infrared spectrometer. The interference infrared light passes through the focusing lens, diaphragm, objective lens and other optical elements of the infrared microscope to generate a micron-level high-brightness focused light spot at the sample stage.

[0055] Use heavy water to prepare a regenerated silk protein solution with a concentration of about 50 mg / ml, and let it stand for a period of time to allow the protein in the solution to undergo sufficient hydrogen-deuterium exchange. The high-voltage generation module uses a type IIa diamond with an anvil surface of 400 μμm. The upper and lower diamond anvils are fixed on two pads respectively and aligned before use. The gasket is made of stainless steel with a central hole diameter of 400 μm. Assemble the sleeve, pad and diamond anvil in sequence, place the stainless steel gasket with a hole in the center of the diamond anvil surface, and support it with plasticine around it. Use a syringe to absorb a certain amount of regenerated silk protein solution and transfer it to the center of the gasket hole; use a tungsten needle to transfer barium sulfate to the center of another diamond anvil surface. Align the center positions of the upper and lower sleeves, press them together, and tighten the four screws.

[0056] Install the rectangular sample holder on the sample stage of the infrared microscope, and place the high-voltage generating module loaded with samples on the rectangular sample holder. After the focused light spot passes through the high-voltage generating module, the infrared light carrying the sample information is collected by the condenser of the microscope and then incident on the infrared light detector after passing through optical elements such as the aperture and focusing mirror. The data processing module performs spectral acquisition and two-dimensional spatial imaging to obtain single-point infrared spectra and two-dimensional infrared spectral imaging.

[0057] In this embodiment, the single-point spectrum acquisition resolution is 4 cm -1 , scanning times 64 times, aperture size 30x30μm; protein aggregate particle imaging aperture size 20x20μm. Adjust the tightness of the four screws of the high pressure generation module to control the pressure of the system to obtain a series of single-point infrared spectra at different pressures. The pressure-sensitive band of barium sulfate is at 983cm -1 Nearby, the displacement through this band is based on the formula P = aΔv 2 +bΔv+c calibrates the system pressure, where a=0.031, b=2.097, c=0.0. Figure 2 The single-point infrared spectrum shown is smoothed based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group; then the second-order derivative spectrum is calculated to obtain the following Figure 3 The second-order derivative spectra of different pressures are shown. The number of smoothing points can be 5 points, and the second-order derivative calculation can be done using a third-order polynomial and 9-point smoothing. Under the action of pressure, the center of the amide I' band of regenerated silk protein moves to a low wave number, which shows that the secondary structure of the protein has changed; from the second-order derivative spectrum, it can be seen that the β conformation increases and the random coil conformation decreases. Select the single-point infrared spectrum amide I' band (1700-1600cm -1 ) for baseline correction, and then the peak fitting was performed to obtain the ratio of each conformation. The obtained single-point infrared spectrum was smoothed based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group, and then a two-dimensional correlation calculation was performed to obtain Figure 4 and Figure 5 During the pressure increase process shown, 1700-1600 cm -1 The synchronous and asynchronous two-dimensional correlation spectra at 1700-1600 cm were used to identify the movement order of the secondary structure under pressure disturbance. -1 The peak area is integrated and then assembled according to the spatial position of each pixel point into Figure 6 The two-dimensional infrared spectral imaging shown shows the differences in composition and structure between protein aggregated particles and bulk solution under pressure.

[0058] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A protein solution conformational change analysis system, characterized in that: It includes synchrotron radiation infrared light source module, Fourier transform infrared spectrometer, infrared microscope and high voltage generation module, data processing module and conformation analysis and two-dimensional spatial distribution analysis module; The synchrotron radiation infrared light source module is used to provide a synchrotron radiation infrared light source; The Fourier transform infrared spectrometer is used to convert the parallel incident synchrotron radiation infrared light source into interference infrared light; The infrared microscope and high-voltage generating module include an infrared microscope and a high-voltage generating module, which are used to apply high voltage to the protein solution based on the high-voltage generating module, convert the interfering infrared light into a focused light spot based on the infrared microscope, and collect infrared signals after the focused light spot is projected onto the protein solution to which high voltage is applied; The data processing module is used to obtain the single-point infrared spectrum and two-dimensional infrared spectrum imaging of the protein solution based on the infrared signal; The conformation analysis and two-dimensional spatial distribution analysis module is used to perform conformation analysis and two-dimensional spatial distribution analysis on the protein solution based on single-point infrared spectroscopy and two-dimensional infrared spectroscopy imaging.

2. The protein solution conformation change analysis system according to claim 1, characterized in that: The high-voltage generating module is arranged on the sample stage of the infrared microscope. Two diamond anvils in the high-voltage generating module are arranged on a sleeve opposite to each other and separated by a gasket in the middle. The hole wall of the gasket and the anvil surfaces of the two diamond anvils form a closed space. The closed space contains a pressure transmission medium, a standard pressure substance and the protein solution.

3. The protein solution conformation change analysis system according to claim 2, characterized in that: The anvil is made of IIa type diamond, the gasket is made of stainless steel or rhenium, the pressure transmission medium is D2O, and the standard pressure substance is barium sulfate or α-quartz.

4. The protein solution conformation change analysis system according to claim 1, characterized in that: The resolution of the single-point infrared spectrum is 4 cm -1 , the scanning number is 64 times, the single-point scanning aperture size is 30x30μm; the scanning aperture size of two-dimensional infrared spectral imaging is 20x20μm, and the step size is 3x3μm.

5. The protein solution conformation change analysis system according to claim 1, characterized in that: The conformation analysis and two-dimensional spatial distribution analysis module includes a solution system pressure calibration module, a second-order derivative spectrum calculation module, a peak fitting module, a two-dimensional correlation analysis and a two-dimensional spatial distribution analysis module; The solution system pressure calibration module is used to calculate the system pressure of the protein solution based on the peak shift of the standard pressure substance; The second-order derivative spectrum calculation module is used to calculate the second-order derivative spectrum of the protein solution based on the single-point infrared spectrum under different pressures to analyze the changes in the secondary structure of the protein in the protein solution; The peak fitting module is used to perform baseline correction and peak fitting based on the single-point infrared spectrum amide I' band to obtain the relative content changes of each secondary structure of the protein in the protein solution; The two-dimensional correlation analysis and two-dimensional spatial distribution analysis module is used to analyze the movement sequence of the secondary structure of the protein in the protein solution under the action of pressure based on the two-dimensional correlation calculation of the single-point infrared spectrum.

6. The protein solution conformation change analysis system according to claim 5, characterized in that: The system pressure P = aΔv 2 +bΔv+c, wherein Δv is the peak displacement of the standard pressure substance in the pressure sensitive spectral band, and a, b and c are preset coefficients.

7. The protein solution conformation change analysis system according to claim 5, characterized in that: The second-order derivative spectrum calculation module first performs a smoothing process on the single-point infrared spectrum based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group; and then performs a second-order derivative spectrum calculation on the smoothed spectrum group to obtain the second-order derivative spectrum.

8. The protein solution conformation change analysis system according to claim 5, characterized in that: The peak fitting module performs baseline correction and peak fitting on the single-point infrared spectrum amide I' band, and then calculates the ratio of each secondary structure peak area to the total peak area to obtain the relative content change of each secondary structure.

9. The protein solution conformation change analysis system according to claim 5, characterized in that: The two-dimensional correlation analysis and two-dimensional spatial distribution analysis module first performs a smoothing process on the single-point infrared spectrum based on the Savitzky-Golay smoothing algorithm to obtain a smoothed spectrum group, then performs a two-dimensional correlation calculation on the smoothed spectrum group, and finally analyzes the movement sequence of the protein secondary structure under pressure through the results of synchronous and asynchronous two-dimensional correlation spectra.

10. The protein solution conformation change analysis system according to claim 5, characterized in that: The two-dimensional correlation analysis and two-dimensional spatial distribution analysis module adds the relative content of the protein secondary structure at each pixel point according to the pixel position in the infrared spectrum imaging image to obtain a protein secondary structure content distribution map.

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